Glenn King

The neurotoxic mode of action of venoms from the spider family theraphosidae

Publisher: Springer Berlin Heidelberg

Date: 07-12-2013

DOI: 10.1007/978-3-642-33989-9_15

A rational nomenclature for naming peptide toxins from spiders and other venomous animals

Publisher: Elsevier BV

Date: 08-2008

DOI: 10.1016/J.TOXICON.2008.05.020

Abstract: Molecular toxinology research was initially driven by an interest in the small subset of animal toxins that are lethal to humans. However, the realization that many venomous creatures possess a complex repertoire of bioactive peptide toxins with potential pharmaceutical and agrochemical applications has led to an explosion in the number of new peptide toxins being discovered and characterized. Unfortunately, this increased awareness of peptide-toxin ersity has not been matched by the development of a generic nomenclature that enables these toxins to be rationally classified, catalogued, and compared. In this article, we introduce a rational nomenclature that can be applied to the naming of peptide toxins from spiders and other venomous animals.

Clawing through Evolution: Toxin Diversification and Convergence in the Ancient Lineage Chilopoda (Centipedes)

Publisher: Oxford University Press (OUP)

Date: 20-05-2014

DOI: 10.1093/MOLBEV/MSU162

The Tarantula Toxin ω-Avsp1a Specifically Inhibits Human CaV3.1 and CaV3.3 via the Extracellular S3-S4 Loop of the Domain 1 Voltage-Sensor

Publisher: MDPI AG

Date: 04-05-2022

DOI: 10.3390/BIOMEDICINES10051066

Abstract: Inhibition of T-type calcium channels (CaV3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of CaV3. However, subtype-specific CaV3 inhibitors for therapeutic purposes or for studying the physiological roles of CaV3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. (“Amazonas Purple”, Peru) tarantula venom inhibited specific T-type CaV channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of CaV3.1 and CaV3.3, but only 25% of CaV3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive CaV3.2 and more sensitive CaV3.3. Our results suggest that domain-1 of CaV3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.

Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels.

Publisher: Springer Science and Business Media LLC

Date: 28-09-2016

DOI: 10.1038/SREP34333

Abstract: Voltage-sensor domains (VSDs) are modular transmembrane domains of voltage-gated ion channels that respond to changes in membrane potential by undergoing conformational changes that are coupled to gating of the ion-conducting pore. Most spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state. To understand the molecular basis underlying this mode of action, we used nuclear magnetic resonance to delineate the atomic details of the interaction between the VSD of the voltage-gated potassium channel KvAP and the spider-venom peptide VSTx1. Our data reveal that the toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating and we propose that this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels.

Cyclization of Peptides by using Selenolanthionine Bridges

Publisher: Wiley

Date: 18-09-2012

DOI: 10.1002/ANIE.201204229

No gain, no pain: Nav1.7 as an analgesic target

Publisher: American Chemical Society (ACS)

Date: 11-08-2014

DOI: 10.1021/CN500171P

Abstract: Chronic pain is one of the most complex and difficult to manage clinical problems, with the therapeutic utility of current-generation analgesics restricted by problems such as dose-limiting side effects, tolerance, and the potential for addiction. The voltage-gated sodium channel NaV1.7 plays a key role in setting the threshold for action potential generation in primary sensory neurons, and humans that lack this channel are completely insensitive to pain. In this Viewpoint, we examine the potential of NaV1.7 as an analgesic target a well as the challenges involved in developing therapeutically useful subtype-selective inhibitors of this ion channel.

Venom landscapes: Mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.TOXICON.2006.01.018

Abstract: The complexity of Australian funnel-web spider venoms has been explored via the combined use of MALDI-TOF mass spectrometry coupled with chromatographic separation and the analysis of venom-gland cDNA libraries. The results show that these venoms are far more complex than previously realized. We show that the venoms of Australian funnel-web spiders contain many hundreds of peptides that follow a bimodal distribution, with about 75% of the peptides having a mass of 3000-5000 Da. The mass spectral data were validated by matching the experimentally observed masses with those predicted from peptide sequences derived from analysis of venom-gland cDNA libraries. We show that multiple isoforms of these peptides are found in small chromatographic windows, which suggests that the wide distribution of close molecular weights among the chromatographic fractions probably reflects a ersity of structures and physicochemical properties. The combination of all predicted and measured parameters permits the interpretation of three-dimensional 'venom landscapes' derived from LC-MALDI analysis. We propose that these venom landscapes might have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms.

Site-specific pK a determination of selenocysteine residues in selenovasopressin by using 77Se NMR spectroscopy

Publisher: Wiley

Date: 14-10-2011

DOI: 10.1002/ANIE.201104169

Direct NMR evidence that prolidase is specific for the trans isomer of imidodipeptide substrates.

Publisher: American Chemical Society (ACS)

Date: 11-03-1986

DOI: 10.1021/BI00353A016

Abstract: The in vitro hydrolysis by porcine kidney prolidase of the imidodipeptide L-alanyl-L-proline was monitored by using 1H high-resolution NMR spectroscopy. The dipeptide exists as an equilibrium mixture of isomers with cis or trans conformation about the peptide bond. The 13C and 1H NMR spectra of the dipeptide displayed well-resolved resonances for each isomer. Inversion-transfer NMR spectroscopy, with a recently developed pulse sequence, was used with a range of temperatures to calculate the unitary rate constants for the exchange between isomers. A new analytical procedure was introduced for directly obtaining estimates of the unitary rate constants from inversion-transfer data. Arrhenius analysis yielded an activation energy for the isomerization of 87.0 +/- 4.1 kJ mol-1. 1H NMR time courses of the prolidase-catalyzed hydrolysis of L-alanyl-L-proline showed a faster removal of the trans isomer as the [enzyme]/[substrate] ratio was increased. The transient-kinetic information coupled with the steady-state kinetic parameters of the enzyme was used to develop two possible models of the overall hydrolytic reaction. Numerical integration of the relevant differential equations using the experimentally determined rate constants gave simulated progress curves that enabled selection of one of the proposed schemes as being the most likely this proposal entailed absolute specificity of prolidase for the trans isomer of L-alanyl-L-proline. Finally, on the basis of the present work, and information from the literature, we have proposed a new model of the active site of the enzyme.

Pharmacological Inhibition of the Voltage-Gated Sodium Channel Na_V1.7 Alleviates Chronic Visceral Pain in a Rodent Model of Irritable Bowel Syndrome

Publisher: American Chemical Society (ACS)

Date: 07-06-2021

DOI: 10.1021/ACSPTSCI.1C00072

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor.

Publisher: Springer Science and Business Media LLC

Date: 10-2010

DOI: 10.1038/NATURE09448

Abstract: The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 's le' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.

Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain

Publisher: Elsevier BV

Date: 03-2012

DOI: 10.1016/J.BIOCHI.2011.09.012

Abstract: The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform s ling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation h ered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.

Site-directed mutants of RTP of Bacillus subtilis and the mechanism of replication fork arrest

Publisher: Elsevier BV

Date: 03-1999

DOI: 10.1006/JMBI.1999.2553

Abstract: DNA replication fork arrest during the termination phase of chromosome replication in Bacillus subtilis is brought about by the replication terminator protein (RTP) bound to specific DNA terminator sequences (Ter sites) distributed throughout the terminus region. An attractive suggestion by others was that crucial to the functioning of the RTP-Ter complex is a specific interaction between RTP positioned on the DNA and the helicase associated with the approaching replication fork. In support of this was the behaviour of two site-directed mutants of RTP. They appeared to bind Ter DNA normally but were ineffective in fork arrest as ascertained by in vitro Escherichia coli DnaB helicase and replication assays. We describe here a system for assessing the fork-arrest behaviour of RTP mutants in a bona fide in vivo assay in B. subtilis. One of the previously studied mutants, RTP.Y33N, was non-functional in fork arrest in vivo, as predicted. But through extensive analyses, this RTP mutant was shown to be severely defective in binding to Ter DNA, contrary to expectation. Taken in conjunction with recent findings on the other mutant (RTP.E30K), it is concluded that there is as yet no substantive evidence from the behaviour of RTP mutants to support the RTP-helicase interaction model for fork arrest. In an extension of the present work on RTP.Y33N, we determined the dissociation rates of complexes formed by wild-type (wt) RTP and another RTP mutant with various terminator sequences. The functional wtRTP-TerI complex was quite stable (half-life of 182 minutes), reminiscent of the great stability of the E. coli Tus-Ter complex. More significant were the exceptional stabilities of complexes comprising wtRTP and an RTP double-mutant (E39K.R42Q) bound to some particular terminator sequences. From the measurement of in vivo fork-arrest activities of the various complexes, it is concluded that the stability (half-life) of the whole RTP-Ter complex is not the overriding determinant of arrest, and that the RTP-Ter complex must be actively disrupted, or RTP removed, by the action of the approaching replication fork.

Determination of the Solution Structure of a Platelet-Adhesion Peptide of von Willebrand Factor

Publisher: American Chemical Society (ACS)

Date: 11-1992

DOI: 10.1021/BI00160A028

Abstract: Two-dimensional nuclear magnetic resonance (NMR) spectroscopy in combination with distance geometry (DG) and dynamical simulated annealing (DSA) calculations have been used to determine the tertiary solution structure of a synthetic 29-residue fragment of von Willebrand factor (vWF). This fragment (D514-E542) represents an adhesion site on vWF for its platelet receptor, the glycoprotein Ib-IX complex (GP Ib-IX). The NMR data yielded 109 interproton distance measurements and two chi 1 dihedral angle constraints for use in DG and DSA calculations. Most prominent in the calculated family of solution structures was an hipathic, right-handed alpha-helix in the C-terminal segment of the peptide. We propose that this highly structured region may be important for the specific molecular interaction of vWF with the GP Ib-IX complex.

Structural basis of the potency and selectivity of Urotoxin, a potent Kv1 blocker from scorpion venom

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.BCP.2019.113782

Abstract: Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC

The venom optimization hypothesis revisited

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.TOXICON.2012.11.022

Abstract: Animal venoms are complex chemical mixtures that typically contain hundreds of proteins and non-proteinaceous compounds, resulting in a potent weapon for prey immobilization and predator deterrence. However, because venoms are protein-rich, they come with a high metabolic price tag. The metabolic cost of venom is sufficiently high to result in secondary loss of venom whenever its use becomes non-essential to survival of the animal. The high metabolic cost of venom leads to the prediction that venomous animals may have evolved strategies for minimizing venom expenditure. Indeed, various behaviors have been identified that appear consistent with frugality of venom use. This has led to formulation of the "venom optimization hypothesis" (Wigger et al. (2002) Toxicon 40, 749-752), also known as "venom metering", which postulates that venom is metabolically expensive and therefore used frugally through behavioral control. Here, we review the available data concerning economy of venom use by animals with either ancient or more recently evolved venom systems. We conclude that the convergent nature of the evidence in multiple taxa strongly suggests the existence of evolutionary pressures favoring frugal use of venom. However, there remains an unresolved dichotomy between this economy of venom use and the lavish biochemical complexity of venom, which includes a high degree of functional redundancy. We discuss the evidence for biochemical optimization of venom as a means of resolving this conundrum.

NMR methods for determining disulfide-bond connectivities

Publisher: Elsevier BV

Date: 11-2010

DOI: 10.1016/J.TOXICON.2010.06.018

Abstract: Animal toxins are the major class of secreted disulfide-rich proteins, with approximately 70% containing two or more disulfide bonds. Incorrect pairing of these disulfide bonds typically leads to a non-native three-dimensional fold accompanied by a loss of protein function. Determination of the native disulfide-bond framework is therefore a key component in the structural characterization of toxins. In this article, we review NMR approaches for elucidation of disulfide-bond connectivities. A major advantage of these NMR approaches is that they are non-invasive, leaving the s le intact at the end of the analysis for use in other studies.

Venom composition of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae) and functional characterization of a major venom peptide

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.TOXICON.2021.09.002

Calculation of symmetric multimer structures from NMR data using a priori knowledge of the monomer structure, co-monomer restraints, and interface mapping: The case of leucine zippers

Publisher: Springer Science and Business Media LLC

Date: 09-1996

DOI: 10.1007/BF00211165

Development of a sensitive peptide-based immunoassay: Application to detection of the Jun and Fos oncoproteins

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI952817O

Venom Peptides with Dual Modulatory Activity on the Voltage-Gated Sodium Channel Na_V1.1 Provide Novel Leads for Development of Antiepileptic Drugs

Publisher: American Chemical Society (ACS)

Date: 25-11-2019

DOI: 10.1021/ACSPTSCI.9B00079

Backbone and side chain NMR assignments of Geobacillus stearothermophilus ZapA allow identification of residues that mediate the interaction of ZapA with FtsZ

Publisher: Springer Science and Business Media LLC

Date: 13-05-2015

DOI: 10.1007/S12104-015-9615-1

Abstract: Bacterial ision begins with the formation of a contractile protein ring at midcell, which constricts the bacterial envelope to generate two daughter cells. The central component of the ision ring is FtsZ, a tubulin-like protein capable of self-assembling into filaments which further associate into a higher order structure known as the Z ring. Proteins that bind to FtsZ play a crucial role in the formation and regulation of the Z ring. One such protein is ZapA, a widely conserved 21 kDa homodimeric protein that associates with FtsZ filaments and promotes their bundling. Although ZapA was discovered more than a decade ago, the structural details of its interaction with FtsZ remain unknown. In this work, backbone and side chain NMR assignments for the Geobacillus stearothermophilus ZapA homodimer are described. We titrated FtsZ into (15)N(2)H-ZapA and mapped ZapA residues whose resonances are perturbed upon FtsZ binding. This information provides a structural understanding of the interaction between FtsZ and ZapA.

Resampling and Network Theory

Publisher: Institute of Electrical and Electronics Engineers (IEEE)

Date: 2022

DOI: 10.1109/TSIPN.2022.3146051

It Takes Two: Dimerization Is Essential for the Broad-Spectrum Predatory and Defensive Activities of the Venom Peptide Mp1a from the Jack Jumper Ant Myrmecia pilosula

Publisher: MDPI AG

Date: 30-06-2020

DOI: 10.3390/BIOMEDICINES8070185

Abstract: Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and erse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula, exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus, the fruit fly Drosophila melanogaster, and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure–activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types.

Two Novel Mosquitocidal Peptides Isolated from the Venom of the Bahia Scarlet Tarantula (Lasiodora klugi)

Publisher: MDPI AG

Date: 27-06-2023

DOI: 10.3390/TOXINS15070418

Abstract: Effective control of diseases transmitted by Aedes aegypti is primarily achieved through vector control by chemical insecticides. However, the emergence of insecticide resistance in A. aegypti undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquitocidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD50 of 38.3 pmol/g when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot motif. U-TRTX-Lk2a has an LD50 of 45.4 pmol/g against adult A. aegypti and its predicted structure conforms to the disulfide-directed β-hairpin motif. These spider-venom peptides represent potential leads for the development of novel control agents for A. aegypti.

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.BBAMEM.2016.02.002

Abstract: Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.

Scanning Mutagenesis of ω-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels

Publisher: Elsevier BV

Date: 10-2004

DOI: 10.1074/JBC.M404006200

The generation of 1H-NMR-Detectable mobile lipid in stimulated lymphocytes: Relationship to cellular activation, the cell cycle, and phosphatidylcholine-specific phospholipase C

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1006/BBRC.1997.7566

Abstract: Mobile lipids detected using 1H-NMR in stimulated lymphocytes were correlated with cell cycle phase, expression of the interleukin-2 receptor alpha and proliferation to assess the activation status of the lymphocytes. Mobile lipid levels, IL-2R alpha expression and proliferation increased after treatment with PMA and ionomycin. PMA or ionomycin stimulation alone induced increased IL-2R alpha expression but not proliferation. PMA- but not ionomycin-stimulation generated mobile lipid. Treatment with anti-CD3 antibody did not increase IL-2R alpha expression or proliferation but did generate increased amounts of mobile lipid. The cell cycle status of thymocytes treated with anti-CD3, PMA or ionomycin alone indicated an accumulation of the cells in the G1 phase of the cell cycle. The generation of mobile lipid was abrogated in anti-CD3 antibody-stimulated thymic lymphocytes but not in splenic lymphocytes, using a phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor which blocked cells in the G1/S phase of the cell cycle. This suggests that the 1H-NMR-detectable mobile lipid may be generated in anti-CD3 antibody-stimulated thymic lymphocytes by the action of PC-PLC activity via the catabolism of PC, in the absence of classical signs of activation.

Conformation of sarafotoxin-6b in aqueous solution determined by NMR spectroscopy and distance geometry

Publisher: Wiley

Date: 06-05-1991

DOI: 10.1016/0014-5793(91)80488-O

Abstract: The solution structure of sarafotoxin-6b in water has been determined using high-resolution NMR spectroscopy. 127 proton-proton distance measurements and three phi dihedral angle constraints derived from NMR spectra were used to calculate the solution structure using a combination of distance geometry and restrained molecular dynamics. The major structural feature of the resulting family of five structures was a right-handed alpha-helix extending from K9 to Q17. In contrast, the C-terminal region of the peptide appears not to adopt a preferred conformation in aqueous solution. The present structure is compared with those previously determined for endothelin peptides in non-aqueous solvents.

Discovery and Structure of a Potent and Highly Specific Blocker of Insect Calcium Channels

Publisher: Elsevier BV

Date: 10-2001

DOI: 10.1074/JBC.M105206200

Combination of Ambiguous and Unambiguous Data in the Restraint-driven Docking of Flexible Peptides with HADDOCK: The Binding of the Spider Toxin PcTx1 to the Acid Sensing Ion Channel (ASIC) 1a.

Publisher: American Chemical Society (ACS)

Date: 24-12-2015

DOI: 10.1021/ACS.JCIM.5B00529

Abstract: Peptides that bind to ion channels have attracted much interest as potential lead molecules for the development of new drugs and insecticides. However, the structure determination of large peptide-channel complexes using experimental methods is challenging. Thus structural models are often derived from combining experimental information with restraint-driven docking approaches. Using the complex formed by the venom peptide PcTx1 and the acid sensing ion channel (ASIC) 1a as a case study, we have examined the effect of different combinations of restraints and input structures on the statistical likelihood of (a) correctly predicting the structure of the binding interface and (b) the ability to predict which residues are involved in specific pairwise peptide-channel interactions. For this, we have analyzed over 200,000 water-refined docked structures obtained with various amounts and types of restraints of the peptide-channel complex predicted using the docking program HADDOCK. We found that increasing the number of restraints or even the use of pairwise interaction data resulted in only a modest improvement in the likelihood of finding a structure within a given accuracy. This suggests that shape complementarity and the force field make a large contribution to the accuracy of the predicted structure. The results also showed that there are large variations in the accuracy of the predicted structure depending on the precise combination of residues used as restraints. Finally, we reflect on the limitations of relying on geometric criteria such as root-mean square deviations to assess the accuracy of docking procedures. We propose that in addition to currently used measures, the likelihood of finding a structure within a given level of accuracy should be also used to evaluate docking methods.

Isolation of a funnel-web spider polypeptide with homology to mamba intestinal toxin 1 and the embryonic head inducer Dickkopf-1

Publisher: Elsevier BV

Date: 03-2000

DOI: 10.1016/S0041-0101(99)00174-9

Abstract: We have isolated and determined the amino acid sequence of a novel peptide component from the venom of the Australian funnel-web spider Hadronyche versuta. This 68-residue toxin, ACTX-Hvf17, does not function like classical neurotoxins in modulating ion channel function as evidenced by its lack of insecticidal activity and its inability to affect vertebrate smooth or skeletal muscle contractility. The peptide shows significant sequence homology with mamba intestinal toxin 1 (MIT1) and to a lesser extent with a variety of colipases. The strong structural homology between MIT1 and porcine colipase leads us to propose that ACTX-Hvf17 also adopts the MIT1/colipase three-dimensional fold. However, we show that ACTX-Hvf17 has no colipase activity and does not stimulate muscle contractility like MITI. We also show that MIT1 and ACTX-Hvf17 display significant sequence homology with the C-terminal cysteine-rich domain of the Dickkopf-1 family of proteins that induce head formation in developing embryos, which leads us to propose that this domain of Dickkopf-1 also adopts the MIT1 colipase fold.

A proton n.m.r. study of iminodipeptide transport and hydrolysis in the human erythrocyte. Possible physiological roles for the coupled system.

Publisher: Portland Press Ltd.

Date: 06-1984

DOI: 10.1042/BJ2200553

Abstract: The first description of a saturable iminodipeptide transport system present in human erythrocytes is given. The 1H-n.m.r. spectra of glycyl-L-proline and those of free glycine and L-proline are significantly different. This enabled the non-invasive monitoring by 1H-n.m.r. spectroscopy of the hydrolysis of the dipeptide in human erythrocytes and their lysates. The concentration-dependence of the rate of glycyl-L-proline hydrolysis by haemolysates was described by the Michaelis-Menten expression with Km = 14.1 +/- 2.4 mmol/litre and Vmax. = 130 +/- 10 mmol/h per litre of cell water. At concentrations of the dipeptide that saturated prolidase, hydrolysis of glycyl-L-proline by whole cells was approximately 130 times slower than by lysates. This rate difference indicated that transport is the rate-determining step in peptide hydrolysis by whole cells, and thus the concentration-dependence of the transport rate was determined. The membrane transport system was found to be saturable and could be described by the Michaelis-Menten expression with Kt = 4.7 +/- 0.4 mmol/litre and Vmax. = 0.997 +/- 0.026 mmol/h per litre of cell water. Numerical integration of a consistent set of differential rate equations that described a minimal model of the coupled transport-hydrolysis system successfully described prolonged time courses of peptide hydrolysis by whole cells. The simulations showed very low steady-state levels of dipeptide in the erythrocyte and very small lag periods (less than 5 min) in the progress curve describing the appearance of free amino acid inside the cells. The rates of transport of glycyl-L-proline into erythrocytes and kidney proximal-tubular epithelium were compared and the possible importance of erythrocyte prolidase in whole-body prolyl-peptide turnover is discussed.

Recombinant Expression of Margatoxin and Agitoxin-2 in Pichia pastoris: An Efficient Method for Production of KV1.3 Channel Blockers

Publisher: Public Library of Science (PLoS)

Date: 26-12-2012

DOI: 10.1371/JOURNAL.PONE.0052965

Cloning and activity of a novel α-latrotoxin from red-back spider venom

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BCP.2011.09.024

Abstract: The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110-140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve-muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.

The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider

Publisher: Springer Science and Business Media LLC

Date: 07-1997

DOI: 10.1038/NSB0797-559

Abstract: A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

A Strategy for production of correctly folded disulfide-rich peptides in the periplasm of E. coli

Publisher: Springer New York

Date: 2017

DOI: 10.1007/978-1-4939-6887-9_10

Abstract: Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold

Publisher: Springer Science and Business Media LLC

Date: 14-08-2018

DOI: 10.1007/S00018-018-2897-6

Abstract: Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K

Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of NaV1.7

Publisher: MDPI AG

Date: 19-02-2020

DOI: 10.3390/BIOMEDICINES8020037

Abstract: Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (NaV). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate NaV channels. Pme1a is a 35 residue peptide that inhibits NaV1.7 peak current (IC50 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits NaV1.7 peak current (EC50 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at NaV1.7 (IC50 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at NaV channels in the future.

Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1016/J.JPROT.2013.01.002

Abstract: Spider venoms represent vast sources of bioactive molecules whose ersity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~43,000 extant spider species. The present study investigated inter- and intra-species venom complexity in 18 s les collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~800 peptides in female venoms while male venoms contained ~400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200-5400m/z range and to a lesser extent the 6600-8200m/z range, consistent with previously described hexatoxins. These findings highlight the extensive ersity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~800 peptides in female venoms and ~400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species variation in venom peptide masses. We show that peptide mass distributions in all 18 venoms were centered around the 3200-5400 m/z range and to a lesser extent the 6600-8200 m/z range, consistent with the small number of previously described hexatoxins from these spiders. These findings highlight the extensive ersity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. The present study has greatly expanded our understanding of peptide variety and complexity in these lethal mygalomorph spiders. Specifically it highlights both the utility of LC-MALDI-TOF in spider taxonomy and the massive combinatorial peptide libraries that spider venoms offer the pharmaceutical and agrochemical industry.

Aphicidal efficacy of scorpion- and spider-derived neurotoxins

Publisher: Elsevier BV

Date: 08-2013

DOI: 10.1016/J.TOXICON.2013.04.015

Abstract: Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD₅₀ values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management.

Understanding the molecular basis of toxin promiscuity: The analgesic sea anemone peptide APETx2 interacts with acid-sensing ion channel 3 and hERG channels via overlapping pharmacophores

Publisher: American Chemical Society (ACS)

Date: 04-11-2014

DOI: 10.1021/JM501400P

Abstract: The sea anemone peptide APETx2 is a potent and selective blocker of acid-sensing ion channel 3 (ASIC3). APETx2 is analgesic in a variety of rodent pain models, but the lack of knowledge of its pharmacophore and binding site on ASIC3 has impeded development of improved analogues. Here we present a detailed structure-activity relationship study of APETx2. Determination of a high-resolution structure of APETx2 combined with scanning mutagenesis revealed a cluster of aromatic and basic residues that mediate its interaction with ASIC3. We show that APETx2 also inhibits the off-target hERG channel by reducing the maximal current litude and shifting the voltage dependence of activation to more positive potentials. Electrophysiological screening of selected APETx2 mutants revealed partial overlap between the surfaces on APETx2 that mediate its interaction with ASIC3 and hERG. Characterization of the molecular basis of these interactions is an important first step toward the rational design of more selective APETx2 analogues.

Backbone and side-chain 1H, 15N, and 13C assignments for chick cofilin.

Publisher: Springer Science and Business Media LLC

Date: 2002

DOI: 10.1023/A:1014227808686

Structural basis for the modulation of voltage-gated sodium channels by animal toxins

Publisher: American Association for the Advancement of Science (AAAS)

Date: 19-10-2018

DOI: 10.1126/SCIENCE.AAU2596

Abstract: In “excitable” cells, like neurons and muscle cells, a difference in electrical potential is used to transmit signals across the cell membrane. This difference is regulated by opening or closing ion channels in the cell membrane. For ex le, mutations in human voltage-gated sodium (Na v ) channels are associated with disorders such as chronic pain, epilepsy, and cardiac arrhythmia. Pan et al. report the high-resolution structure of a human Na v channel, and Shen et al. report the structures of an insect Na v channel bound to the toxins that cause pufferfish and shellfish poisoning in humans. Together, the structures give insight into the molecular basis of sodium ion permeation and provide a path toward structure-based drug discovery. Science , this issue p. eaau2486 , p. eaau2596

Holistic profiling of the venom from the lethal spiderPhoneutria nigriventerby combining high-throughput ion channel screens with venomics

Publisher: Cold Spring Harbor Laboratory

Date: 18-11-2022

DOI: 10.1101/2022.11.17.516848

Abstract: Spider venoms are a unique source of bioactive peptides, many of which display remarkable biological stability and neuroactivity. Phoneutria nigriventer , often referred to as the Brazilian wandering spider, banana spider or “armed” spider, is endemic to South America and amongst the most dangerous venomous spiders in the world. There are 4,000 envenomation accidents with P. nigriventer each year in Brazil, which can lead to symptoms including priapism, hypertension, blurred vision, sweating, and vomiting. In addition to its clinical relevance, P. nigriventer venom contains peptides that provide therapeutic effects in a range of disease models. In this study, we explored the neuroactivity and molecular ersity P. nigriventer venom using fractionation-guided high-throughput cellular assays coupled to proteomics and multi-pharmacology activity to broaden the knowledge about this venom and its therapeutic potential and provide a proof-of-concept for an investigative pipeline to study spider-venom derived neuroactive peptides. We coupled proteomics with ion channel assays using a neuroblastoma cell line to identify venom compounds that modulate the activity of voltage-gated sodium and calcium channels, as well as the nicotinic acetylcholine receptor. Our data revealed that P. nigriventer venom is highly complex compared to other neurotoxin-rich venoms and contains potent modulators of voltage-gated ion channels which were classified into four families of neuroactive peptides based on their activity and structures. In addition to the reported P. nigriventer neuroactive peptides, we identified at least 27 novel cysteine-rich venom peptides for which their activity and molecular target remains to be determined. Our findings provide a platform for studying the bioactivity of known and novel neuroactive components in the venom of P. nigriventer and other spiders and suggests that our discovery pipeline can be used to identify ion channel-targeting venom peptides with potential as pharmacological tools and to drug leads.

Backbone and Side-Chain 1H, 15N and 13C Assignments for the cis Conformer of the β Domain of the Bacterial Cell Division Protein DivIB

Publisher: Springer Science and Business Media LLC

Date: 10-2005

DOI: 10.1007/S10858-005-2590-6

Three peptide modulators of the human voltage-gated sodium channel 1.7, an important analgesic target, from the venom of an Australian Tarantula

Publisher: MDPI AG

Date: 30-06-2015

DOI: 10.3390/TOXINS7072494

Venomics: A new paradigm for natural products-based drug discovery

Publisher: Springer Science and Business Media LLC

Date: 24-02-2011

DOI: 10.1007/S00726-010-0516-4

Abstract: The remarkable potency and pharmacological ersity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical ersity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular ersity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.

Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.

Publisher: Springer Science and Business Media LLC

Date: 24-11-2015

DOI: 10.1038/NG.3153

Abstract: Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six in iduals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.

The lethal toxin from Australian funnel-web spiders is encoded by an intronless gene

Publisher: Public Library of Science (PLoS)

Date: 22-08-2012

DOI: 10.1371/JOURNAL.PONE.0043699

The Toxicogenomic Multiverse: Convergent Recruitment of Proteins Into Animal Venoms

Publisher: Annual Reviews

Date: 09-2009

DOI: 10.1146/ANNUREV.GENOM.9.081307.164356

Abstract: Throughout evolution, numerous proteins have been convergently recruited into the venoms of various animals, including centipedes, cephalopods, cone snails, fish, insects (several independent venom systems), platypus, scorpions, shrews, spiders, toxicoferan reptiles (lizards and snakes), and sea anemones. The protein scaffolds utilized convergently have included AVIT/colipase rokineticin, CAP, chitinase, cystatin, defensins, hyaluronidase, Kunitz, lectin, lipocalin, natriuretic peptide, peptidase S1, phospholipase A 2 , sphingomyelinase D, and SPRY. Many of these same venom protein types have also been convergently recruited for use in the hematophagous gland secretions of invertebrates (e.g., fleas, leeches, kissing bugs, mosquitoes, and ticks) and vertebrates (e.g., v ire bats). Here, we discuss a number of overarching structural, functional, and evolutionary generalities of the protein families from which these toxins have been frequently recruited and propose a revised and expanded working definition for venom. Given the large number of striking similarities between the protein compositions of conventional venoms and hematophagous secretions, we argue that the latter should also fall under the same definition.

Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex

Publisher: Oxford University Press (OUP)

Date: 02-1997

DOI: 10.1093/NAR/25.3.590

Abstract: Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Spider toxins: A new group of potassium channel modulators

Publisher: Springer Science and Business Media LLC

Date: 1999

DOI: 10.1023/A:1017039418046

NMR spectroscopy and X-ray crystallography provide complementary information on the structure and dynamics of leucine zippers.

Publisher: Elsevier BV

Date: 08-1996

DOI: 10.1016/S0006-3495(96)79317-7

Tentacle Morphological Variation Coincides with Differential Expression of Toxins in Sea Anemones

Publisher: MDPI AG

Date: 29-06-2021

DOI: 10.3390/TOXINS13070452

Abstract: Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails.

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.TOXICON.2016.10.013

Functional expression in Escherichia coli of the disulfide-richsea anemone peptide APETx2, a potent blocker of acid-sensing ion channel 3

Publisher: MDPI AG

Date: 23-07-2012

DOI: 10.3390/MD10071605

Were arachnids the first to use combinatorial peptide libraries?

Publisher: Elsevier BV

Date: 2005

DOI: 10.1016/J.PEPTIDES.2004.07.016

Abstract: Spiders, scorpions, and cone snails are remarkable for the extent and ersity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide ersity.

The Bacillus subtilis cell division proteins FtsL and DivIC are intrinsically unstable and do not interact with one another in the absence of other septasomal components

Publisher: Wiley

Date: 05-2002

DOI: 10.1046/J.1365-2958.2002.02920.X

Abstract: The bacterial septum appears to comprise a macromolecular assembly of essential cell ision proteins (the 'septasome') that are responsible for physically iding the cell during cytokinesis. FtsL and DivIC are essential components of this ision machinery in Bacillus subtilis. We used yeast two-hybrid analysis as well as a variety of biochemical and biophysical methods to examine the proposed interaction between Bacillus subtilis FtsL and DivIC. We show that FtsL and DivIC are thermodynamically unstable proteins that are likely to be unfolded and therefore targeted for degradation unless stabilized by interactions with other components of the septasome. However, we show that this stabilization does not result from a direct interaction between FtsL and DivIC. We propose that the observed interdependence of DivIC and FtsL stability is a result of indirect interactions that are mediated by other septasomal proteins.

Role of interfacial hydrophobic residues in the stabilization of the leucine zipper structures of the transcription factors c-Fos and c-Jun

Publisher: Elsevier BV

Date: 2002

DOI: 10.1074/JBC.M104556200

Isolation, synthesis and characterization of ω-TRTX-Cc1a, a novel tarantula venom peptide that selectively targets L-type CaV channels

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.BCP.2014.02.008

Abstract: Spider venoms are replete with peptidic ion channel modulators, often with novel subtype selectivity, making them a rich source of pharmacological tools and drug leads. In a search for subtype-selective blockers of voltage-gated calcium (CaV) channels, we isolated and characterized a novel 39-residue peptide, ω-TRTX-Cc1a (Cc1a), from the venom of the tarantula Citharischius crawshayi (now Pelinobius muticus). Cc1a is 67% identical to the spider toxin ω-TRTX-Hg1a, an inhibitor of CaV2.3 channels. We assembled Cc1a using a combination of Boc solid-phase peptide synthesis and native chemical ligation. Oxidative folding yielded two stable, slowly interconverting isomers. Cc1a preferentially inhibited Ba(2+) currents (IBa) mediated by L-type (CaV1.2 and CaV1.3) CaV channels heterologously expressed in Xenopus oocytes, with half-maximal inhibitory concentration (IC50) values of 825nM and 2.24μM, respectively. In rat dorsal root ganglion neurons, Cc1a inhibited IBa mediated by high voltage-activated CaV channels but did not affect low voltage-activated T-type CaV channels. Cc1a exhibited weak activity at NaV1.5 and NaV1.7 voltage-gated sodium (NaV) channels stably expressed in mammalian HEK or CHO cells, respectively. Experiments with modified Cc1a peptides, truncated at the N-terminus (ΔG1-E5) or C-terminus (ΔW35-V39), demonstrated that the N- and C-termini are important for voltage-gated ion channel modulation. We conclude that Cc1a represents a novel pharmacological tool for probing the structure and function of L-type CaV channels.

Trends in peptide drug discovery

Publisher: Springer Science and Business Media LLC

Date: 03-02-2021

DOI: 10.1038/S41573-020-00135-8

Abstract: Since the introduction of insulin almost a century ago, more than 80 peptide drugs have reached the market for a wide range of diseases, including diabetes, cancer, osteoporosis, multiple sclerosis, HIV infection and chronic pain. In this Perspective, we summarize key trends in peptide drug discovery and development, covering the early efforts focused on human hormones, elegant medicinal chemistry and rational design strategies, peptide drugs derived from nature, and major breakthroughs in molecular biology and peptide chemistry that continue to advance the field. We emphasize lessons from earlier approaches that are still relevant today as well as emerging strategies such as integrated venomics and peptide-display libraries that create new avenues for peptide drug discovery. We also discuss the pharmaceutical landscape in which peptide drugs could be particularly valuable and analyse the challenges that need to be addressed for them to reach their full potential.

The solution structure of the N-terminal zinc finger of GATA-1 reveals a specific binding face for the transcriptional co-factor FOG

Publisher: Springer Science and Business Media LLC

Date: 1999

DOI: 10.1023/A:1008309602929

Abstract: Zinc fingers (ZnFs) are generally regarded as DNA-binding motifs. However, a number of recent reports have implicated particular ZnFs in the mediation of protein-protein interactions. The N-terminal ZnF of GATA-1 (NF) is one such finger, having been shown to interact with a number of other proteins, including the recently discovered transcriptional co-factor FOG. Here we solve the three-dimensional structure of the NF in solution using multidimensional 1H/15N NMR spectroscopy, and we use 1H/15N spin relaxation measurements to investigate its backbone dynamics. The structure consists of two distorted beta-hairpins and a single alpha-helix, and is similar to that of the C-terminal ZnF of chicken GATA-1. Comparisons of the NF structure with those of other C4-type zinc binding motifs, including hormone receptor and LIM domains, also reveal substantial structural homology. Finally, we use the structure to map the spatial locations of NF residues shown by mutagenesis to be essential for FOG binding, and demonstrate that these residues all lie on a single face of the NF. Notably, this face is well removed from the putative DNA-binding face of the NF, an observation which is suggestive of simultaneous roles for the NF that is, stabilisation of GATA-1 DNA complexes and recruitment of FOG to GATA-1-controlled promoter regions.

Evaluation of Chemical Strategies for Improving the Stability and Oral Toxicity of Insecticidal Peptides

Publisher: MDPI AG

Date: 28-08-2018

DOI: 10.3390/BIOMEDICINES6030090

Abstract: Spider venoms are a rich source of insecticidal peptide toxins. Their development as bioinsecticides has, however, been h ered due to concerns about potential lack of stability and oral bioactivity. We therefore systematically evaluated several synthetic strategies to increase the stability and oral potency of the potent insecticidal spider-venom peptide ω-HXTX-Hv1a (Hv1a). Selective chemical replacement of disulfide bridges with diselenide bonds and N- to C-terminal cyclization were anticipated to improve Hv1a resistance to proteolytic digestion, and thereby its activity when delivered orally. We found that native Hv1a is orally active in blowflies, but 91-fold less potent than when administered by injection. Introduction of a single diselenide bond had no effect on the susceptibility to scrambling or the oral activity of Hv1a. N- to C-terminal cyclization of the peptide backbone did not significantly improve the potency of Hv1a when injected into blowflies and it led to a significant decrease in oral activity. We show that this is likely due to a dramatically reduced rate of translocation of cyclic Hv1a across the insect midgut, highlighting the importance of testing bioavailability in addition to toxin stability.

Characterization of peptide fluxes into human erythrocytes. A proton-n.m.r. study.

Publisher: Portland Press Ltd.

Date: 04-1990

DOI: 10.1042/BJ2670141

Abstract: A new protocol for measuring cellular uptake of dipeptides was developed in which the problem of peptide hydrolysis is obviated by introduction into the cell suspension of a membrane-permeant peptidase inhibitor. The uptake of unlabelled dipeptide is readily monitored so long as some analytical technique is available for measuring the intracellular peptide concentration in this study we used n.m.r. spectroscopy. Using this protocol, we demonstrated that dipeptide uptake by human erythrocytes occurs by simple diffusion through the lipid bilayer and not via a high-capacity protein-mediated transport system. Substantiating evidence includes demonstration that: (a) the fluxes are slow compared with known protein-mediated transport processes in human erythrocytes (b) the uptake is not stereospecific (c) the uptake does not display saturation kinetics (d) the fluxes are significantly enhanced by butanol (e) a distinct correlation exists between the size-corrected permeability coefficients of the dipeptides and their calculated n-octanol/water partition coefficients. It is calculated that under normal physiological conditions the diffusive fluxes of circulating plasma peptides into human erythrocytes are too small for these cells to play a significant role in dipeptide catabolism.

Horizontal gene transfer underlies the painful stings of asp caterpillars (Lepidoptera: Megalopygidae)

Publisher: Proceedings of the National Academy of Sciences

Date: 10-07-2023

DOI: 10.1073/PNAS.2305871120

Abstract: Larvae of the genus Megalopyge (Lepidoptera: Zygaenoidea: Megalopygidae), known as asp or puss caterpillars, produce defensive venoms that cause severe pain. Here, we present the anatomy, chemistry, and mode of action of the venom systems of caterpillars of two megalopygid species, the Southern flannel moth Megalopyge opercularis and the black-waved flannel moth Megalopyge crispata . We show that megalopygid venom is produced in secretory cells that lie beneath the cuticle and are connected to the venom spines by canals. Megalopygid venoms consist of large aerolysin-like pore-forming toxins, which we have named megalysins, and a small number of peptides. The venom system differs markedly from those of previously studied venomous zygaenoids of the family Limacodidae, suggestive of an independent origin. Megalopygid venom potently activates mammalian sensory neurons via membrane permeabilization and induces sustained spontaneous pain behavior and paw swelling in mice. These bioactivities are ablated by treatment with heat, organic solvents, or proteases, indicating that they are mediated by larger proteins such as the megalysins. We show that the megalysins were recruited as venom toxins in the Megalopygidae following horizontal transfer of genes from bacteria to the ancestors of ditrysian Lepidoptera. Megalopygids have recruited aerolysin-like proteins as venom toxins convergently with centipedes, cnidarians, and fish. This study highlights the role of horizontal gene transfer in venom evolution.

Domain architecture and structure of the bacterial cell division protein DivIB

Publisher: Proceedings of the National Academy of Sciences

Date: 25-04-2006

DOI: 10.1073/PNAS.0601397103

Abstract: Bacterial cytokinesis requires the coordinated assembly of a complex of proteins, collectively known as the isome, at the incipient ision site. DivIB/FtsQ is a conserved component of the isome in bacteria with cell walls, suggesting that it plays a role in synthesis and/or remodeling of septal peptidoglycan. We demonstrate that the extracytoplasmic region of DivIB comprises three discrete domains that we designate α, β, and γ from the N to C terminus. The α-domain is proximal to the cytoplasmic membrane and coincident with the polypeptide transport-associated domain that was proposed previously to function as a molecular chaperone. The β-domain has a unique 3D fold, with no eukaryotic counterpart, and we show that it interconverts between two discrete conformations via cis–trans isomerization of a Tyr–Pro peptide bond. We propose that this isomerization might modulate protein–protein interactions of the flanking α- and γ-domains. The C-terminal γ-domain is unstructured in the absence of other isomal proteins, but we show that it is critical for DivIB function.

Proteome of urticating setae of Ochrogaster lunifer, a processionary caterpillar of medical and veterinary importance, including primary structures of putative toxins

Publisher: Wiley

Date: 08-2023

DOI: 10.1002/PMIC.202300204

Abstract: Ochrogaster lunifer (Lepidoptera: Notodontidae) is an Australian processionary caterpillar with detachable urticating setae that have a defensive function. These true setae induce inflammation when they contact human skin, and equine foetal loss syndrome if they are accidentally ingested by gravid horses. We used transcriptomics and proteomics to identify proteins and peptides present in and on urticating setae, which may include toxins that contribute to inflammation and/or foetal loss syndromes. This process identified 37 putative toxins, including multiple homologues of the honeybee venom peptide secapin, and proteins with similarity to odorant binding proteins, arylphorins, and the insect immune modulator Diedel. This work identifies candidate molecules that may contribute to the adverse effects of processionary caterpillar setae on human and animal health.

The future of venoms-based drug discovery: an interview with Glenn King

Publisher: Future Science Ltd

Date: 10-2014

DOI: 10.4155/FMC.14.102

Abstract: Interview by Hannah Coaker (Commissioning Editor, Future Science Group). Glenn King secured a PhD from the University of Sydney before doing his postdoctoral studies at the University of Oxford. After academic stints at the University of Sydney and the University of Connecticut Health Center, he joined the Institute for Molecular Bioscience at The University of Queensland in 2007, where he is currently an NHMRC Principal Research Fellow. The current focus of King's research is venom-based drug discovery, in particular the development of analgesics and anti-stroke therapies. Several molecules discovered in his lab have already progressed to preclinical studies. King currently serves on the Scientific Advisory Board of three companies and the Editorial Board of four journals. He is a former President of the Australian Society for Biophysics, current President of the Australian & New Zealand Society for Magnetic Resonance and a member of the Executive Council of the International Society for Toxinology. In addition, King is a Fellow of the American Academy of Microbiology, and in 2013, he received the Sir Bob Robertson Award from the Australian Society for Biophysics and the Beckman Coulter Discovery Science Award from the Australian Society for Biochemistry & Molecular Biology.

Spider venomics: Implications for drug discovery

Publisher: Future Science Ltd

Date: 10-2014

DOI: 10.4155/FMC.14.103

Abstract: Over a period of more than 300 million years, spiders have evolved complex venoms containing an extraordinary array of toxins for prey capture and defense against predators. The major components of most spider venoms are small disulfide-bridged peptides that are highly stable and resistant to proteolytic degradation. Moreover, many of these peptides have high specificity and potency toward molecular targets of therapeutic importance. This unique combination of bioactivity and stability has made spider-venom peptides valuable both as pharmacological tools and as leads for drug development. This review describes recent advances in spider-venom-based drug discovery pipelines. We discuss spider-venom-derived peptides that are currently under investigation for treatment of a erse range of pathologies including pain, stroke and cancer.

Structural and molecular diversification of the anguimorpha lizard mandibular venom gland system in the arboreal species abronia graminea

Publisher: Springer Science and Business Media LLC

Date: 17-11-2012

DOI: 10.1007/S00239-012-9529-9

Abstract: In the past, toxinological research on reptiles has focused principally on clinically important species. As a result, our understanding of the evolution of the reptile venom system is limited. Here, for the first time, we describe the structural and molecular evolutionary features of the mandibular toxin-secreting gland of Abronia graminea, a representative of one of the poorly known and entirely arboreal lineages of anguimorph lizards. We show that the mandibular gland is robust and serous, characters consistent with those expected of a toxin-secreting gland in active use. A wide array of transcripts were recovered that were homologous to those encoded by the indisputably venomous helodermatid lizards. We show that some of these toxin transcripts are evolving under active selection and show evidence of rapid ersification. Helokinestatin peptides in particular are revealed to have accumulated residues that have undergone episodic ersifying selections. Conversely, the natriuretic peptides have evolved under tremendous evolutionary constraints despite being encoded in tandem with helokinestatins by the same gene precursor. Of particular note is the sequencing for the first time of kunitz peptides from a lizard toxin-secreting gland. Not only are kunitz peptides shown to be an ancestral toxicoferan toxin, the ancestral state of this peptide is revealed to be a dual domain encoding precursor. This research provides insight into the evolutionary history of the ancient toxicoferan reptile venom system. In addition, it shows that even 'clinically irrelevant' species can be a rich source of novel venom components, worthy of investigation for drug design and biomedical research.

The solution structure of the leucine zipper motif of the Jun oncoprotein homodimer.

Publisher: Wiley

Date: 06-1993

DOI: 10.1111/J.1432-1033.1993.TB17937.X

Abstract: Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost all proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled-coil arrangement of parallel alpha-helical strands. The rates of exchange of in idual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.

Differential hydrolysis of erythrocyte and mitochondrial membrane phospholipids by two phospholipase A2 isoenzymes (NK-PLA 2-I and NK-PLA2-II) from the venom of the Indian monocled cobra Naja kaouthia

Publisher: Elsevier BV

Date: 05-2004

DOI: 10.1016/J.ABB.2004.02.007

Olfactory bulb‐targeted quantum dot (QD) bioconjugate and Kv1.3 blocking peptide improve metabolic health in obese male mice

Publisher: Wiley

Date: 20-10-2020

DOI: 10.1111/JNC.15200

Abstract: The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the in idual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage‐dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon‐like peptide‐1. Because gene‐targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal‐histidine coordination to self‐assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch‐cl electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC 50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet‐induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini‐pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses. image

Selective Na _V 1.1 activation rescues Dravet syndrome mice from seizures and premature death

Publisher: Proceedings of the National Academy of Sciences

Date: 03-08-2018

DOI: 10.1073/PNAS.1804764115

Abstract: Spider venom is a rich source of peptides, many targeting ion channels. We assessed a venom peptide, Hm1a, as a potential targeted therapy for Dravet syndrome, the genetic epilepsy linked to a mutation in the gene encoding the sodium channel alpha subunit Na V 1.1. Cell-based assays showed Hm1a was selective for hNa V 1.1 over other sodium and potassium channels. Utilizing a mouse model of Dravet syndrome, Hm1a restored inhibitory neuron function and significantly reduced seizures and mortality in heterozygote mice. Evidence from the structure of Hm1a and modeling suggest Hm1a interacts with Na V 1.1 inactivation domains, providing a structural correlate of the functional mechanisms. This proof-of-concept study provides a promising strategy for future drug development in genetic epilepsy and other neurogenetic disorders.

The Vps4 C-terminal helix is a critical determinant for assembly and ATPase activity and has elements conserved in other members of the meiotic clade of AAA ATPases.

Publisher: Wiley

Date: 11-02-2008

DOI: 10.1111/J.1742-4658.2008.06300.X

Abstract: Sorting of membrane proteins into intralumenal endosomal vesicles, multivesicular body (MVB) sorting, is critical for receptor down regulation, antigen presentation and enveloped virus budding. Vps4 is an AAA ATPase that functions in MVB sorting. Although AAA ATPases are oligomeric, mechanisms that govern Vps4 oligomerization and activity remain elusive. Vps4 has an N‐terminal microtubule interacting and trafficking domain required for endosome recruitment, an AAA domain containing the ATPase catalytic site and a β domain, and a C‐terminal α helix positioned close to the catalytic site in the 3D structure. Previous attempts to identify the role of the C‐terminal helix have been unsuccessful. Here, we show that the C‐terminal helix is important for Vps4 assembly and ATPase activity in vitro and function in vivo , but not endosome recruitment or interactions with Vta1 or ESCRT‐III. Unlike the β domain, which is also important for Vps4 assembly, the C‐terminal helix is not required in vivo for Vps4 homotypic interaction or dominant‐negative effects of Vps4–E233Q, carrying a mutation in the ATP hydrolysis site. Vta1 promotes assembly of hybrid complexes comprising Vps4–E233Q and Vps4 lacking an intact C‐terminal helix in vitro . Formation of catalytically active hybrid complexes demonstrates an intersubunit catalytic mechanism for Vps4. One end of the C‐terminal helix lies in close proximity to the second region of homology (SRH), which is important for assembly and intersubunit catalysis in AAA ATPases. We propose that Vps4 SRH function requires an intact C‐terminal helix. Co‐evolution of a distinct Vps4 SRH and C‐terminal helix in meiotic clade AAA ATPases supports this possibility.

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer

Publisher: Elsevier BV

Date: 06-1996

DOI: 10.1074/JBC.271.23.13663

Squeezers and leaf-cutters: Differential diversification and degeneration of the venom system in toxicoferan reptiles

Publisher: Elsevier BV

Date: 07-2013

DOI: 10.1074/MCP.M112.023143

Molecular evolution of vertebrate neurotrophins: Co-option of the highly conserved nerve growth factor gene into the advanced snake venom arsenalf

Publisher: Public Library of Science (PLoS)

Date: 29-11-2013

DOI: 10.1371/JOURNAL.PONE.0081827

Insect-active toxins with promiscuous pharmacology from the African theraphosid spider Monocentropus balfouri

Publisher: MDPI AG

Date: 05-05-2017

DOI: 10.3390/TOXINS9050155

Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2

Publisher: Springer Science and Business Media LLC

Date: 11-1995

DOI: 10.1007/BF00197813

Abstract: Changes in dopaminergic neural function can be induced by an acute inflammatory state that, by altering the integrity of the neurovasculature, induces neuronal stress, cell death and causes functional deficits. Effectively blocking these effects of inflammation could, therefore, reduce both neuronal and functional decline. To test this hypothesis, we inhibited vascular adhesion protein 1 (VAP-1), a membrane-bound protein expressed on the endothelial cell surface, that mediates leukocyte extravasation and induces oxidative stress. We induced dopaminergic neuronal loss by infusing lipopolysaccharide (LPS) directly into the substantia nigra (SN) in rats and administered the VAP-1 inhibitor, PXS-4681A, daily. LPS produced: an acute inflammatory response, the loss of dopaminergic neurons in the SN, reduced the dopaminergic projection to SN target regions, particularly the dorsolateral striatum (DLS), and a deficit in habit learning, a key function of the DLS. In an attempt to protect SN neurons from this inflammatory response we found that VAP-1 inhibition not only reduced neutrophil infiltration in the SN and striatum, but also reduced the associated striatal microglia and astrocyte response. We found VAP-1 inhibition protected dopamine neurons in the SN, their projections to the striatum and promoted the functional recovery of habit learning. Thus, we reversed the loss of habitual actions, a function usually dependent on dopamine release in DLS and sensitive to striatal dysfunction. We establish, therefore, that VAP-1 inhibition has an anti-inflammatory profile that may be beneficial in the treatment of dopamine neuron dysfunction caused by an acute inflammatory state in the brain.

Nuclear Magnetic Resonance Characterization of the Jun Leucine Zipper Domain: Unusual Properties of Coiled-Coil Interfacial Polar Residues

Publisher: American Chemical Society (ACS)

Date: 09-05-1995

DOI: 10.1021/BI00018A020

Abstract: Leucine zippers constitute a widely observed structural motif which serves to promote both homo- and heterodimerization in a number of DNA-binding proteins. As part of our ongoing efforts to characterize both the structure and the dynamical properties of this dimerization domain as they relate to biological function, we report here the secondary structure in solution of a recombinant dimeric peptide (rJunLZ) comprising residues Arg276-Asn314 of the leucine zipper domain of c-Jun. Two- and three-dimensional homo- and heteronuclear NMR experiments have allowed definition of the secondary structure of rJunLZ and have provided a total of approximately 1500 interproton distance and 62 phi dihedral angle constraints for tertiary structure calculations. Amide proton protection factors, calculated from hydrogen-deuterium exchange experiments, have identified 62 hydrogen bonds in the rJunLZ dimer. We have also examined the role of Asn22, the only polar residue situated at the hydrophobic dimer interface. Virtually all leucine zipper sequences contain such a polar residue (usually Asn) near the center of the motif. X-ray crystallographic studies showed that, in the case of the GCN4 homodimer, the polar residue (Asn) adopts an asymmetric conformation in an otherwise essentially symmetric structure. In contrast, all NMR studies of leucine zipper homodimers to date have suggested that the dimers are completely symmetric in solution. We present evidence that the side-chain amide protons of Asn22 are hydrogen-bonded in solution and that this side chain exchanges rapidly between two distinct conformations. On the basis of these observations, we propose a dynamic model which can explain the apparent differences in symmetry observed in NMR and X-ray crystallographic studies of leucine zipper homodimers. We show that mutation of Asn22 to a hydrophobic Leu residue markedly increases the thermal stability of the rJunLZ homodimer, consistent with a destabilizing role for this residue. However, at temperatures below 30 degrees C, the Asn22-->Leu mutant rearranges to form oligomers larger than the dimer, as was previously observed for the corresponding Asn-->Val mutation in the GCN4 leucine zipper. These results are consistent with the hypothesis that the polar Asn residue commonly observed at the interface of leucine zippers imposes specificity for the dimer structure at the expense of stability [Harbury, P.B., Zhang, T., Kim, P.S., & Alber, T. (1993) Science 262, 1401-1407].

No evidence for bradykinin hydrolysis in human erythrocyte suspensions: 1H NMR studies.

Publisher: Wiley

Date: 06-1987

DOI: 10.1002/AJH.2830250208

Abstract: In view of their permeability to small peptides, it has been postulated that human erythrocytes may play a role in terminating the action of some circulating peptide hormones. Work using classical paper chromatographic techniques for detecting free amino acids indicated that the octapeptide, des-(Arg9)-bradykinin, enters these cells and its amino-terminal arginine residue is released by cytosolic aminopeptidase-P. We have used 1H NMR to monitor directly the release of arginine from bradykinin. The hydrolytic reaction rate in hemolysates, with an initial peptide concentration of 13.0 mmol l-1 was 6.5 mmol (1 packed red cell)-1 h-1. But no reaction was evident after a 4.5-h incubation with intact cells, thus contradicting the earlier suggestion that erythrocytes are involved in the primary inactivation of this hormone. This is consistent with our previous findings that the pentapeptide leu-enkephalin fails to enter human erythrocytes but that its lower-order degradation products may do so.

Improved efficacy of an arthropod toxin expressing fungus against insecticide-resistant malaria-vector mosquitoes.

Publisher: Springer Science and Business Media LLC

Date: 13-06-2017

DOI: 10.1038/S41598-017-03399-0

Abstract: The continued success of malaria control efforts requires the development, study and implementation of new technologies that circumvent insecticide resistance. We previously demonstrated that fungal pathogens can provide an effective delivery system for mosquitocidal or malariacidal biomolecules. Here we compared genes from arthropod predators encoding insect specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under control of a hemolymph-specific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA approved Hybrid (Ca ++ /K + channel blocker), was studied for pre-lethal effects. These included reduced blood feeding behavior, with almost 100% of insects infected with ~6 spores unable to transmit malaria within 5 days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na + channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in 5 days as single toxin strains. Our results identify a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain

Publisher: Springer Science and Business Media LLC

Date: 30-01-2014

DOI: 10.1038/NCOMMS4165

Abstract: Poor oral availability and susceptibility to reduction and protease degradation is a major hurdle in peptide drug development. However, drugable receptors in the gut present an attractive niche for peptide therapeutics. Here we demonstrate, in a mouse model of chronic abdominal pain, that oxytocin receptors are significantly upregulated in nociceptors innervating the colon. Correspondingly, we develop chemical strategies to engineer non-reducible and therefore more stable oxytocin analogues. Chemoselective selenide macrocyclization yields stabilized analogues equipotent to native oxytocin. Ultra-high-field nuclear magnetic resonance structural analysis of native oxytocin and the seleno-oxytocin derivatives reveals that oxytocin has a pre-organized structure in solution, in marked contrast to earlier X-ray crystallography studies. Finally, we show that these seleno-oxytocin analogues potently inhibit colonic nociceptors both in vitro and in vivo in mice with chronic visceral hypersensitivity. Our findings have potentially important implications for clinical use of oxytocin analogues and disulphide-rich peptides in general.

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency

Publisher: American Chemical Society (ACS)

Date: 20-10-2020

DOI: 10.1021/ACS.JMEDCHEM.0C01107

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider

Publisher: Proceedings of the National Academy of Sciences

Date: 24-01-2022

DOI: 10.1073/PNAS.2110932119

Abstract: Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.

A sequence similarity between proteins involved in initiation and termination of bacterial chromosome replication.

Publisher: Portland Press Ltd.

Date: 05-1991

DOI: 10.1042/BJ2750823

The Janus-faced atracotoxins are specific blockers of invertebrate K Ca channels

Publisher: Wiley

Date: 28-07-2008

DOI: 10.1111/J.1742-4658.2008.06545.X

Abstract: The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.

Dimethylsulphoxide and trimethylamine-N-oxide as bacterial electron transport acceptors: use of nuclear magnetic resonance to assay and characterise the reductase system in Rhodobacter capsulatus

Publisher: Springer Science and Business Media LLC

Date: 11-1987

DOI: 10.1007/BF00423135

SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

Publisher: Public Library of Science (PLoS)

Date: 22-07-2013

DOI: 10.1371/JOURNAL.PONE.0066279

The cystine knot is responsible for the exceptional stability of the insecticidal spider toxin ω-Hexatoxin-Hv1a

Publisher: MDPI AG

Date: 26-10-2015

DOI: 10.3390/TOXINS7104366

Venomics as a drug discovery platform

Publisher: Informa UK Limited

Date: 06-2009

DOI: 10.1586/EPR.09.45

Letter to the Editor: Backbone and side-chain 1H, 15N, and 13C assignments for the ? domain of the bacterial cell division protein DivIB

Publisher: Springer Science and Business Media LLC

Date: 03-2005

DOI: 10.1007/S10858-005-0178-9

Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/S1097-2765(04)00084-X

Abstract: Histidine kinases are used extensively in prokaryotes to monitor and respond to changes in cellular and environmental conditions. In Bacillus subtilis, sporulation-specific gene expression is controlled by a histidine kinase phosphorelay that culminates in phosphorylation of the Spo0A transcription factor. Sda provides a developmental checkpoint by inhibiting this phosphorelay in response to DNA damage and replication defects. We show that Sda acts at the first step in the relay by inhibiting autophosphorylation of the histidine kinase KinA. The structure of Sda, which we determined using NMR, comprises a helical hairpin. A cluster of conserved residues on one face of the hairpin mediates an interaction between Sda and the KinA dimerization hosphotransfer domain. This interaction stabilizes the KinA dimer, and the two proteins form a stable heterotetramer. The data indicate that Sda forms a molecular barricade that inhibits productive interaction between the catalytic and phosphotransfer domains of KinA.

Fifteen years of Na_V1.7 channels as an analgesic target: Why has excellent in vitro pharmacology not translated into in vivo analgesic efficacy?

Publisher: Wiley

Date: 18-12-2020

DOI: 10.1111/BPH.15327

Abstract: In 2006, humans with a congenital insensitivity to pain (CIP) were found to lack functional Na V 1.7 channels. In the subsequent 15 years there was a rush to develop selective inhibitors of Na V 1.7 channels with the goal of producing broadly effective analgesics without the problems of addiction and tolerance associated with opioids. Pharmacologically, this mission has been highly successful, leading to a number of highly potent and selective inhibitors of Na V 1.7 channels. However, there are very few ex les where these inhibitors have yielded effective analgesia in preclinical pain models or human clinical trials. In this review, we summarise the role of the Na V 1.7 channel in nociception, its history as a therapeutic target and the quest to develop potent inhibitors of this channel. Finally, we discuss possible reasons why the pain‐free state seen in humans with CIP has been so difficult to replicate pharmacologically. This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit oi/10.1111/bph.v179.14/issuetoc

The unusual conformation of cross-strand disulfide bonds is critical to the stability of β-hairpin peptides.

Publisher: Wiley

Date: 16-10-2019

DOI: 10.1002/PROT.25828

Abstract: The cross-strand disulfides (CSDs) found in β-hairpin antimicrobial peptides (β-AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα-Cα distance. While the sequence and disulfide bond connectivity of disulfide-rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of β-AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the β-AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide-rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα-Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide-rich peptides from the Protein Data Bank revealed that right-handed and left-handed rotamers are equally likely in CSDs. The previously reported preference for right-handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X-ray crystallography. Furthermore, data from MD simulations showed that the short Cα-Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of β-hairpin fold over long simulation times, restraints on the torsion angles might be required.

Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases

Publisher: Proceedings of the National Academy of Sciences

Date: 21-04-2011

DOI: 10.1073/PNAS.1017110108

Abstract: Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

NMR structure and dynamics of inhibitory repeat domain variant 12, a plant protease inhibitor from Capsicum annuum, and its structural relationship to other plant protease inhibitors

Publisher: Informa UK Limited

Date: 30-04-2019

DOI: 10.1080/07391102.2019.1607559

Abstract: Although several plant protease inhibitors have been structurally characterized using X-ray crystallography, very few have been studied using NMR techniques. Here, we report an NMR study of the solution structure and dynamics of an inhibitory repeat domain (IRD) variant 12 from the wound-inducible Pin-II type proteinase inhibitor from

Revisiting venom of the sea anemone Stichodactyla haddoni: Omics techniques reveal the complete toxin arsenal of a well-studied sea anemone genus

Publisher: Elsevier BV

Date: 08-2017

DOI: 10.1016/J.JPROT.2017.07.007

Abstract: More than a century of research on sea anemone venoms has shown that they contain a ersity of biologically active proteins and peptides. However, recent omics studies have revealed that much of the venom proteome remains unexplored. We used, for the first time, a combination of proteomic and transcriptomic techniques to obtain a holistic overview of the venom arsenal of the well-studied sea anemone Stichodactyla haddoni. A purely search-based approach to identify putative toxins in a transcriptome from tentacles regenerating after venom extraction identified 508 unique toxin-like transcripts grouped into 63 families. However, proteomic analysis of venom revealed that 52 of these toxin families are likely false positives. In contrast, the combination of transcriptomic and proteomic data enabled positive identification of 23 families of putative toxins, 12 of which have no homology known proteins or peptides. Our data highlight the importance of using proteomics of milked venom to correctly identify venom proteins eptides, both known and novel, while minimizing false positive identifications from non-toxin homologues identified in transcriptomes of venom-producing tissues. This work lays the foundation for uncovering the role of in idual toxins in sea anemone venom and how they contribute to the envenomation of prey, predators, and competitors. Proteomic analysis of milked venom combined with analysis of a tentacle transcriptome revealed the full extent of the venom arsenal of the sea anemone Stichodactyla haddoni. This combined approach led to the discovery of 12 entirely new families of disulfide-rich peptides and proteins in a genus of anemones that have been studied for over a century.

Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.BCP.2021.114693

Localization of Na_v1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function

Publisher: Informa UK Limited

Date: 03-2012

DOI: 10.4161/CHAN.19484

Abstract: Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Nav 1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Nav 1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Nav 1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Nav 1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Nav 1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.

Macromolecular NMR spectroscopy for the non-spectroscopist: Beyond macromolecular solution structure determination

Publisher: Wiley

Date: 28-01-2011

DOI: 10.1111/J.1742-4658.2011.08005.X

Abstract: A strength of NMR spectroscopy is its ability to monitor, on an atomic level, molecular changes and interactions. In this review, which is intended for non-spectroscopist, we describe major uses of NMR in protein science beyond solution structure determination. After first touching on how NMR can be used to quickly determine whether a mutation induces structural perturbations in a protein, we describe the unparalleled ability of NMR to monitor binding interactions over a wide range of affinities, molecular masses and solution conditions. We discuss the use of NMR to measure the dynamics of proteins at the atomic level and over a wide range of timescales. Finally, we outline new and expanding areas such as macromolecular structure determination in multicomponent systems, as well as in the solid state and in vivo.

Towards a generic prototyping approach for therapeutically-relevant peptides and proteins in a cell-free translation system

Publisher: Springer Science and Business Media LLC

Date: 11-01-2022

DOI: 10.1038/S41467-021-27854-9

Abstract: Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli -based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.

Entomo-venomics: The evolution, biology and biochemistry of insect venoms

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.TOXICON.2018.09.004

Abstract: The insects are a hyper erse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyper erse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.

Venom Profiling of a Population of the Theraphosid Spider Phlogius crassipes Reveals Continuous Ontogenetic Changes from Juveniles through Adulthood

Publisher: MDPI AG

Date: 25-03-2017

DOI: 10.3390/TOXINS9040116

Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6

Publisher: Wiley

Date: 28-04-2014

DOI: 10.1111/FEBS.12800

Abstract: Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I. Here, we present the backbone dynamics of apo-IL-6 as determined by analysis of NMR relaxation data with the extended model-free formalism of Lipari and Szabo. To alleviate significant resonance overlap in the HSQC-type spectra, cell-free protein synthesis was used to selectively (15) N-label residues, thereby ensuring a complete set of residue-specific dynamics. The calculated order parameters [square of the generalized model-free order parameter (S(2))] showed significant conformational heterogeneity among clusters of residues in IL-6. In particular, the N-terminal region of the long AB-loop, which corresponds spatially to one of the gp130 receptor binding epitopes (i.e. site III), experiences substantial fluctuations along the conformation of the main chain (S(2) = 0.3-0.8) that are not observed at the other two epitopes or in other cytokines. Thus, we postulate that dynamic properties of the AB-loop are responsible for inhibiting the interaction of IL-6 with gp130 in the absence of the IL-6R, and that binding of IL-6R at site I shifts the dynamic equilibrium to favor interaction with gp130 at site III. In addition, molecular dynamics simulations corroborated the NMR-derived dynamics, and showed that the BC-loop adopts different substates that possibly play a role in facilitating receptor assembly.

The Structure of the KinA-Sda Complex Suggests an Allosteric Mechanism of Histidine Kinase Inhibition

Publisher: Elsevier BV

Date: 04-2007

DOI: 10.1016/J.JMB.2007.01.064

Abstract: The Bacillus subtilis histidine kinase KinA controls activation of the transcription factor governing sporulation, Spo0A. The decision to sporulate involves KinA phosphorylating itself on a conserved histidine residue, after which the phosphate moiety is relayed via two other proteins to Spo0A. The DNA-damage checkpoint inhibitor Sda halts this pathway by binding KinA and blocking the autokinase reaction. We have performed small-angle X-ray scattering and neutron contrast variation studies on the complex formed by KinA and Sda. The data show that two Sda molecules bind to the base of the DHp dimerization domain of the KinA dimer. In this position Sda does not appear to be able to sterically block the catalytic domain from accessing its target histidine, as previously proposed, but rather may effect an allosteric mode of inhibition involving transmission of the inhibitory signal via the four-helix bundle that forms the DHp domain.

Weaponisation ‘on the fly’: Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.IBMB.2019.103310

Abstract: Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the K

A process of convergent amplification and tissue‐specific expression dominates the evolution of toxin and toxin‐like genes in sea anemones

Publisher: Wiley

Date: 05-2019

DOI: 10.1111/MEC.15084

Abstract: Members of phylum Cnidaria are an ancient group of venomous animals and rely on a number of specialized tissues to produce toxins in order to fulfil a range of ecological roles including prey capture, defence against predators, digestion and aggressive encounters. However, limited comprehensive analyses of the evolution and expression of toxin genes currently exist for cnidarian species. In this study, we use genomic and transcriptomic sequencing data to examine gene copy number variation and selective pressure on toxin gene families in phylum Cnidaria. Additionally, we use quantitative RNA-seq and mass spectrometry imaging to understand expression patterns and tissue localization of toxin production in sea anemones. Using genomic data, we demonstrate that the first large-scale expansion and ersification of known toxin genes occurs in phylum Cnidaria, a process we also observe in other venomous lineages, which we refer to as convergent lification. Our analyses of selective pressure on sea anemone toxin gene families reveal that purifying selection is the dominant mode of evolution for these genes and that phylogenetic inertia is an important determinant of toxin gene complement in this group. The gene expression and tissue localization data revealed that specific genes and proteins from toxin gene families show strong patterns of tissue and developmental-phase specificity in sea anemones. Overall, convergent lification and phylogenetic inertia have strongly influenced the distribution and evolution of the toxin complement observed in sea anemones, while the production of venoms with different compositions across tissues is related to the functional and ecological roles undertaken by each tissue type.

NaV1.1 inhibition can reduce visceral hypersensitivity

Publisher: American Society for Clinical Investigation

Date: 07-06-2018

DOI: 10.1172/JCI.INSIGHT.121000

Potent neuroprotection after stroke afforded by a double-knot spider-venom peptide that inhibits acid-sensing ion channel 1a

Publisher: Proceedings of the National Academy of Sciences

Date: 20-03-2017

DOI: 10.1073/PNAS.1614728114

Abstract: Six million people die each year from stroke, and 5 million survivors are left with a permanent disability. Moreover, the neuronal damage caused by stroke often triggers a progressive decline in cognitive function that doubles the risk of dementia for stroke survivors. Despite this massive global disease burden, there are no approved drugs for treating the neuronal injury caused to the brain by the oxygen deprivation occurring during an ischemic stroke. The precipitous drop in brain pH resulting from stroke activates acid-sensing ion channel 1a. We show that inhibition of these channels using a “double-knot” spider venom peptide massively attenuates brain damage after stroke and improves behavioral outcomes, even when the peptide is administered 8 h after stroke onset.

A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain

Publisher: Elsevier BV

Date: 09-2019

DOI: 10.1016/J.CELL.2019.07.014

Molecular phylogeny and evolution of the proteins encoded by coleoid (cuttlefish, octopus, and squid) posterior venom glands

Publisher: Springer Science and Business Media LLC

Date: 02-03-2013

DOI: 10.1007/S00239-013-9552-5

Abstract: In this study, we report for the first time a detailed evaluation of the phylogenetic history and molecular evolution of the major coleoid toxins: CAP, carboxypeptidase, chitinase, metalloprotease GON-domain, hyaluronidase, pacifastin, PLA2, SE-cephalotoxin and serine proteases, with the carboxypeptidase and GON-domain documented for the first time in the coleoid venom arsenal. We show that although a majority of sites in these coleoid venom-encoding genes have evolved under the regime of negative selection, a very small proportion of sites are influenced by the transient selection pressures. Moreover, nearly 70 % of these episodically adapted sites are confined to the molecular surface, highlighting the importance of variation of the toxin surface chemistry. Coleoid venoms were revealed to be as complex as other venoms that have traditionally been the recipient of the bulk of research efforts. The presence of multiple peptide rotein types in coleoids similar to those present in other animal venoms identifies a convergent strategy, revealing new information as to what characteristics make a peptide rotein type amenable for recruitment into chemical arsenals. Coleoid venoms have significant potential not only for understanding fundamental aspects of venom evolution but also as an untapped source of novel toxins for use in drug design and discovery.

A model genetic system for testing the in vivo function of peptide toxins

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.PEPTIDES.2006.08.026

Abstract: We have developed a model genetic system for analyzing the function of peptide toxins from animal venoms. We engineered and propagated strains of Drosophila melanogaster expressing heat-inducible transgenes encoding either kappa-ACTX-Hv1c or omega-ACTX-Hv1a, two insect-specific neurotoxic peptides found in the venom of the Australian funnel-web spider Hadronyche versuta. Heat induction of transgene expression for 20 min was sufficient to kill all transgenic flies, indicating that the ion channels targeted by these toxins are viable insecticide targets. The unusual phenotype of flies induced to express omega-ACTX-Hv1a recapitulates that of a hypomorphic allele of the high-voltage-activated calcium channel Dmca1D, suggesting that this is likely to be the target of omega-ACTX-Hv1a.

Dipteran toxicity assays for determining the oral insecticidal activity of venoms and toxins

Publisher: Elsevier BV

Date: 08-2018

DOI: 10.1016/J.TOXICON.2018.06.077

Abstract: The growing world population is placing an increasing demand on food production. In addition, abuse and misuse of chemical insecticides has led to the evolution of resistance in insect pests as well as environmental damage. Together, these developments have created a demand for new insecticidal compounds to facilitate global food production. Arachnid venom peptides provide an environmentally-friendly alternative as potential bioinsecticides given their advantages of being fully biodegradable, highly potent, and phyletically selective. However, the use of arachnid venom peptides as bioinsecticides has been questioned due to their presumed lack of oral toxicity. Thus, the aim of this work was to develop screens for oral insecticidal activity. Based on the high susceptibility of dipterans to venom peptides, fruit flies (Drosophila melanogaster) and sheep blowflies (Lucilia cuprina) were selected for screening 56 arachnid venoms. 71.4% of these venoms caused 50% or higher mortality in Drosophila, whereas 30.4% were lethal to blowflies at oral doses of 1 or 30 μg/fly, respectively. We used these assays to compare the oral and injection activity of four well-known spider venom peptides (Hv1a, Hv1c, Dc1a and Ta1a). Hv1c and Ta1a only showed weak or no oral activity in both species, while Hv1a and Dc1a showed higher oral activity in blowflies than Drosophila. Overall, we have established screens for oral toxicity in two dipteran insects. Our results indicate that oral insecticidal activity is more widespread in arachnid venoms than expected, and that some arachnid venoms and venom peptides exhibit phyletic differences in oral toxicity.

Direct Visualization of Disulfide Bonds through Diselenide Proxies Using ⁷⁷Se NMR Spectroscopy

Publisher: Wiley

Date: 17-11-2009

DOI: 10.1002/ANIE.200905206

Unique scorpion toxin with a putative ancestral fold provides insight into evolution of the inhibitor cystine knot motif.

Publisher: Proceedings of the National Academy of Sciences

Date: 13-06-2011

DOI: 10.1073/PNAS.1103501108

Abstract: The three-disulfide inhibitor cystine knot (ICK) motif is a fold common to venom peptides from spiders, scorpions, and aquatic cone snails. Over a decade ago it was proposed that the ICK motif is an elaboration of an ancestral two-disulfide fold coined the disulfide-directed β-hairpin (DDH). Here we report the isolation, characterization, and structure of a novel toxin [U 1 -liotoxin-Lw1a (U 1 -LITX-Lw1a)] from the venom of the scorpion Liocheles waigiensis that is the first ex le of a native peptide that adopts the DDH fold. U 1 -LITX-Lw1a not only represents the discovery of a missing link in venom protein evolution, it is the first member of a fourth structural fold to be adopted by scorpion-venom peptides. Additionally, we show that U 1 -LITX-Lw1a has potent insecticidal activity across a broad range of insect pest species, thereby providing a unique structural scaffold for bioinsecticide development.

Inhibition and active‐site modelling of prolidase

Publisher: Wiley

Date: 03-1989

DOI: 10.1111/J.1432-1033.1989.TB14659.X

Abstract: Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM) its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions.

Centipede venoms as a source of drug leads.

Publisher: Informa UK Limited

Date: 19-09-2016

DOI: 10.1080/17460441.2016.1235155

Abstract: Centipedes are one of the oldest and most successful lineages of venomous terrestrial predators. Despite their use for centuries in traditional medicine, centipede venoms remain poorly studied. However, recent work indicates that centipede venoms are highly complex chemical arsenals that are rich in disulfide-constrained peptides that have novel pharmacology and three-dimensional structure. Areas covered: This review summarizes what is currently known about centipede venom proteins, with a focus on disulfide-rich peptides that have novel or unexpected pharmacology that might be useful from a therapeutic perspective. The authors also highlight the remarkable ersity of constrained three-dimensional peptide scaffolds present in these venoms that might be useful for bioengineering of drug leads. Expert opinion: Like most arthropod predators, centipede venoms are rich in peptides that target neuronal ion channels and receptors, but it is also becoming increasingly apparent that many of these peptides have novel or unexpected pharmacological properties with potential applications in drug discovery and development.

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

Publisher: American Society for Microbiology

Date: 12-2010

DOI: 10.1128/JB.00783-10

Abstract: Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the isome. The Escherichia coli isome is comprised of at least 10 essential proteins whose in idual functions are mostly unknown. Most isomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the isomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the β domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the β domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.

The structure, dynamics and selectivity profile of a NaV1.7 potency-optimised huwentoxin-IV variant

Publisher: Public Library of Science (PLoS)

Date: 16-03-2017

DOI: 10.1371/JOURNAL.PONE.0173551

A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa , reveals a hyperdiverse hymenopteran toxin gene family

Publisher: American Association for the Advancement of Science (AAAS)

Date: 07-09-2018

DOI: 10.1126/SCIADV.AAU4640

Abstract: Characterization of the venom of an ant reveals a erse and multifunctional hymenopteran toxin superfamily.

The antitrypanosomal diarylamidines, diminazene and pentamidine, show anthelmintic activity against Haemonchus contortus in vitro

Publisher: Elsevier BV

Date: 06-2019

DOI: 10.1016/J.VETPAR.2019.05.008

Abstract: Parasitic nematodes pose a major threat to livestock production worldwide. The blood-feeding parasite Haemonchus contortus is a key small-ruminant pathogen that causes anaemia, and thereby seriously impacts animal health and production. Control of this parasite relies largely upon broad-spectrum anthelmintics, but new drugs are urgently needed to combat the threat of widespread multidrug resistance. Repurposing drugs can accelerate the development pipeline by reducing costs and risks, and can be an effective way of quickly bringing new antiparasitic drugs to market. Diarylamidine compounds such as pentamidine and diminazene have been employed in the treatment of trypanosomiasis and leishmaniasis in both human and veterinary settings, but their activity against parasitic worms has not yet been reported. We screened a small panel of diarylamidine compounds against H. contortus to assess their potential to be repurposed as anthelmintic drugs. Pentamidine and diminazene inhibited H. contortus larval development at low micromolar concentrations (IC

Functional Significance of the β-Hairpin in the Insecticidal Neurotoxin ω-Atracotoxin-Hv1a

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1074/JBC.M102199200

The insecticidal potential of venom peptides

Publisher: Springer Science and Business Media LLC

Date: 23-03-2013

DOI: 10.1007/S00018-013-1315-3

Abstract: Pest insect species are a burden to humans as they destroy crops and serve as vectors for a wide range of diseases including malaria and dengue. Chemical insecticides are currently the dominant approach for combating these pests. However, the de-registration of key classes of chemical insecticides due to their perceived ecological and human health risks in combination with the development of insecticide resistance in many pest insect populations has created an urgent need for improved methods of insect pest control. The venoms of arthropod predators such as spiders and scorpions are a promising source of novel insecticidal peptides that often have different modes of action to extant chemical insecticides. These peptides have been optimized via a prey-predator arms race spanning hundreds of millions of years to target specific types of insect ion channels and receptors. Here we review the current literature on insecticidal venom peptides, with a particular focus on their structural and pharmacological ersity, and discuss their potential for deployment as insecticides.

Production and packaging of a biological arsenal: Evolution of centipede venoms under morphological constraint

Publisher: Proceedings of the National Academy of Sciences

Date: 16-03-2015

DOI: 10.1073/PNAS.1424068112

Abstract: Venom peptides have attracted considerable attention because of their value as pharmacological tools and their potential for development as novel pharmaceuticals and bioinsecticides. There is also a growing interest in venoms as model evolutionary systems, particularly for understanding antagonistic coevolutionary processes. We previously demonstrated that although centipede venoms are rich in novel proteins and peptides, there are considerable differences in venom complexity between high-order taxa. We show that this disparity appears to stem from morphological limitations of the venom gland, and that most centipede venoms likely evolve under constraints imposed by low-complexity toxin production facilities. Thus, the centipede venom apparatus should be a useful model system for gaining insight into the impact of morphological constraints on venom evolution.

Australian funnel-web spiders evolved human-lethal δ-hexatoxins for defense against vertebrate predators

Publisher: Proceedings of the National Academy of Sciences

Date: 21-09-2020

DOI: 10.1073/PNAS.2004516117

Abstract: The venom of Australian funnel-web spiders contains δ-hexatoxins (δ-HXTXs) that exert fatal neurotoxic effects in humans by inhibiting inactivation of voltage-gated sodium channels, but their precise ecological role remains unclear. Sequencing of venom-gland transcriptomes from 10 funnel-web species uncovered 22 δ-HXTXs. Evolutionary analysis revealed extreme conservation of these toxins, despite their ancient origin. We isolated the lethal δ-HXTX from venom of the Sydney funnel-web spider and showed that it induces pain in mice, suggesting a role in predator deterrence. Although humans are not the target of δ-HXTXs, these toxins likely evolved to deter vertebrate predators commonly encountered by these spiders, such as bandicoots, birds, and lizards. Thus, the lethal potency of δ-HXTXs against humans is an unfortunate evolutionary coincidence.

Dracula's children: Molecular evolution of vampire bat venom

Publisher: Elsevier BV

Date: 08-2013

DOI: 10.1016/J.JPROT.2013.05.034

Abstract: While v ire bat oral secretions have been the subject of intense research, efforts have concentrated only on two components: DSPA (Desmodus rotundus salivary plasminogen activator) and Draculin. The molecular evolutionary history of DSPA has been elucidated, while conversely draculin has long been known from only a very small fragment and thus even the basic protein class was not even established. Despite the fact that v ire bat venom has a multitude of effects unaccounted by the documented bioactivities of DSPA and draculin, efforts have not been made to establish what other bioactive proteins are secreted by their submaxillary gland. In addition, it has remained unclear whether the anatomically distinct anterior and posterior lobes of the submaxillary gland are evolving on separate gene expression trajectories or if they remain under the shared genetic control. Using a combined proteomic and transcriptomic approach, we show that identical proteins are simultaneously expressed in both lobes. In addition to recovering the known structural classes of DSPA, we recovered a novel DSPA isoform as well as obtained a very large sequence stretch of draculin and thus established that it is a mutated version of the lactotransferrin scaffold. This study reveals a much more complex secretion profile than previously recognised. In addition to obtaining novel versions of scaffolds convergently recruited into other venoms (allergen-like, CRiSP, kallikrein, Kunitz, lysozyme), we also documented novel expression of small peptides related to calcitonin, PACAP, and statherin. Other overexpressed protein types included BPI-fold, lacritin, and secretoglobin. Further, we investigate the molecular evolution of various v ire bat venom-components and highlight the dominant role of positive selection in the evolution of these proteins. Conspicuously many of the proteins identified in the proteome were found to be homologous to proteins with known activities affecting vasodilation and platelet aggregation. We show that v ire bat venom proteins possibly evade host immune response by the mutation of the surface chemistry through focal mutagenesis under the guidance of positive Darwinian selection. These results not only contribute to the body of knowledge regarding haematophagous venoms but also provide a rich resource for novel lead compounds for use in drug design and development. These results have direct implications in understanding the molecular evolutionary history of v ire bat venom. The unusual peptides discovered reinforce the value of studying such neglected taxon for biodiscovery.

Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.

Publisher: Proceedings of the National Academy of Sciences

Date: 12-05-2020

DOI: 10.1073/PNAS.1914536117

Abstract: Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a erse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute % of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: Implications for human prolidase

Publisher: Springer Science and Business Media LLC

Date: 10-1998

DOI: 10.1007/S007750050257

Macromolecular NMR spectroscopy for the non-spectroscopist

Publisher: Wiley

Date: 28-01-2011

DOI: 10.1111/J.1742-4658.2011.08004.X

Abstract: NMR spectroscopy is a powerful tool for studying the structure, function and dynamics of biological macromolecules. However, non-spectroscopists often find NMR theory daunting and data interpretation nontrivial. As the first of two back-to-back reviews on NMR spectroscopy aimed at non-spectroscopists, the present review first provides an introduction to the basics of macromolecular NMR spectroscopy, including a discussion of typical s le requirements and what information can be obtained from simple NMR experiments. We then review the use of NMR spectroscopy for determining the 3D structures of macromolecules and examine how to judge the quality of NMR-derived structures.

Chemical synthesis and folding of APETx2, a potent and selective inhibitor of acid sensing ion channel 3

Publisher: Elsevier BV

Date: 07-2009

DOI: 10.1016/J.TOXICON.2009.03.014

Abstract: Acid sensing ion channels (ASICs) are pH-sensitive channels that are distributed in the central and peripheral nervous system and which are believed to play a key role in pain perception. APETx2, a 42-residue peptide toxin isolated from the sea anemone Anthopleura elegantissima, is the only known selective inhibitor of ASIC3 channels. Here we describe the total chemical synthesis of APETx2 by solid-phase peptide synthesis and native chemical ligation. The folded synthetic toxin had an IC(50) of 57 nM for inhibition of rat ASIC3 channels expressed in Xenopus oocytes, in agreement with the IC(50) reported for the native toxin (63 nM). The native chemical ligation approach should provide an efficient route for synthesis of other pharmacologically useful disulfide-rich toxins from venomous animals.

Backbone dynamics of the c-Jun leucine zipper: 15N NMR relaxation studies.

Publisher: American Chemical Society (ACS)

Date: 1996

DOI: 10.1021/BI952761Y

Structural and Biochemical Studies of Human Galanin: NMR Evidence for Nascent Helical Structures in Aqueous Solution

Publisher: American Chemical Society (ACS)

Date: 11-04-1995

DOI: 10.1021/BI00014A006

Abstract: The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3-11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha-region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein

Publisher: Wiley

Date: 10-1998

DOI: 10.1046/J.1365-2958.1998.01059.X

Abstract: MinE is an oligomeric protein that, in conjunction with other Min proteins, is required for the proper placement of the cell ision site of Escherichia coli. We have examined the self-association properties of MinE by analytical ultracentrifugation and by studies of hetero-oligomer formation in non-denaturing polyacrylamide gels. The self-association properties of purified MinE predict that cytoplasmic MinE is likely to exist as a mixture of monomers and dimers. Consistent with this prediction, the C-terminal MinE22-88 fragment forms hetero-oligomers with MinE+ when the proteins are co-expressed. In contrast, the MinE36-88 fragment does not form MinE+/MinE36-88 hetero-oligomers, although MinE36-88 affects the topological specificity of septum placement as shown by its ability to induce minicell formation when co-expressed with MinE+ in wild-type cells. Therefore, hetero-oligomer formation is not necessary for the induction of minicelling by expression of MinE36-88 in wild-type cells. The interference with normal septal placement is ascribed to competition between MinE36-88 and the corresponding domain in the complete MinE protein for a component required for the topological specificity of septal placement.

Multifunctional warheads: Diversification of the toxin arsenal of centipedes via novel multidomain transcripts

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.JPROT.2014.02.024

Abstract: Arthropod toxins are almost invariably encoded by transcripts encoding prepropeptides that are posttranslationally processed to yield a single mature toxin. In striking contrast to this paradigm, we used a complementary transcriptomic, proteomic and MALDI-imaging approach to identify four classes of multidomain centipede-toxin transcripts that each encodes multiple mature toxins. These multifunctional warheads comprise either: (1) repeats of linear peptides (2) linear peptides preceding cysteine-rich peptides (3) cysteine-rich peptides preceding linear peptides or (4) repeats of linear peptides preceding cysteine-rich peptides. MALDI imaging of centipede venom glands revealed that these peptides are posttranslationally liberated from the original gene product in the venom gland and not by proteases following venom secretion. These multidomain transcripts exhibit a remarkable conservation of coding sequences, in striking contrast to monodomain toxin transcripts from related centipede species, and we demonstrate that they represent a rare class of predatory toxins that have evolved under strong negative selection. We hypothesize that the peptide toxins liberated from multidomain precursors might have synergistic modes of action, thereby allowing negative selection to dominate as the toxins encoded by the same transcript become increasingly interdependent. These results have direct implications for understanding the evolution of centipede venoms, and highlight the importance of taking a multidisciplinary approach for the investigation of novel venoms. The potential synergistic actions of the mature peptides are also of relevance to the growing biodiscovery efforts aimed at centipede venom. We also demonstrate the application of MALDI imaging in providing a greater understanding of toxin production in venom glands. This is the first MALDI imaging data of any venom gland.

On the venom system of centipedes (Chilopoda), a neglected group of venomous animals

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.TOXICON.2011.01.004

Abstract: Centipedes are among the oldest extant terrestrial arthropods and are an ecologically important group of soil and leaf litter predators. Despite their abundance and frequent, often painful, encounters with humans, little is known about the venom and venom apparatus of centipedes, although it is apparent that these are both quite different from other venomous lineages. The venom gland can be regarded as an invaginated cuticle and epidermis, consisting of numerous epithelial secretory units each with its own unique valve-like excretory system. The venom contains several different enzymes, but is strikingly different to most other arthropods in that metalloproteases appear to be important. Myotoxic, cardiotoxic, and neurotoxic activities have been described, most of which have been attributed to high molecular weight proteins. Neurotoxic activities are also unusual in that G-protein coupled receptors often seem to be involved, either directly as targets of neurotoxins or indirectly by activating endogenous agonists. These relatively slow responses may be complemented by the rapid effects caused by histamines present in the venom and from endogenous release of histamines induced by venom cytotoxins. The differences probably reflect the ancient and independent evolutionary history of the centipede venom system, although they may also be somewhat exaggerated by the paucity of information available on this largely neglected group.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans

Publisher: Proceedings of the National Academy of Sciences

Date: 23-04-2021

DOI: 10.1073/PNAS.2023815118

Abstract: Venoms of limacodid caterpillars evolved independently to those of previously studied animals, and analysis of their venom therefore provides an opportunity to examine patterns of molecular convergence and ergence underlying the evolution of venom use. We report remarkable convergence in the recruitment of venom peptides between limacodids and other venomous taxa. Like scorpions and spiders, limacodids have weaponized immune system peptides to deter potential predators, although these cecropin-derived venom peptides are more similar in structure and mode of action to the venom peptides of hymenopteran insects. This study reveals a venom system with a unique combination of features not previously reported in other venomous animals and provides insights into the functional basis of venoms of the Limacodidae.

Modulation of insect Cav channels by peptidic spider toxins

Publisher: Elsevier BV

Date: 03-2007

DOI: 10.1016/J.TOXICON.2006.11.012

Abstract: Insects have a much smaller repertoire of voltage-gated calcium (Ca(V)) channels than vertebrates. Drosophila melanogaster harbors only a single ortholog of each of the vertebrate Ca(V)1, Ca(V)2, and Ca(V)3 subtypes, although its basal inventory is expanded by alternative splicing and editing of Ca(V) channel transcripts. Nevertheless, there appears to be little functional plasticity within this limited panel of insect Ca(V) channels, since severe loss-of-function mutations in genes encoding the pore-forming alpha1 subunits in Drosophila are embryonic lethal. Since the primary role of spider venom is to paralyze or kill insect prey, it is not surprising that most, if not all, spider venoms contain peptides that potently modify the activity of these functionally critical insect Ca(V) channels. Unfortunately, it has proven difficult to determine the precise ion channel subtypes recognized by these peptide toxins since insect Ca(V) channels have significantly different pharmacology to their vertebrate counterparts, and cloned insect Ca(V) channels are not available for electrophysiological studies. However, biochemical and genetic studies indicate that some of these spider toxins might ultimately become the defining pharmacology for certain subtypes of insect Ca(V) channels. This review focuses on peptidic spider toxins that specifically target insect Ca(V) channels. In addition to providing novel molecular tools for ion channel characterization, some of these toxins are being used as leads to develop new methods for controlling insect pests.

Letter to the editor: Backbone and side-chain 1H, 15N, and 13C assignments for the topological specificity domain of the MinE cell division protein [2]

Publisher: Springer Science and Business Media LLC

Date: 1999

DOI: 10.1023/A:1008386622578

Role of the structurally disordered N- and C-terminal residues in the Janus-faced atracotoxins

Publisher: Elsevier BV

Date: 09-2002

DOI: 10.1016/S0041-0101(02)00154-X

Abstract: The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory toxins isolated from the venom of Australian funnel-web spiders (genera Atrax and Hadronyche). In addition to a classical cystine knot motif, these toxins contain a rare vicinal disulfide bond. While the vicinal disulfide is known to be critical for insecticidal activity, the role of other residues in toxin function remains to be determined. In this study, we probed the role of the structurally disordered N- and C-terminal residues using a panel of recombinant mutants of the prototypic family member J-ACTX-Hv1c. We found that the structurally disordered C-terminal residues (Glu 36 and Pro 37) were dispensable for toxin function. However, whereas deletion of Ala 1 had minimal impact on toxin function, deletion of both Ala 1 and Ile 2 decreased insecticidal activity more than 70-fold. We propose that Ile 2 forms a part of the target binding site of J-ACTX-Hv1c.

Theoretical and practical aspects of NMR studies of cells

Publisher: Elsevier BV

Date: 04-1994

DOI: 10.1006/IMMU.1994.1011

Abstract: Various theoretical and practical aspects of biological nuclear magnetic resonance (NMR) spectroscopy that are relevant to the study of immune cells are discussed as a prelude to the following papers in this issue of ImmunoMethods. We explain some of the salient features of modern Fourier-transform NMR spectroscopy, including spectral acquisition, Fourier transformation, signal averaging, apodization, and relaxation phenomena. The major features of the one-dimensional NMR spectrum are summarized prior to a brief description of two-dimensional NMR spectroscopy. We consider various practical questions, such as which NMR-receptive nuclide might be most useful for discerning the metabolic information being sought in particular, the relative advantages and disadvantages of 1H, 13C, 19F, and 31P NMR spectroscopy are discussed in the context of elucidating metabolic information. The relative merits and pitfalls of using cell extracts, cell suspensions, and perfused immobilized cells for studies of immune cell activation are also considered.

A Proteomics and Transcriptomics investigation of the venom from the Barychelid spider Trittame loki (brush-foot trapdoor)

Publisher: MDPI AG

Date: 13-12-2013

DOI: 10.3390/TOXINS5122488

Two-Dimensional 1H NMR Studies of Membrane Changes during the Activation of Primary T Lymphocytes

Publisher: Elsevier BV

Date: 04-1994

DOI: 10.1006/IMMU.1994.1014

Abstract: Two-dimensional 1H NMR spectroscopy was used to quantify the level of "mobile" plasma membrane triglyceride and the intracellular concentrations of water-soluble phospholipid precursors during the activation of both mature and immature primary T lymphocytes. The concentration of mobile triglyceride in the plasma membrane was seen to increase approximately 35-fold during 72 h of activation of murine thymic and splenic T lymphocytes with ionomycin and phorbol 12-myristate 13-acetate. This dramatic increase in mobile plasma membrane triglyceride during the activation of both mature and immature T-lymphocyte populations supports the hypothesis that immune cell activation is associated with increased plasma membrane fluidity. The intracellular concentrations of various phospholipid precursors were shown to increase during the early stages of T-lymphocyte activation and then remain at levels above those in resting cells. This may facilitate de novo phospholipid biosynthesis, which is presumably necessary since cell volume, and hence the plasma membrane surface area, was demonstrated to increase significantly during T-lymphocyte activation. Various models that might explain the origin of the NMR-visible plasma membrane triglyceride that is observed during immune cell activation and malignant transformation are examined.

Tandem use of selective insecticides and natural enemies for effective, reduced-risk pest management

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.BIOCONTROL.2009.07.012

Bimodal Imaging of Mouse Peripheral Nerves with Chlorin Tracers

Publisher: American Chemical Society (ACS)

Date: 06-01-2021

DOI: 10.1021/ACS.MOLPHARMACEUT.0C00946

Centipede venom: Recent discoveries and current state of knowledge

Publisher: MDPI AG

Date: 25-02-2015

DOI: 10.3390/TOXINS7030679

The ω-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels

Publisher: Elsevier BV

Date: 08-2007

DOI: 10.1016/J.BCP.2007.05.017

Abstract: The omega-atracotoxins (omega-ACTX) are a family of arthropod-selective peptide neurotoxins from Australian funnel-web spider venoms (Hexathelidae: Atracinae) that are candidates for development as biopesticides. We isolated a 37-residue insect-selective neurotoxin, omega-ACTX-Ar1a, from the venom of the Sydney funnel-web spider Atrax robustus, with high homology to several previously characterized members of the omega-ACTX-1 family. The peptide induced potent excitatory symptoms, followed by flaccid paralysis leading to death, in acute toxicity tests in house crickets. Using isolated smooth and skeletal nerve-muscle preparations, the toxin was shown to lack overt vertebrate toxicity at concentrations up to 1 microM. To further characterize the target of the omega-ACTXs, voltage-cl analysis using the whole-cell patch-cl technique was undertaken using cockroach dorsal unpaired median neurons. It is shown here for the first time that omega-ACTX-Ar1a, and its homolog omega-ACTX-Hv1a from Hadronyche versuta, reversibly block both mid-low- (M-LVA) and high-voltage-activated (HVA) insect calcium channel (Ca(v)) currents. This block occurred in the absence of alterations in the voltage-dependence of Ca(v) channel activation, and was voltage-independent, suggesting that omega-ACTX-1 family toxins are pore blockers rather than gating modifiers. At a concentration of 1 microM omega-ACTX-Ar1a failed to significantly affect global K(v) channel currents. However, 1 microM omega-ACTX-Ar1a caused a modest 18% block of insect Na(v) channel currents, similar to the minor block of Na(v) channels reported for other insect Ca(v) channel blockers such as omega-agatoxin IVA. These findings validate both M-LVA and HVA Ca(v) channels as potential targets for insecticides.

Key residues characteristic of GATA N-fingers are recognized by FOG

Publisher: Elsevier BV

Date: 12-1998

DOI: 10.1074/JBC.273.50.33595

Determination of the structure of symmetric coiled-coil proteins from nmr data: application of the leucine zipper proteins jun and gcn4

Publisher: Oxford University Press (OUP)

Date: 1993

DOI: 10.1093/PROTEIN/6.6.557

Abstract: Previous attempts to determine the solution structures of homodimeric 'leucine zippers' using nuclear magnetic resonance (NMR) spectroscopy have been impeded by the complete symmetry of these coiled-coil molecules, which makes it impossible a priori to distinguish between intra- and intermonomer dipolar connectivities. Consequently, a number of ad hoc approaches have been used in an attempt to derive tertiary solution structures of these molecules from the NMR data. In this paper we present a more rigorous approach for analysing the NMR spectra of symmetric coiled-coil proteins. This analysis is based on calculations of intra- and intermonomer interproton distances in the recently determined crystal structure of the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. (1991) Science, 254, 539-543] and in symmetric coiled-coil models of the leucine zippers of GCN4 and the human oncoprotein Jun which we constructed using a dynamic simulated annealing approach. This analysis has enabled the formulation of a set of rules for interpreting the NMR spectra of symmetric coiled-coil proteins and has also led to the prediction of novel dipolar connectivities which we demonstrate in a 2-D NMR spectrum of the homodimeric Jun leucine zipper.

Histidine kinases as antimicrobial targets: Prospects and pitfalls

Publisher: Bentham Science Publishers Ltd.

Date: 11-2007

DOI: 10.2174/138955707782331759

Abstract: Histidine kinases are ubiquitous molecular sensors that are used by bacteria to detect and respond to a myriad of environmental signals. They are attractive antimicrobial targets because of their roles in mediating the virulence of pathogenic organisms, as well as the ability of bacteria to resist host defenses and develop resistance to antibiotics. In this review, we discuss the challenges involved in developing specific inhibitors of this highly erse group of kinases.

Venom peptides as therapeutics: advances, challenges and the future of venom-peptide discovery

Publisher: Informa UK Limited

Date: 13-09-2017

DOI: 10.1080/14789450.2017.1377613

Abstract: Animal venoms are complex chemical arsenals. Most venoms are rich in bioactive peptides with proven potential as research tools, drug leads and drugs. Areas covered: We review recent advances in venom-peptide discovery, particularly the adoption of combined transcriptomic roteomic approaches for the exploration of venom composition. Expert commentary: Advances in transcriptomics and proteomics have dramatically altered the manner and rate of venom-peptide discovery. The increasing trend towards a toxin-driven approach, as opposed to traditional target-based screening of venoms, is likely to expedite the discovery of venom-peptides with novel structures and new and unanticipated mechanisms of action. At the same time, these advances will drive the development of higher-throughput approaches for target identification. Taken together, these approaches should enhance our understanding of the natural ecological function of venom peptides and increase the rate of identification of novel venom-derived pharmacological tools, drug leads and drugs.

Modulatory features of the novel spider toxin μ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus

Publisher: Wiley

Date: 27-06-2017

DOI: 10.1111/BPH.13865

The structural conformation of the tachykinin domain drives the anti-tumoural activity of an octopus peptide in melanoma BRAF^V600E.

Publisher: Wiley

Date: 29-07-2022

DOI: 10.1111/BPH.15923

Abstract: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF‐mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF‐melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin‐peptide from the Octopus kaurna , Octpep‐1 in melanoma. We evaluated the cytotoxicity of Octpep‐1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep‐1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high‐throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). Octpep‐1 selectively reduced the proliferative capacity of human melanoma BRAF V600E ‐mutated cells with minimal effects on fibroblasts. In melanoma‐treated cells, Octpep‐1 increased ROS with unaltered mitochondrial membrane potential and promoted non‐mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep‐1 depends upon the α‐helix and polyproline conformation in the C‐terminal region of the peptide. A truncated form of the C‐terminal end of Octpep‐1 displayed enhanced potency and efficacy against melanoma. Octpep‐1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. We unravel the intrinsic anti‐tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF‐mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.

A tarantula-venom peptide antagonizes the TRPA1 nociceptor Ion channel by binding to the S1-S4 gating domain

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1016/J.CUB.2014.01.013

Regulation of RhoGEF activity by intramolecular and intermolecular SH3 domain interactions

Publisher: Elsevier BV

Date: 07-2006

DOI: 10.1074/JBC.M512482200

ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures

Publisher: Oxford University Press (OUP)

Date: 29-10-2011

DOI: 10.1093/NAR/GKQ1058

Periplasmic Expression of 4/7 α-Conotoxin TxIA Analogs in E. coli Favors Ribbon Isomer Formation – Suggestion of a Binding Mode at the α7 nAChR

Publisher: Frontiers Media SA

Date: 31-05-2019

DOI: 10.3389/FPHAR.2019.00577

Intraspecific venom variation in the medically significant Southern Pacific Rattlesnake (Crotalus oreganus helleri): Biodiscovery, clinical and evolutionary implications

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1016/J.JPROT.2014.01.013

Abstract: Due to the extreme variation of venom, which consequently results in drastically variable degrees of neutralization by CroFab antivenom, the management and treatment of envenoming by Crotalus oreganus helleri (the Southern Pacific Rattlesnake), one of the most medically significant snake species in all of North America, has been a clinician's nightmare. This snake has also been the subject of sensational news stories regarding supposed rapid (within the last few decades) evolution of its venom. This research demonstrates for the first time that variable evolutionary selection pressures sculpt the intraspecific molecular ersity of venom components in C. o. helleri. We show that myotoxic β-defensin peptides (aka: crotamines/small basic myotoxic peptides) are secreted in large amounts by all populations. However, the mature toxin-encoding nucleotide regions evolve under the constraints of negative selection, likely as a result of their non-specific mode of action which doesn't enforce them to follow the regime of the classic predator-prey chemical arms race. The hemorrhagic and tissue destroying snake venom metalloproteinases (SVMPs) were secreted in larger amounts by the Catalina Island and Phelan rattlesnake populations, in moderate amounts in the Loma Linda population and in only trace levels by the Idyllwild population. Only the Idyllwild population in the San Jacinto Mountains contained potent presynaptic neurotoxic phospholipase A2 complex characteristic of Mohave Rattlesnake (Crotalus scutulatus) and Neotropical Rattlesnake (Crotalus durissus terrificus). The derived heterodimeric lectin toxins characteristic of viper venoms, which exhibit a ersity of biological activities, including anticoagulation, agonism/antagonism of platelet activation, or procoagulation, appear to have evolved under extremely variable selection pressures. While most lectin α- and β-chains evolved rapidly under the influence of positive Darwinian selection, the β-chain lectin of the Catalina Island population appears to have evolved under the constraint of negative selection. Both lectin chains were conspicuously absent in both the proteomics and transcriptomics of the Idyllwild population. Thus, we not only highlight the tremendous biochemical ersity in C. o. helleri's venom-arsenal, but we also show that they experience remarkably variable strengths of evolutionary selection pressures, within each toxin class among populations and among toxin classes within each population. The mapping of geographical venom variation not only provides additional information regarding venom evolution, but also has direct medical implications by allowing prediction of the clinical effects of rattlesnake bites from different regions. Such information, however, also points to these highly variable venoms as being a rich source of novel toxins which may ultimately prove to be useful in drug design and development. These results have direct implications for the treatment of envenomed patients. The variable venom profile of Crotalus oreganus helleri underscores the biodiscovery potential of novel snake venoms.

Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug Platymeris rhadamanthus

Publisher: MDPI AG

Date: 18-11-2019

DOI: 10.3390/TOXINS11110673

Abstract: Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their in idual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus, a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom s les collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom s les induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

Publisher: International Union of Crystallography (IUCr)

Date: 15-05-2009

DOI: 10.1107/S090744490901169X

The assimilation of tri- and tetrapeptides by human erythrocytes

Publisher: Elsevier BV

Date: 07-1985

DOI: 10.1016/0167-4889(85)90118-1

Abstract: Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F. and Kuchel, P.W. (1985) Biochem. J. 227, 833-842) but that the transmembrane flux rates for tetrapeptides are considerably less. 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo. Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave Km = 2.11 +/- 0.08 and 23.4 +/- 0.9 mmol/l and Vmax = 307 +/- 3 and 905 +/- 22 mmol/h per 1 packed cells, respectively. In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide. Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport the values were Km = 11.6 +/- 1.1 and 56 +/- 18 mmol/l and Vmax = 12.9 +/- 3.0 and 36.4 +/- 3.2 mmol/h per 1 packed cells, respectively, for the previously mentioned peptides. The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides. The above mentioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo.

Mapping the Phosphoinositide-Binding Site on Chick Cofilin Explains How PIP2 Regulates the Cofilin-Actin Interaction

Publisher: Elsevier BV

Date: 11-2006

DOI: 10.1016/J.MOLCEL.2006.10.007

Abstract: Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (PIs). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound PIs abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity.

Chemical punch packed in venoms makes centipedes excellent predators

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1074/MCP.M112.018853

Assimilation of alpha-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis.

Publisher: Portland Press Ltd.

Date: 05-1985

DOI: 10.1042/BJ2270833

Abstract: Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.

The Deadly Toxin Arsenal of the Tree-Dwelling Australian Funnel-Web Spiders

Publisher: MDPI AG

Date: 28-10-2022

DOI: 10.3390/IJMS232113077

Abstract: Australian funnel-web spiders are amongst the most dangerous venomous animals. Their venoms induce potentially deadly symptoms, including hyper- and hypotension, tachycardia, bradycardia and pulmonary oedema. Human envenomation is more frequent with the ground-dwelling species, including the infamous Sydney funnel-web spider (Atrax robustus) although, only two tree-dwelling species induce more severe envenomation. To unravel the mechanisms that lead to this stark difference in clinical outcomes, we investigated the venom transcriptome and proteome of arboreal Hadronyche cerberea and H. formidabilis. Overall, Hadronyche venoms comprised 44 toxin superfamilies, with 12 being exclusive to tree-dwellers. Surprisingly, the major venom components were neprilysins and uncharacterized peptides, in addition to the well-known ω- and δ-hexatoxins and double-knot peptides. The insecticidal effects of Hadronyche venom on sheep blowflies were more potent than Atrax venom, and the venom of both tree- and ground-dwelling species potently modulated human voltage-gated sodium channels, particularly NaV1.2. Only the venom of tree-dwellers exhibited potent modulation of voltage-gated calcium channels. H. formidabilis appeared to be under less ersifying selection pressure compared to the newly adapted tree-dweller, H. cerberea. Thus, this study contributes to unravelling the fascinating molecular and pharmacological basis for the severe envenomation caused by the Australian tree-dwelling funnel-web spiders.

Toxin structures as evolutionary tools: Using conserved 3D folds to study the evolution of rapidly evolving peptides

Publisher: Wiley

Date: 11-05-2016

DOI: 10.1002/BIES.201500165

Abstract: Three-dimensional (3D) structures have been used to explore the evolution of proteins for decades, yet they have rarely been utilized to study the molecular evolution of peptides. Here, we highlight areas in which 3D structures can be particularly useful for studying the molecular evolution of peptide toxins. Although we focus our discussion on animal toxins, including one of the most widespread disulfide-rich peptide folds known, the inhibitor cystine knot, our conclusions should be widely applicable to studies of the evolution of disulfide-constrained peptides. We show that conserved 3D folds can be used to identify evolutionary links and test hypotheses regarding the evolutionary origin of peptides with extremely low sequence identity construct accurate multiple sequence alignments and better understand the evolutionary forces that drive the molecular evolution of peptides. Also watch the video abstract.

The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens

Publisher: Springer Science and Business Media LLC

Date: 22-02-2018

DOI: 10.1038/S41467-018-03091-5

Abstract: The assassin bug venom system plays erse roles in prey capture, defence and extra-oral digestion, but it is poorly characterised, partly due to its anatomical complexity. Here we demonstrate that this complexity results from numerous adaptations that enable assassin bugs to modulate the composition of their venom in a context-dependent manner. Gland reconstructions from multimodal imaging reveal three distinct venom gland lumens: the anterior main gland (AMG) posterior main gland (PMG) and accessory gland (AG). Transcriptomic and proteomic experiments demonstrate that the AMG and PMG produce and accumulate distinct sets of venom proteins and peptides. PMG venom, which can be elicited by electrostimulation, potently paralyses and kills prey insects. In contrast, AMG venom elicited by harassment does not paralyse prey insects, suggesting a defensive role. Our data suggest that assassin bugs produce offensive and defensive venoms in anatomically distinct glands, an evolutionary adaptation that, to our knowledge, has not been described for any other venomous animal.

Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel Na_V1.7

Publisher: American Chemical Society (ACS)

Date: 27-08-2018

DOI: 10.1021/ACS.BIOCONJCHEM.8B00505

Abstract: Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (Na

Discovery of a selective Na _V 1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models

Publisher: Proceedings of the National Academy of Sciences

Date: 30-09-2013

DOI: 10.1073/PNAS.1306285110

Abstract: The economic burden of chronic pain in the United States is currently ∼$600 billion per annum, which exceeds the combined annual cost of cancer, heart disease, and diabetes. Few drugs are available for treating chronic pain, and many have limited efficacy and dose-limiting side-effects. Humans with inheritable loss-of-function mutations in the voltage-gated sodium channel Na V 1.7 are indifferent to all types of pain, and therefore drugs that block this channel should be useful analgesics for treating many pain conditions. Herein we describe Ssm6a, a peptide from centipede venom that potently and selectively blocks the human Na V 1.7 channel. Ssm6a proved to be more analgesic than morphine in rodent pain models and did not cause any side-effects.

Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli : an Interspecies Approach to the Study of Pro

Publisher: American Society for Microbiology

Date: 15-09-2008

DOI: 10.1128/JB.00462-08

Abstract: Bacterial cell ision is mediated by a set of proteins that assemble to form a large multiprotein complex called the isome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell ision proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell ision proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis isomal proteins in E. coli , namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell ision proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell ision proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

Mapping the MinE site involved in interaction with the MinD division site selection protein of Escherichia coli

Publisher: American Society for Microbiology

Date: 15-08-2003

DOI: 10.1128/JB.185.16.4948-4955.2003

Abstract: Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli ision septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.

Venoms to the rescue

Publisher: American Association for the Advancement of Science (AAAS)

Date: 31-08-2018

DOI: 10.1126/SCIENCE.AAU7761

Abstract: Insights into the evolutionary biology of venoms are leading to therapeutic advances

Venom-derived modulators of epilepsy-related ion channels.

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1016/J.BCP.2020.114043

Identification and functional characterization of sugarcane invertase inhibitor (ShINH1): a potential candidate for reducing pre- and post-harvest loss of sucrose in sugarcane.

Publisher: Frontiers Media SA

Date: 03-05-2018

DOI: 10.3389/FPLS.2018.00598

Peptide toxins that selectively target insect NaV and Ca V channels

Publisher: Informa UK Limited

Date: 05-03-2008

DOI: 10.4161/CHAN.2.2.6022

Abstract: Numerous metazoans express venoms for the purpose of defense, competitor deterrence or prey capture. Peptide neurotoxins are particularly well represented in the venoms of arachnids, cnidarians and mollusks and these toxins often possess high affinity and specificity for particular classes of ion channels. Some of these toxins have become the defining pharmacology for certain vertebrate ion channel subtypes. Unfortunately, due to differences in the structure, pharmacology and ion selectivity of insect voltage-gated sodium (Na(V)) and calcium (Ca(V)) channels compared with their vertebrate counterparts, these peptide toxins have proven less useful for the characterization of insect ion channels. Despite these disparities in channel structure and function, the armament of peptide toxins that specifically modulate the activity of insect ion channels is slowly expanding. This review focuses on insect-selective peptide toxins and their utility for the study of insect Na(V) and Ca(V) channels. The high affinity and selectivity of some of these neurotoxins means that they have the potential to become the defining pharmacology for specific subtypes of insect ion channels. In addition, it might be possible to exploit the phyletic specificity of these toxins as the basis for rational development of novel classes of ion channel insecticides.

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa

Publisher: MDPI AG

Date: 12-12-2019

DOI: 10.3390/MD17120701

Abstract: Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite ergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified in idual residue contributions to the inhibitor’s stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular ersity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain.

Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy.

Publisher: Elsevier BV

Date: 05-1986

DOI: 10.1016/0003-2697(86)90221-6

Abstract: The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque s les. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration.

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms.

Publisher: Wiley

Date: 09-2020

DOI: 10.1002/PMIC.201900324

A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

Publisher: Springer Science and Business Media LLC

Date: 11-07-2014

DOI: 10.1038/NCOMMS5350

A selective Na_V1.1 activator with potential for treatment of Dravet syndrome epilepsy.

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1016/J.BCP.2020.113991

Widespread convergence in toxin resistance by predictable molecular evolution.

Publisher: Proceedings of the National Academy of Sciences

Date: 08-09-2015

DOI: 10.1073/PNAS.1511706112

Abstract: Convergence has strong bearing on the fundamental debate about whether evolution is stochastic and unpredictable or subject to constraints. Here we show that, in certain circumstances, evolution can be highly predictable. We demonstrate that several lineages of insects, hibians, reptiles, and mammals have utilized the same molecular solution, via the process of convergence, to evolve resistance to toxic cardiac glycosides produced defensively by plants and bufonid toads. The repeatability of this process across the animal kingdom demonstrates that evolution can be constrained to proceed along highly predictable pathways at molecular and functional levels. Our study has important implications for conservation biology by providing a predictive framework for assessing the vulnerability of native fauna to the introduction of invasive toxic toads.

Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge

Publisher: Springer Science and Business Media LLC

Date: 06-2000

DOI: 10.1038/75921

Abstract: We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

A spider-venom peptide with multitarget activity on sodium and calcium channels alleviates chronic visceral pain in a model of irritable bowel syndrome

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 17-08-2021

DOI: 10.1097/J.PAIN.0000000000002041

Spider-Venom Peptides: Structure, Pharmacology, and Potential for Control of Insect Pests

Publisher: Annual Reviews

Date: 07-01-2013

DOI: 10.1146/ANNUREV-ENTO-120811-153650

Abstract: Spider venoms are an incredibly rich source of disulfide-rich insecticidal peptides that have been tuned over millions of years to target a wide range of receptors and ion channels in the insect nervous system. These peptides can act in idually, or as part of larger toxin cabals, to rapidly immobilize envenomated prey owing to their debilitating effects on nervous system function. Most of these peptides contain a unique arrangement of disulfide bonds that provides them with extreme resistance to proteases. As a result, these peptides are highly stable in the insect gut and hemolymph and many of them are orally active. Thus, spider-venom peptides can be used as stand-alone bioinsecticides, or transgenes encoding these peptides can be used to engineer insect-resistant crops or enhanced entomopathogens. We critically review the potential of spider-venom peptides to control insect pests and highlight their advantages and disadvantages compared with conventional chemical insecticides.

Enkephalin degradation by human erythrocytes and hemolysates studied using 1H NMR spectroscopy

Publisher: Elsevier BV

Date: 11-1985

DOI: 10.1016/0003-9861(85)90238-3

Abstract: High resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to monitor the degradation of leucine-enkephalin, and peptide fragments of it, by human erythrocytes and hemolysates. We showed that leucine-enkephalin is rapidly degraded by the cytosolic peptidases of the human erythrocyte, and we have elucidated the most probable pathway of degradation. Computer simulations of the proposed pathway, using a model incorporating the experimentally derived steady-state kinetic parameters obtained for the in idual enzyme steps, showed close agreement with the experimental results. From a methodological perspective, the work demonstrates the value of 1H spin-echo NMR spectroscopy for rapidly elucidating, both qualitatively and quantitatively, an entire peptide-degradation pathway as it operates in situ.

A tale of two terminators: Crystal structures sharpen the debate on DNA replication fork arrest mechanisms

Publisher: Elsevier BV

Date: 1997

DOI: 10.1016/S0969-2126(97)00160-3

Abstract: The structure of the Tus-Ter DNA replication fork arrest complex of Escherichia coli reveals a novel architecture for the bound Tus protein and a new type of DNA-binding motif. The structure of the complex may explain how Tus can block movement of a replication fork approaching from one direction and not the other.

Pharmacological characterisation of the highly Na v 1.7 selective spider venom peptide Pn3a

Publisher: Springer Science and Business Media LLC

Date: 20-01-2017

DOI: 10.1038/SREP40883

Abstract: Human genetic studies have implicated the voltage-gated sodium channel Na V 1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula P hobeteus nigricolor, potently inhibits Na V 1.7 (IC 50 0.9 nM) with at least 40–1000-fold selectivity over all other Na V subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na V 1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na V 1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na V 1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na V 1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

Structure-function studies of ω-atracotoxin, a potent antagonist of insect voltage-gated calcium channels

Publisher: Wiley

Date: 09-1999

DOI: 10.1046/J.1432-1327.1999.00646.X

Abstract: The omega-atracotoxins are a family of 36 to 37-residue peptide neurotoxins that block insect but not mammalian voltage-gated calcium channels. The high phylogenetic specificity of these toxins recommends them as lead compounds for targeting insects that have developed resistance to chemical pesticides. We have begun to examine structure-function relationships in the omega-atracotoxins in order to explore the molecular basis of their activity and phylogenetic specificity. By probing the venom of the Blue Mountains funnel-web spider, Hadronyche versuta, for insecticidal toxins with masses close to that of omega-atracotoxin-Hv1a (omega-ACTX-Hv1a), we have isolated and sequenced five additional omega-atracotoxins. Five of the six omega-atracotoxins isolated from the venom of H. versuta (omega-ACTX-Hv1a to -Hv1e) differ from one another by only 1-3 residues and have similar insecticidal potencies. In contrast, omega-ACTX-Hv1f differs from the other toxins by up to 10 residues and it has markedly reduced insecticidal potency, thus providing information on key functional residues. The new atracotoxin sequences have revealed that the three N-terminal residues are highly conserved. Despite the fact that these residues are structurally disordered in solution we show here, by a series of N-terminal truncations, that they contribute significantly to insecticidal potency. However, loss of activity does not correlate with deletion of highly conserved residues, which leads us to propose that the disposition of the N-terminal charge, rather than the chemical properties of the N-terminal residues themselves, may be critical for the activity of omega-atracotoxin on insect calcium channels.

The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites

Publisher: Wiley

Date: 15-01-2008

DOI: 10.1111/J.1365-2958.2008.06114.X

Abstract: Bacterial cytokinesis is orchestrated by an assembly of essential cell ision proteins that form a supramolecular structure known as the isome. DivIB and its orthologue FtsQ are essential members of the isome in Gram-positive and Gram-negative bacteria respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. A molecular dissection approach was used to determine which of these domains are essential for recruitment of DivIB to incipient ision sites and for its cell ision functions. We show that DivIB has three molecular epitopes that mediate its localization to ision septa two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other isomal proteins, and we have used this information to develop a model of the way in which DivIB and FtsQ are integrated into the isome. Remarkably, two of the three DivIB localization epitopes are dispensable for vegetative cell ision this suggests that the isome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be in idually non-essential.

Diversification of a single ancestral gene into a successful toxin superfamily in highly venomous Australian funnel-web spiders

Publisher: Springer Science and Business Media LLC

Date: 05-03-2014

DOI: 10.1186/1471-2164-15-177

Isolation of an Orally Active Insecticidal Toxin from the Venom of an Australian Tarantula

Publisher: Public Library of Science (PLoS)

Date: 11-09-2013

DOI: 10.1371/JOURNAL.PONE.0073136

Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids

Publisher: Springer Science and Business Media LLC

Date: 08-12-2014

DOI: 10.1038/NBT.2753

Abstract: The sap-sucking insects (order Hemiptera), including aphids, planthoppers, whiteflies and stink bugs, present one of the greatest challenges for pest management in global agriculture. Insect neurotoxins offer an alternative to chemical insecticides for controlling these pests, but require delivery into the insect hemocoel. Here we use the coat protein of a luteovirus, an aphid-vectored plant virus, to deliver a spider-derived, insect-specific toxin that acts within the hemocoel. The luteovirid coat protein is sufficient for delivery of fused proteins into the hemocoel of pea aphids, Acyrthosiphon pisum, without virion assembly. We show that when four aphid pest species-A. pisum, Rhopalosiphum padi, Aphis glycines and Myzus persicae-feed on a recombinant coat protein-toxin fusion, either in an experimental membrane sachet or in transgenic Arabidopsis plants, they experience significant mortality. Aphids fed on these fusion proteins showed signs of neurotoxin-induced paralysis. Luteovirid coat protein-insect neurotoxin fusions represent a promising strategy for transgenic control of aphids and potentially other hemipteran pests.

Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug (Havinthus rufovarius)

Publisher: MDPI AG

Date: 22-12-2020

DOI: 10.3390/TOXINS13010003

Abstract: Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (Havinthus rufovarius), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (Lucilia cuprina). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with erse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug Pristhesancus plagipennis, their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the H. rufovarius venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.

Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms

Publisher: American Chemical Society (ACS)

Date: 18-09-2019

DOI: 10.1021/ACS.JNATPROD.9B00410

Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1074/JBC.RA118.002553

SYMMETRY AND SECONDARY STRUCTURE OF THE REPLICATION TERMINATOR PROTEIN OF BACILLUS-SUBTILIS - SEDIMENTATION EQUILIBRIUM AND CIRCULAR DICHROIC, INFRARED, AND NMR SPECTROSCOPIC STUDIES

Publisher: American Chemical Society (ACS)

Date: 28-09-1993

DOI: 10.1021/BI00089A043

Abstract: We have used analytical ultracentrifugation in combination with a number of spectroscopic techniques to analyze the symmetry and secondary structure of the DNA-binding replication terminator protein (RTP) of Bacillus subtilis. Sedimentation equilibrium studies confirm that RTP is a dimer in solution under the conditions used for spectroscopic analysis, whereas the number of cross peaks displayed in 1H-15N HSQC NMR spectra of uniformly 15N-labeled RTP are consistent with the primary structure of the monomer. These two results in combination lead to the conclusion that RTP is a symmetric dimer in solution. Circular dichroic and Fourier-transform infrared spectra reveal, in contrast to the results obtained from a number of commonly used secondary structure prediction algorithms, that RTP contains 20-30% alpha-helical and 40-50% beta-sheet/beta-turn secondary structure and that the conformation of the protein remains unchanged over the pH range 5-8. It is proposed on the basis of protein folding-class prediction algorithms, in combination with various physical properties of RTP, that it belongs to the alpha + beta protein-folding class.

Venoms of Heteropteran Insects: A Treasure Trove of Diverse Pharmacological Toolkits.

Publisher: MDPI AG

Date: 12-02-2016

DOI: 10.3390/TOXINS8020043

Do Vicinal Disulfide Bridges Mediate Functionally Important Redox Transformations in Proteins?

Publisher: Mary Ann Liebert Inc

Date: 12-2013

DOI: 10.1089/ARS.2013.5365

Construction of a hypervirulent and specific mycoinsecticide for locust control

Publisher: Springer Science and Business Media LLC

Date: 05-12-2014

DOI: 10.1038/SREP07345

The tale of a resting gland: Transcriptome of a replete venom gland from the scorpion Hottentotta judaicus

Publisher: Elsevier BV

Date: 04-2011

DOI: 10.1016/J.TOXICON.2011.02.001

Abstract: cDNA libraries are increasingly being used for high-throughput interrogation of animal venomes. Most previous studies have focused on discovery of new venom toxins, whereas the dynamics of toxin transcription and associated cellular processes have received much less attention. Here we provide, for the first time, an analysis of a transcriptome from the venom gland of a scorpion (Hottentotta judaicus) that is not actively engaged in regenerating its venom. We demonstrate a low abundance of toxin-encoding transcripts coupled with a previously unobserved proliferation of protease sequences. Additionally, we identified several low abundance, toxin-like sequences that may represent decommissioned toxins that are unlikely to be translated. These sequences are not evenly distributed across all toxin families, but rather appear more frequently in transcripts related to α-toxins and β-toxins that are known to target voltage-gated sodium channels. The transcriptomic profile of the replete venom gland is very different to that obtained previously from scorpion venom glands actively engaged in venom regeneration, and it highlights our lack of knowledge as to how the dynamics of transcription changes as the gland progresses from venom regeneration to a "resting" state. This study therefore provides an important foundation for future studies into the dynamics of transcription in the venom glands of scorpions and other venomous animals.

Fluorescence Imaging of Peripheral Nerves by a Na_v1.7-Targeted Inhibitor Cystine Knot Peptide

Publisher: American Chemical Society (ACS)

Date: 24-10-2019

DOI: 10.1021/ACS.BIOCONJCHEM.9B00612

A non-uniformly sampled 4D HCC(CO)NH-TOCSY experiment processed using maximum entropy for rapid protein sidechain assignment

Publisher: Elsevier BV

Date: 05-2010

DOI: 10.1016/J.JMR.2010.02.012

The wonderful world of spiders: Preface to the special Toxicon issue on spider venoms

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.TOXICON.2004.02.001

Gomesin peptides prevent proliferation and lead to the cell death of devil facial tumour disease cells

Publisher: Springer Science and Business Media LLC

Date: 14-02-2018

DOI: 10.1038/S41420-018-0030-0

Abstract: The Tasmanian devil faces extinction due to devil facial tumour disease (DFTD), a highly transmittable clonal form of cancer without available treatment. In this study, we report the cell-autonomous antiproliferative and cytotoxic activities exhibited by the spider peptide gomesin (AgGom) and gomesin-like homologue (HiGom) in DFTD cells. Mechanistically, both peptides caused a significant reduction at G0/G1 phase, in correlation with an augmented expression of the cell cycle inhibitory proteins p53, p27, p21, necrosis, exacerbated generation of reactive oxygen species and diminished mitochondrial membrane potential, all hallmarks of cellular stress. The screening of a novel panel of AgGom-analogues revealed that, unlike changes in the hydrophobicity and electrostatic surface, the cytotoxic potential of the gomesin analogues in DFTD cells lies on specific arginine substitutions in the eight and nine positions and alanine replacement in three, five and 12 positions. In conclusion, the evidence supports gomesin as a potential antiproliferative compound against DFTD disease.

Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.

Publisher: Springer Science and Business Media LLC

Date: 09-02-2018

DOI: 10.1007/S00018-018-2768-1

Abstract: True Bugs (Insecta: Heteroptera) produce venom or saliva with erse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins underlying these biological activities. We examined venom of the giant fish-killing water bug Lethocerus distinctifemur (Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and ersified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.

The dimerization function of MinC resides in a structurally autonomous C-terminal domain

Publisher: American Society for Microbiology

Date: 15-11-2001

DOI: 10.1128/JB.183.22.6684-6687.2001

Abstract: Limited proteolysis of the Escherichia coli cell ision inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain. We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.

Discovery and mode of action of a novel analgesic β-toxin from the African spider Ceratogyrus darlingi

Publisher: Public Library of Science (PLoS)

Date: 07-09-2017

DOI: 10.1371/JOURNAL.PONE.0182848

Involvement of the N-finger in the self-association of GATA-1

Publisher: Elsevier BV

Date: 11-1998

DOI: 10.1074/JBC.273.46.30560

Venoms as a platform for human drugs: Translating toxins into therapeutics

Publisher: Informa Healthcare

Date: 23-09-2011

DOI: 10.1517/14712598.2011.621940

Abstract: An extraordinarily erse range of animals have evolved venoms for predation, defence, or competitor deterrence. The major components of most venoms are peptides and proteins that are often protease-resistant due to their disulfide-rich architectures. Some of these toxins have become valuable as pharmacological tools and/or therapeutics due to their extremely high specificity and potency for particular molecular targets. There are currently six FDA-approved drugs derived from venom peptides or proteins. This article surveys the current pipeline of venom-derived therapeutics and critically examines the potential of peptide and protein drugs derived from venoms. Emerging trends are identified, including an increasing industry focus on disulfide-rich venom peptides and the use of a broader array of molecular targets in order to develop venom-based therapeutics for treating a wider range of clinical conditions. Key technical advances in combination with a renewed industry-wide focus on biologics have converged to provide a larger than ever pipeline of venom-derived therapeutics. Disulfide-rich venom peptides obviate some of the traditional disadvantages of therapeutic peptides and some may be suitable for oral administration. Moreover, some venom peptides can breach the blood brain barrier and translocate across cell membranes, which opens up the possibility of exploiting molecular targets not previously accessible to peptide drugs.

The Bacillus subtilis DNA replication terminator

Publisher: Elsevier BV

Date: 07-1996

DOI: 10.1006/JMBI.1996.0381

Abstract: The recent discovery of the Bacillus subtilis plasmid terminator TerLS20 with bidirectional fork arrest activity has provided the opportunity to probe further the structural and functional features of B. subtilis replication terminators in general. The minimal TerI and TerLS20 terminators each comprise two 13 nt segments flanking a central trinucleotide, which is almost completely conserved in all terminators. It corresponds to the region of overlap of the two RTP binding sites (A and B) on the DNA. It has been shown that, despite this conservation, considerable variation in this trinucleotide region still allows fork arrest activity. Thus, the productive interaction of the RTP dimers, which presumably occurs in the vicinity of this trinucleotide region, is not dependent upon stringently defined contacts with the bases in this region. A completely synthetic and highly symmetrical terminator was constructed by replacing the 13 nt segment of the A site of TerI with an opposed segment identical to that in the B site. The efficient bidirectional activity of this new terminator, TerSymB, established more firmly the need for two opposed RTP binding sites in a functional terminator. TerSymB was used to investigate the effect of sequence deviation in one of the 13 nt segments, from that in the B site, on bidirectionality of the terminator. It was found that the deviations introduced converted the terminator significantly towards polarity of action. The partial symmetry within each of the 13 nt segments of TerSymB, and the presumed recognition of this symmetry in the binding of a symmetrical dimer of RTP to each overlapping site, suggest that the bound dimers are centred over positions in the DNA sequence separated by 15 nt. This separation distance has been used in conjunction with the mode of binding of RTP to DNA proposed by Bussiere et al., based on their crystal structure for RTP, to model the interaction of the two dimers of RTP with unbent B-form DNA. Increased separation of the two binding sites of TerSymB was performed by inserting an extra three, seven or ten nucleotides centrally within the TerSymB sequence. The effects of these insertions on RTP binding and fork arrest activity were consistent with the proposed positioning of the RTP dimers within the terminator sequence, and interaction between the dimers bound to TerSymB. A model to account for the generation of RTP-terminator complexes with bidirectional or polar fork arrest activity utilising TerSymB or TerI-VI is presented.

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects.

Publisher: MyJove Corporation

Date: 21-04-2018

DOI: 10.3791/57729

The MinD membrane targeting sequence is a transplantable lipid-binding helix

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1074/JBC.M306876200

Selective inhibition of ASIC1a confers functional and morphological neuroprotection following traumatic spinal cord injury

Publisher: F1000 Research Ltd

Date: 07-12-2016

DOI: 10.12688/F1000RESEARCH.9094.2

Abstract: Tissue loss after spinal trauma is biphasic, with initial mechanical/haemorrhagic damage at the time of impact being followed by gradual secondary expansion into adjacent, previously unaffected tissue. Limiting the extent of this secondary expansion of tissue damage has the potential to preserve greater residual spinal cord function in patients. The acute tissue hypoxia resulting from spinal cord injury (SCI) activates acid-sensing ion channel 1a (ASIC1a). We surmised that antagonism of this channel should provide neuroprotection and functional preservation after SCI. We show that systemic administration of the spider-venom peptide PcTx1, a selective inhibitor of ASIC1a, improves locomotor function in adult Sprague Dawley rats after thoracic SCI. The degree of functional improvement correlated with the degree of tissue preservation in descending white matter tracts involved in hind limb locomotor function. Transcriptomic analysis suggests that PcTx1-induced preservation of spinal cord tissue does not result from a reduction in apoptosis, with no evidence of down-regulation of key genes involved in either the intrinsic or extrinsic apoptotic pathways. We also demonstrate that trauma-induced disruption of blood-spinal cord barrier function persists for at least 4 days post-injury for compounds up to 10 kDa in size, whereas barrier function is restored for larger molecules within a few hours. This temporary loss of barrier function provides a “ treatment window ” through which systemically administered drugs have unrestricted access to spinal tissue in and around the sites of trauma. Taken together, our data provide evidence to support the use of ASIC1a inhibitors as a therapeutic treatment for SCI. This study also emphasizes the importance of objectively grading the functional severity of initial injuries (even when using standardized impacts) and we describe a simple scoring system based on hind limb function that could be adopted in future studies.

Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms

Publisher: American Chemical Society (ACS)

Date: 06-10-2020

DOI: 10.1021/ACSPTSCI.0C00119

Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection

Publisher: Wiley

Date: 11-08-2004

DOI: 10.1111/J.1365-2958.2004.04265.X

The β3‐subunit modulates the effect of venom peptides ProTx‐II and OD1 on Na_V1.7 gating

Publisher: Wiley

Date: 12-04-2023

DOI: 10.1002/JCP.31018

Abstract: The voltage‐gated sodium channel Na V 1.7 is involved in various pain phenotypes and is physiologically regulated by the Na V ‐β3‐subunit. Venom toxins ProTx‐II and OD1 modulate Na V 1.7 channel function and may be useful as therapeutic agents and/or research tools. Here, we use patch‐cl recordings to investigate how the β3‐subunit can influence and modulate the toxin‐mediated effects on Na V 1.7 function, and we propose a putative binding mode of OD1 on Na V 1.7 to rationalise its activating effects. The inhibitor ProTx‐II slowed the rate of Na V 1.7 activation, whilst the activator OD1 reduced the rate of fast inactivation and accelerated recovery from inactivation. The β3‐subunit partially abrogated these effects. OD1 induced a hyperpolarising shift in the V 1/2 of steady‐state activation, which was not observed in the presence of β3. Consequently, OD1‐treated Na V 1.7 exhibited an enhanced window current compared with OD1‐treated Na V 1.7‐β3 complex. We identify candidate OD1 residues that are likely to prevent the upward movement of the DIV S4 helix and thus impede fast inactivation. The binding sites for each of the toxins and the predicted location of the β3‐subunit on the Na V 1.7 channel are distinct. Therefore, we infer that the β3‐subunit influences the interaction of toxins with Na V 1.7 via indirect allosteric mechanisms. The enhanced window current shown by OD1‐treated Na V 1.7 compared with OD1‐treated Na V 1.7‐β3 is discussed in the context of differing cellular expressions of Na V 1.7 and the β3‐subunit in dorsal root ganglion (DRG) neurons. We propose that β3, as the native binding partner for Na V 1.7 in DRG neurons, should be included during screening of molecules against Na V 1.7 in relevant analgesic discovery c aigns.

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Publisher: Springer Science and Business Media LLC

Date: 06-1988

DOI: 10.1007/BF00425152

Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts

Publisher: Proceedings of the National Academy of Sciences

Date: 07-11-2002

DOI: 10.1073/PNAS.232590599

Abstract: MinD is a widely conserved ATPase that has been demonstrated to play a pivotal role in selection of the ision site in eubacteria and chloroplasts. It is a member of the large ParA superfamily of ATPases that are characterized by a deviant Walker-type ATP-binding motif. MinD localizes to the cytoplasmic face of the inner membrane in Escherichia coli , and its association with the inner membrane is a prerequisite for membrane recruitment of the septation inhibitor MinC. However, the mechanism by which MinD associates with the membrane has proved enigmatic it seems to lack a transmembrane domain and the amino acid sequence is devoid of hydrophobic tracts that might predispose the protein to interaction with lipids. In this study, we show that the extreme C-terminal region of MinD contains a highly conserved 8- to 12-residue sequence motif that is essential for membrane localization of the protein. We provide evidence that this motif forms an hipathic helix that most likely mediates a direct interaction between MinD and membrane phospholipids. A model is proposed whereby the membrane-targeting motif mediates the rapid cycles of membrane attachment–release–reattachment that are presumed to occur during pole-to-pole oscillation of MinD in E. coli .

Australian funnel-web spiders: Master insecticide chemists

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.TOXICON.2004.02.010

No evidence of high capacity α-glutamyl-dipeptide transport into human erythrocytes.

Publisher: Portland Press Ltd.

Date: 15-02-1987

DOI: 10.1042/BJ2420311

Abstract: Bone resorption is regulated by a complex system of hormones and cytokines that cause osteoblasts/stromal cells and lymphocytes to produce factors including RANKL, that ultimately result in the differentiation and activation of osteoclasts, the bone resorbing cells. We used a microarray approach to identify genes upregulated in RANKL-stimulated osteoclast precursor cells. Osteoclast expression was confirmed by multiple tissue Northern and in situ hybridization analysis. Gene function studies were carried out by siRNA analysis. We identified a novel gene, which we termed nha-oc/NHA2, which is strongly upregulated during RANKL-induced osteoclast differentiation in vitro and in vivo. nha-oc/NHA2 encodes a novel cation-proton antiporter (CPA) and is the mouse orthologue of a human gene identified in a database search: HsNHA2. nha-oc/NHA2 is selectively expressed in osteoclasts. NHA-oc/NHA2 protein localizes to the mitochondria, where it mediates Na(+)-dependent changes in mitochondrial pH and Na(+) acetate induced mitochondrial passive swelling. RNA silencing of nha-oc/nha2 reduces osteoclast differentiation and resorption, suggesting a role for NHA-oc/NHA2 in these processes. nha-oc/NHA2 therefore is a novel member of the CPA family and is the first mitochondrial NHA characterized to date. nha-oc/NHA2 is also unique in that it is the first eukaryotic and tissue-specific CPA2 characterized to date. NHA-oc/NHA2 displays the expected activities of a bona fide CPA and plays a key role(s) in normal osteoclast differentiation and function.

Orally active acaricidal peptide toxins from spider venom

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1016/J.TOXICON.2005.10.011

Abstract: Numerous species of ticks and mites (collectively known as acarines) are serious pests of animals, humans, and crops. There are few commercially available acaricides and major classes of these chemicals continue to be lost from the marketplace due to resistance development or deregistration by regulatory agencies. There is consequently a pressing need to isolate new and safe acaricidal compounds. In this study, we show that two families of peptide neurotoxins isolated from the venom of the Australian funnel-web spider Hadronyche versuta are lethal to the lone star tick Amblyomma americanum. These toxins, which are specific blockers of arthropod voltage-gated calcium channels, induce a pronounced phenotype characterized by an unusual gait that is rapidly followed by paralysis and death. Remarkably, one of these toxins, the calcium channel blocker omega-atracotoxin-Hv1a, is virtually equipotent whether the toxin is injected or fed to A. americanum.

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Publisher: Wiley

Date: 02-1999

DOI: 10.1046/J.1365-2958.1999.01256.X

Abstract: Correct placement of the ision septum in Escherichia coli requires the co-ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non-specific ision inhibitor that blocks septation at all potential ision sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted ision sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin-resistant domain comprising residues 31-88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both alpha and beta secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti-MinCD domain of MinE (residues 1-22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1-22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and alpha-helical conformations. This suggests that the N-terminal region of MinE may be poised to adopt an alpha-helical conformation when it interacts with the target of its anti-MinCD activity, presumably MinD.

The North American Society of Toxinology establishes formal affiliation with Toxicon.

Publisher: Elsevier BV

Date: 02-2018

DOI: 10.1016/J.TOXICON.2017.11.009

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 15-05-2015

DOI: 10.1124/MOL.115.098178

Abstract: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 (hNaV1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNaV1.7 > hNaV1.6 > hNaV1.2 > hNaV1.1 > hNaV1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form (IC50 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNaV1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNaV1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.

Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin

Publisher: Wiley

Date: 27-03-2000

DOI: 10.1016/S0014-5793(00)01339-9

Abstract: delta-Atracotoxins (delta-ACTXs) are peptide toxins isolated from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion alpha-toxins. We have isolated and determined the amino acid sequence of a novel delta-ACTX, designated delta-ACTX-Hv1b, from the venom of the funnel-web spider Hadronyche versuta. This 42 residue toxin shows 67% sequence identity with delta-ACTX-Hv1a previously isolated from the same spider. Under whole-cell voltage-cl conditions, the toxin had no effect on tetrodotoxin (TTX)-resistant sodium currents in rat dorsal root ganglion neurones but exerted a concentration-dependent reduction in peak TTX-sensitive sodium current litude accompanied by a slowing of sodium current inactivation similar to other delta-ACTXs. However, delta-ACTX-Hv1b is approximately 15-30-fold less potent than other delta-ACTXs and is remarkable for its complete lack of insecticidal activity. Thus, the sequence differences between delta-ACTX-Hv1a and -Hv1b provide key insights into the residues that are critical for targeting of these toxins to vertebrate and invertebrate sodium channels.

ArachnoServer: A database of protein toxins from spiders

Publisher: Springer Science and Business Media LLC

Date: 2009

DOI: 10.1186/1471-2164-10-375

Measuring macromolecular diffusion using heteronuclear multiple-quantum pulsed-field-gradient NMR

Publisher: Springer Science and Business Media LLC

Date: 1997

DOI: 10.1023/A:1018339526108

Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels

Publisher: Springer Science and Business Media LLC

Date: 06-10-2015

DOI: 10.1038/SREP14763

Abstract: For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-cl technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-cl fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

Novel venom-derived inhibitors of the human EAG channel, a putative antiepileptic drug target

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.BCP.2018.08.038

Abstract: Recently, we and other groups revealed that gain-of-function mutations in the human ether à go-go voltage-gated potassium channel hEAG1 (K

Ant venoms contain vertebrate-selective pain-causing sodium channel toxins

Publisher: Springer Science and Business Media LLC

Date: 23-05-2023

DOI: 10.1038/S41467-023-38839-1

Abstract: Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (Na V ) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

Sea anemone toxins: A structural overview

Publisher: MDPI AG

Date: 06-2019

DOI: 10.3390/MD17060325

Abstract: Sea anemones produce venoms of exceptional molecular ersity, with at least 17 different molecular scaffolds reported to date. These venom components have traditionally been classified according to pharmacological activity and amino acid sequence. However, this classification system suffers from vulnerabilities due to functional convergence and functional promiscuity. Furthermore, for most known sea anemone toxins, the exact receptors they target are either unknown, or at best incomplete. In this review, we first provide an overview of the sea anemone venom system and then focus on the venom components. We have organised the venom components by distinguishing firstly between proteins and non-proteinaceous compounds, secondly between enzymes and other proteins without enzymatic activity, then according to the structural scaffold, and finally according to molecular target.

Inhibition of acid-sensing ion channels by diminazene and APETx2 evoke partial and highly variable antihyperalgesia in a rat model of inflammatory pain

Publisher: Wiley

Date: 03-01-2018

DOI: 10.1111/BPH.14089

Structural basis for the topological specificity function of MinE

Publisher: Springer Science and Business Media LLC

Date: 11-2000

DOI: 10.1038/80917

Abstract: Correct positioning of the ision septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD ision inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell ision site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel alphabeta sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 21-09-2021

DOI: 10.1161/CIRCULATIONAHA.121.054360

Abstract: Ischemia–reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell–derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell–derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

The structure of versutoxin (δ-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Publisher: Elsevier BV

Date: 11-1997

DOI: 10.1016/S0969-2126(97)00301-8

Abstract: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta-ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 310 helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-I, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Weaponization of a Hormone: Convergent Recruitment of Hyperglycemic Hormone into the Venom of Arthropod Predators

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.STR.2015.05.003

Abstract: Arthropod venoms consist primarily of peptide toxins that are injected into their prey with devastating consequences. Venom proteins are thought to be recruited from endogenous body proteins and mutated to yield neofunctionalized toxins with remarkable affinity for specific subtypes of ion channels and receptors. However, the evolutionary history of venom peptides remains poorly understood. Here we show that a neuropeptide hormone has been convergently recruited into the venom of spiders and centipedes and evolved into a highly stable toxin through ergent modification of the ancestral gene. High-resolution structures of representative hormone-derived toxins revealed they possess a unique structure and disulfide framework and that the key structural adaptation in weaponization of the ancestral hormone was loss of a C-terminal α helix, an adaptation that occurred independently in spiders and centipedes. Our results raise a new paradigm for toxin evolution and highlight the value of structural information in providing insight into protein evolution.

Mutational analysis of ProTx-I and the novel venom peptide Pe1b provide insight into residues responsible for selective inhibition of the analgesic drug target NaV1.7

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1016/J.BCP.2020.114080

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation

Publisher: Public Library of Science (PLoS)

Date: 13-01-2011

DOI: 10.1371/JOURNAL.PONE.0016191

From kinetics to imaging: An NMR odyssey - A festschrift symposium in honour of Philip William Kuchel

Publisher: Springer Science and Business Media LLC

Date: 21-12-2013

DOI: 10.1007/S00249-012-0883-8

Development of a rational nomenclature for naming peptide and protein toxins from sea anemones

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1016/J.TOXICON.2012.05.020

Abstract: Sea anemone toxins are predominantly peptide and proteins that act mainly on sodium and potassium channels, as well as in a variety of target cells causing lysis. Over recent years, the number of sea anemone peptide toxins as well as cytolytic pore-forming proteins and phospholipase A(2) sequences submitted to databases has been rapidly increasing due to the developments in DNA sequencing technology and proteomic approaches. However, the lack of a systematic nomenclature has resulted in multiple names being assigned to the same toxins, toxins from unrelated species being designated by the same name, and ambiguous name designations. Therefore, in this work we propose a systematic nomenclature in which we adopted specific criteria, based on order of discovery and phylogenetic analysis, in order to avoid redundant sea anemone toxin names. Implementation of the nomenclature proposed here not only allowed us to rename the already published 191 anemone toxins without ambiguities, but it can be used to unambiguously name newly discovered toxins whether or not they are related to previously published sea anemone sequences. In the new nomenclature each toxin name contains information about the toxin's biological activity, origin and relationship to known isoforms. Ongoing increases in the speed of DNA sequencing will raise significantly the number of sea anemone toxin sequences in the literature. This will represent a constant challenge in their clear identification and logical classification, which could be solved using the proposed nomenclature.

Structure and function of insecticidal neurotoxins from Australian funnel-web spiders

Publisher: Informa UK Limited

Date: 2002

DOI: 10.1081/TXR-120014410

Solution Structure of Endothelin-3 Determined Using NMR Spectroscopy

Publisher: American Chemical Society (ACS)

Date: 06-1992

DOI: 10.1021/BI00139A030

Abstract: The aqueous solution structure of the 21-residue vasoactive peptide hormone endothelin-3 has been determined using high-resolution NMR spectroscopy. A total of 177 proton-proton distance measurements and 5 chi 1 dihedral angle constraints derived from NMR spectra were used to calculate the structure using a combination of distance geometry and dynamical simulated annealing calculations. The calculations reveal a highly ordered, compact conformation in which a helical region extending from K9 to C15 lies in close apposition with the C-terminal hexapeptide this interaction seems to be largely driven by hydrophobic interactions. Structure-activity studies are interpreted in terms of the conformational features of the calculated endothelin-3 structure.

Venom of the Red-Bellied Black Snake Pseudechis porphyriacus Shows Immunosuppressive Potential

Publisher: MDPI AG

Date: 26-10-2020

DOI: 10.3390/TOXINS12110674

Abstract: Venoms act with remarkable specificity upon a broad ersity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals

Publisher: Proceedings of the National Academy of Sciences

Date: 07-02-2022

DOI: 10.1073/PNAS.2112630119

Abstract: The targeting of mammalian ErbB receptor signaling by a venom toxin to cause hypersensitivity is a mode of action that has not previously been described. Natural selection of a defensive toxin to target ErbB signaling provides compelling independent evidence for a fundamental role of this receptor and its ligands in mammalian pain. The evolution of a toxin in ant venom to mimic a vertebrate nociceptive hormone serves as an ex le of both convergent evolution and molecular mimicry, illustrating how natural selection can shape the gene product of one organism to resemble that of another.

Polar explorers: Membrane proteins that determine division site placement

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0092-8674(01)00432-9

Spider-Venom Peptides as Bioinsecticides

Publisher: MDPI AG

Date: 22-03-2012

DOI: 10.3390/TOXINS4030191

Division site placement in E.coli: Mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains

Publisher: Wiley

Date: 07-2002

DOI: 10.1093/EMBOJ/CDF323

¹H, ¹³C and ¹⁵N NMR assignments of two plant protease inhibitors (IRD7 and IRD12) from the plant Capsicum annuum.

Publisher: Springer Science and Business Media LLC

Date: 18-09-2018

DOI: 10.1007/S12104-018-9846-Z

Abstract: Helicoverpa species are polyphagous pests, with the larval stages causing major damage to economically valuable crops such as cotton, tomato, corn, sorghum, peas, sunflower, wheat and other pulses. Over the years, Helicoverpa armigera has developed resistance to most classes of chemical insecticides, and consequently it is now largely controlled on cotton plants via the use of Bt transgenic crops that express insecticidal Cry toxins which in-turn expedited resistance development in a number of pest species including H. armigera. In a hope to provide other eco-friendly alternatives solutions to counter the effect of the pest, people have identified a number of protease inhibitors (PIs) from the domesticated capsicum species Capsicum annuum, several of which potently inhibited H. armigera gut proteases and impeded growth of H. armigera larva. With a view to explore and enhance the specific nature or properties of these PIs on the mechanism of inhibition, structural and functional characterization of these PIs are inevitable. Towards this goal, we have carried out complete

The insecticidal spider toxin SFI1 is a knottin peptide that blocks the pore of insect voltage-gated sodium channels via a large β-hairpin loop

Publisher: Wiley

Date: 23-01-2015

DOI: 10.1111/FEBS.13189

Abstract: Spider venoms contain a plethora of insecticidal peptides that act on neuronal ion channels and receptors. Because of their high specificity, potency and stability, these peptides have attracted much attention as potential environmentally friendly insecticides. Although many insecticidal spider venom peptides have been isolated, the molecular target, mode of action and structure of only a small minority have been explored. Sf1a, a 46-residue peptide isolated from the venom of the tube-web spider Segesteria florentina, is insecticidal to a wide range of insects, but nontoxic to vertebrates. In order to investigate its structure and mode of action, we developed an efficient bacterial expression system for the production of Sf1a. We determined a high-resolution solution structure of Sf1a using multidimensional 3D/4D NMR spectroscopy. This revealed that Sf1a is a knottin peptide with an unusually large β-hairpin loop that accounts for a third of the peptide length. This loop is delimited by a fourth disulfide bond that is not commonly found in knottin peptides. We showed, through mutagenesis, that this large loop is functionally critical for insecticidal activity. Sf1a was further shown to be a selective inhibitor of insect voltage-gated sodium channels, consistent with its 'depressant' paralytic phenotype in insects. However, in contrast to the majority of spider-derived sodium channel toxins that function as gating modifiers via interaction with one or more of the voltage-sensor domains, Sf1a appears to act as a pore blocker.

Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain.

Publisher: Wiley

Date: 02-1994

DOI: 10.1111/J.1432-1033.1994.TB18569.X

Abstract: Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains. We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs. With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain. A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction lification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini. The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase. Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells. We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices. We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy.

Can we resolve the taxonomic bias in spider venom research?

Publisher: Elsevier BV

Date: 2019

DOI: 10.1016/J.TOXCX.2018.100005

Buzz Kill: Function and Proteomic Composition of Venom from the Giant Assassin Fly Dolopus genitalis (Diptera: Asilidae)

Publisher: MDPI AG

Date: 05-11-2018

DOI: 10.3390/TOXINS10110456

Abstract: Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

Natural born insect killers: Spider-venom peptides and their potential for managing arthropod pests

Publisher: Research Information Ltd.

Date: 02-2013

DOI: 10.1564/V24_FEB_05

Methods for Deployment of Spider Venom Peptides as Bioinsecticides

Publisher: Elsevier

Date: 2014

DOI: 10.1016/B978-0-12-800197-4.00008-7

Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

Publisher: Cold Spring Harbor Laboratory

Date: 10-12-2022

DOI: 10.1101/2022.12.08.518931

Abstract: ShK from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni , characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T . stephensoni . We identified ten SA8 genes in two clusters and six SA8 genes in five clusters for T. stephensoni and A. tenebrosa , respectively. Nine SA8 T. stephensoni genes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T . stephensoni .

1H-NMR visible neutral lipids in activated T lymphocytes: Relationship to phosphatidylcholine cycling

Publisher: Elsevier BV

Date: 10-1996

DOI: 10.1016/0005-2760(96)00104-X

Abstract: Two-dimensional 1H-NMR spectroscopy was used to compare changes in the concentration of isotropically-tumbling neutral lipid during the activation of splenic and thymic T lymphocytes. The concentration of mobile neutral lipid (MNL) was similar in splenic and thymic T cells after 72 h of activation with phorbol myristate acetate and ionomycin. However, after 120 h of activation, MNL concentrations in splenic T cells were more than 3-fold higher than in thymic T cells. An increase in choline (Cho), phosphocholine (PCho) and glycerophosphocholine (GPC) was also observed in both thymic and splenic T cells after 24 h of activation. However, after 72 h of stimulation, Cho and PCho levels had decreased and continued to decline at 96-120 h, while GPC continued to be maintained at elevated levels. The simultaneous increase in MNL and GPC and the decline in Cho and PCho leads us to propose that the synthesis of NMR-visible MNL in activated lymphocytes is linked to the phosphatidylcholine cycle.

Animal toxins - Nature's evolutionary-refined toolkit for basic research and drug discovery.

Publisher: Elsevier BV

Date: 11-2020

DOI: 10.1016/J.BCP.2020.114096

RNA polymerase-induced remodelling of NusA produces a pause enhancement complex

Publisher: Oxford University Press (OUP)

Date: 17-02-2015

DOI: 10.1093/NAR/GKV108

Chemical synthesis, 3D structure, and ASIC binding site of the toxin mambalgin-2

Publisher: Wiley

Date: 09-12-2014

DOI: 10.1002/ANIE.201308898

Abstract: Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.

Proton NMR spectroscopic studies of dipeptidase in human erythrocytes

Publisher: Elsevier BV

Date: 1983

DOI: 10.1016/0006-291X(83)91296-2

Abstract: In studies on human erythrocyte metabolism in situ, high resolution (400 MHz) 1H spin-echo NMR spectroscopy was used to follow the time dependence of hydrolysis of glycylglycine and L-cysteinylglycine in intact cells and their lysates. The concentration dependence of the hydrolysis of L-cysteinylglycine was described by a rectangular hyperbola with Km, 3.5 +/- 0.6 mmol/l lysate and Vmax, 64.2 +/- 3.2 mmol/l lysate/h. We demonstrated that glycylglycine readily enters the erythrocyte and we introduce a means of analysing the data from the coupled reaction sequence the sequence consists of transport followed by enzyme catalysed hydrolysis.

Comparison of the peptidome and insecticidal activity of venom from a taxonomically diverse group of theraphosid spiders

Publisher: Elsevier BV

Date: 04-2009

DOI: 10.1016/J.TOXICON.2009.01.025

Abstract: We screened a panel of theraphosid venoms in two orders of insect in order to determine whether these bioassays would help in the selection of candidate venoms for future discovery of insecticidal toxins. Venoms from six different theraphosid genera were compared with venom from the Australian funnel-web spider Hadronyche infensa (Hexathelidae). The tarantulas included were Coremiocnemis tropix, Selenocosmia crossipes, and Selenotholus foelschei from Australia and Brachypelma albiceps and Brachypelma hamorii from Mexico. The insects assayed, Tenebrio molitor (Coleoptera: Tenebrionidae) and Acheta domesticus (Orthoptera: Gryllidae), were selected because of their relevance as model holometabolous and hemimetabolous insects, respectively, as well as their taxonomic relationship to economically important pest insects. Despite significant differences in their peptide rotein profiles as determined using SDS-PAGE, HPLC, and mass spectrometry, all of the theraphosid venoms exhibited remarkably similar LD50 values of 46-126 microg/g for crickets and 0.5-4.0 microg/g for mealworms. Notably, mealworms were on average 50-fold more susceptible than crickets to each of the crude theraphosid venoms and consequently they provide an excellent bioassay system when venom supply is limited. This study indicates that even closely related spiders have evolved quite different toxin repertoires that nevertheless have comparable efficiency with respect to killing their primary prey, namely insects.

Heterodimeric insecticidal peptide provides new insights into the molecular and functional diversity of ant venoms

Publisher: Cold Spring Harbor Laboratory

Date: 30-07-2020

DOI: 10.1101/2020.07.29.226878

Abstract: Ants use venom for predation, defence and communication, however, the molecular ersity, function and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modelling, stability studies, and a series of in vitro and in vivo bioassays to investigate the molecular ersity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain crosslinked by two disulfide bonds in an antiparallel orientation. We chemically synthesised Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blow fly assays (LD 50 = 3 nM). Molecular modelling and circular dichroism studies revealed strong alpha-helical features, indicating its cytotoxic effects could derive from membrane disruption, which was further supported by insect cell calcium assays. The native heterodimer was also substantially more stable against proteolytic degradation (t 1/2 =13 h) than its homodimers or monomers (t 1/2 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterisation of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom, and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability a concept that is also highly relevant for the design and development of peptide therapeutics, molecular probes and bioinsecticides.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus

Publisher: Wiley

Date: 15-10-2016

DOI: 10.1111/IMB.12268

Abstract: Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U

High resolution 1H NMR study of the solution structure of the S4 segment of the sodium channel protein

Publisher: Wiley

Date: 23-10-1989

DOI: 10.1016/0014-5793(89)81799-5

Abstract: A peptide (S4) of the rat brain sodium channel has been synthesized, studied by high-resolution NMR and its secondary structure modelled by distance geometry and restrained molecular dynamics techniques.

An NMR investigation of the changes in plasma membrane triglyceride and phospholipid precursors during the activation of T-lymphocytes

Publisher: American Chemical Society (ACS)

Date: 22-09-1992

DOI: 10.1021/BI00152A054

Abstract: Two-dimensional 1H-NMR spectroscopy was used to quantify the level of isotropically tumbling plasma membrane triglyceride and the intracellular concentrations of water-soluble phospholipid precursors during the activation of thymic T-lymphocytes. The concentration of "mobile" triglyceride in the plasma membrane was seen to increase 25-fold during 72 h of activation of murine thymic T-lymphocytes with ionomycin and phorbol myristate acetate. This is the first unequivocal demonstration of such a dramatic increase in mobile plasma membrane triglyceride during T-lymphocyte activation and leads to the suggestion that immune cell activation is associated with increased plasma membrane fluidity. The intracellular concentrations of choline- and ethanolamine-based phospholipid precursors were shown to increase during the early stages of T-lymphocyte activation and then remain at levels above those in resting cells. This may facilitate de novo phospholipid biosynthesis, which is presumably necessary since cell volume, and hence the plasma membrane surface area, was demonstrated to increase significantly during thymocyte activation.

Chemical synthesis and structure of the prokineticin Bv8

Publisher: Wiley

Date: 30-08-2010

DOI: 10.1002/CBIC.201000330

Abstract: Bv8, a 77-residue protein isolated from frogs, is the prototypic member of the prokineticin family of cytokines. Prokineticins (PKs) have only recently been identified in vertebrates (including humans), and they are believed to be involved in a number of key physiological processes, such as angiogenesis, neurogenesis, nociception, and tissue development. We used a combination of Boc solid-phase peptide synthesis, native chemical ligation, and in vitro protein folding to establish robust chemical access to this molecule. Synthetic Bv8 was obtained in good yield and exhibited full activity in a human neuroblastoma cell line and rat dorsal root ganglion (DRG) neurons. The 3D structure of the synthetic protein was determined by using NMR spectroscopy and it was found to be homologous with that of mamba intestinal toxin 1, which is the only other known prokineticin structure. Analysis of a truncated mutant lacking five residues at the N terminus that are critical for receptor binding and activation showed no perturbation to the core protein structure. Together with the functional data, this suggests that receptor binding is likely to be a highly cooperative process possibly involving major allosterically driven structural rearrangements. The facile and efficient synthesis presented here will enable preparation of unique chemical analogues of prokineticins, which should be powerful tools for modulating the structure and function of prokineticins and their receptors, and studying the many physiological processes that have been linked to them.

Melt with this kiss: Paralyzing and liquefying venom of the Assassin bug Pristhesancus plagipennis (Hemiptera: Reduviidae)

Publisher: Elsevier BV

Date: 04-2017

DOI: 10.1074/MCP.M116.063321

Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function

Publisher: Elsevier BV

Date: 06-2002

DOI: 10.1074/JBC.M202297200

Tying pest insects in knots: the deployment of spider‐venom‐derived knottins as bioinsecticides

Publisher: Wiley

Date: 31-05-2019

DOI: 10.1002/PS.5452

Abstract: Spider venoms are complex chemical arsenals that contain a rich variety of insecticidal toxins. However, the major toxin class in many spider venoms is disulfide-rich peptides known as knottins. The knotted three-dimensional fold of these mini-proteins provides them with exceptional chemical and thermal stability as well as resistance to proteases. In contrast with other bioinsecticides, which are often slow-acting, spider knottins are fast-acting neurotoxins. In addition to being potently insecticidal, some knottins have exceptional taxonomic selectivity, being lethal to key agricultural pests but innocuous to vertebrates and beneficial insects such as bees. The intrinsic oral activity of these peptides, combined with the ability of aerosolized knottins to penetrate insect spiracles, has enabled them to be developed commercially as eco-friendly bioinsecticides. Moreover, it has been demonstrated that spider-knottin transgenes can be used to engineer faster-acting entomopathogens and insect-resistant crops. © 2019 Society of Chemical Industry.

Fluorescence labeling of a NaV1.7-targeted peptide for near-infrared nerve visualization

Publisher: Springer Science and Business Media LLC

Date: 14-05-2020

DOI: 10.1186/S13550-020-00630-4

High-resolution solution structure of gurmarin, a sweet-taste- suppressing plant polypeptide

Publisher: Wiley

Date: 09-1999

DOI: 10.1046/J.1432-1327.1999.00659.X

Abstract: Gurmarin is a 35-residue polypeptide from the Asclepiad vine Gymnema sylvestre. It has been utilised as a pharmacological tool in the study of sweet-taste transduction because of its ability to selectively inhibit the neural response to sweet tastants in rats. We have chemically synthesised and folded gurmarin and determined its three-dimensional solution structure to high resolution using two-dimensional NMR spectroscopy. Structure calculations utilised 612 interproton-distance, 19 dihedral-angle, and 18 hydrogen-bond restraints. The structure is well defined for residues 3-34, with backbone and heavy atom rms differences of 0.27 +/- 0.09 A and 0.73 +/- 0.09 A, respectively. Gurmarin adopts a compact structure containing an antiparallel beta-hairpin (residues 22-34), several well-defined beta-turns, and a cystine-knot motif commonly observed in toxic and inhibitory polypeptides. Despite striking structural homology with delta-atracotoxin, a spider neurotoxin known to slow the inactivation of voltage-gated Na+ channels, we show that gurmarin has no effect on a variety of voltage-sensitive channels.

Studies of rat brain metabolism using proton nuclear magnetic resonance: Spectral assignments and monitoring of prolidase, acetylcholinesterase, and glutaminase

Publisher: Wiley

Date: 12-1984

DOI: 10.1111/J.1471-4159.1984.TB06079.X

Abstract: The first application of inversion-recovery spin-echo proton nuclear magnetic resonance spectroscopy to the monitoring of reactions in rat brain preparations is presented. The initial report of the assignment of proton spin-echo nuclear magnetic resonance spectra from rabbit brain homogenates (C. R. Middlehurst et al., J. Neurochem. 42, 878-879, 1984) was used to assist in the assignment of spectra acquired from rat brain homogenates that were obtained from animals killed by cervical fracture or focussed microwave irradiation. Microwave-irradiated brains were ided into four major anatomical regions. Differences in metabolite levels were detected when spectra from fresh tissue and from various regions were compared. The in situ steady-state kinetics of prolidase in whole brain homogenate was determined. The procedure relies on the spectral differences between enzyme substrates and reaction products. The concentration dependence of the rate of hydrolysis of glycyl-L-proline was discribable by the Michaelis-Menten expression with a Michaelis constant of 1.90 mmol L-1 and a maximal velocity of 9.30 mumol min-1 mg-1 protein. The reactions catalysed by glutaminase and acetylcholinesterase in the brain were also monitored.

National Health And Medical Research Council

Location: Australia

View Organisation

University Of Queensland

Location: Australia

View Organisation

University Of Sydney

Location: Australia

View Organisation

University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

View Organisation

Linkage Projects - Grant ID: LP0882794

Start Date: 08-2008

End Date: 08-2011

Amount: $439,628.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0878450

Start Date: 2008

End Date: 12-2010

Amount: $545,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100226

Start Date: 07-2010

End Date: 08-2011

Amount: $424,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP110100509

Start Date: 08-2011

End Date: 04-2015

Amount: $315,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE220100060

Start Date: 06-2022

End Date: 06-2023

Amount: $772,676.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0774245

Start Date: 2007

End Date: 12-2009

Amount: $576,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP1095728

Start Date: 2010

End Date: 12-2012

Amount: $560,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0346325

Start Date: 01-2003

End Date: 12-2005

Amount: $135,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160104025

Start Date: 2016

End Date: 12-2018

Amount: $320,700.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100192

Start Date: 05-2017

End Date: 12-2017

Amount: $450,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP160104411

Start Date: 2016

End Date: 12-2018

Amount: $435,700.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110103129

Start Date: 2011

End Date: 12-2014

Amount: $550,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP130101143

Start Date: 04-2014

End Date: 04-2017

Amount: $450,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP120200623

Start Date: 03-2013

End Date: 12-2018

Amount: $1,575,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100186

Start Date: 03-2011

End Date: 03-2012

Amount: $550,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP130103813

Start Date: 2013

End Date: 12-2015

Amount: $325,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP140100832

Start Date: 11-2014

End Date: 11-2017

Amount: $425,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190103787

Start Date: 01-2019

End Date: 01-2022

Amount: $405,000.00

Funder: Australian Research Council

Linkage - International - Grant ID: LX0775899

Start Date: 05-2007

End Date: 06-2009

Amount: $137,771.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE200100190

Start Date: 08-2020

End Date: 12-2020

Amount: $620,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE200100012

Start Date: 01-2021

End Date: 01-2028

Amount: $35,000,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0559396

Start Date: 02-2005

End Date: 12-2009

Amount: $240,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100023

Start Date: 03-2017

End Date: 06-2018

Amount: $650,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100015

Start Date: 03-2012

End Date: 02-2013

Amount: $630,000.00

Funder: Australian Research Council