ORCID Profile
0000-0002-2945-1484
Current Organisation
National Institutes of Health
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Medicinal and Biomolecular Chemistry | Proteins and Peptides | Animal Cell and Molecular Biology | Genome Structure and Regulation | Characterisation of Biological Macromolecules | Biologically Active Molecules | Evolutionary Biology | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Basic Pharmacology | Biological Adaptation
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Biological Sciences | Expanding Knowledge in the Medical and Health Sciences |
Related Links
Publications
PH‐Domain‐driven targeting of collybistin but not Cdc42 activation is required for synaptic gephyrin clustering
Publisher: Wiley
Date: 04-2010
DOI: 10.1111/J.1460-9568.2010.07149.X
Abstract: Collybistin (Cb) is a brain-specific guanine nucleotide exchange factor (GEF) that is essential for the synaptic clustering of gephyrin and GABAA receptors in selected regions of the mammalian central nervous system. It has been previously proposed that Cb regulates gephyrin clustering by activating Cdc42, and thus acts as a signal transducer in a membrane activation process which labels postsynaptic membrane domains for inhibitory synapse formation. Here, we dissected the functional roles of the Dbl-homology (DH) and pleckstrin homology (PH) domains of the constitutively active splice variant Cb II by substituting conserved amino acid residues that are required for GEF activity towards Cdc42 and phosphoinositide binding, respectively. A Cb II mutant lacking any detectable GEF activity towards Cdc42 was still fully active in inducing gephyrin scaffold formation, both in transfected NIH-3T3 cells and in cultured hippoc al neurons. Furthermore, mice with a forebrain-specific inactivation of the Cdc42 gene displayed normal densities of gephyrin and GABA(A) receptor clusters in the hippoc us. In contrast, substitution of Cb II PH-domain residues essential for phosphoinositide binding abolished gephyrin recruitment to synaptic sites. Our results provide evidence that the formation of gephyrin scaffolds at inhibitory synapses requires an intact Cb II PH-domain but is Cdc42-independent.
Synthesis, Structural and Pharmacological Characterizations of CIC, a Novel α-Conotoxin with an Extended N-Terminal Tail
Publisher: MDPI AG
Date: 02-03-2021
DOI: 10.3390/MD19030141
Abstract: Cone snails are venomous marine predators that rely on fast-acting venom to subdue their prey and defend against aggressors. The conotoxins produced in the venom gland are small disulfide-rich peptides with high affinity and selectivity for their pharmacological targets. A dominant group comprises α-conotoxins, targeting nicotinic acetylcholine receptors. Here, we report on the synthesis, structure determination and biological activity of a novel α-conotoxin, CIC, found in the predatory venom of the piscivorous species Conus catus and its truncated mutant Δ-CIC. CIC is a 4/7 α-conotoxin with an unusual extended N-terminal tail. High-resolution NMR spectroscopy shows a major influence of the N-terminal tail on the apparent rigidity of the three-dimensional structure of CIC compared to the more flexible Δ-CIC. Surprisingly, this effect on the structure does not alter the biological activity, since both peptides selectively inhibit α3β2 and α6/α3β2β3 nAChRs with almost identical sub- to low micromolar inhibition constants. Our results suggest that the N-terminal part of α-conotoxins can accommodate chemical modifications without affecting their pharmacology.
Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity
Publisher: Wiley
Date: 24-10-2017
Proteomic Analysis of the Predatory Venom of Conus striatus Reveals Novel and Population-Specific κA-Conotoxin SIVC
Publisher: MDPI AG
Date: 17-11-2022
Abstract: Animal venoms are a rich source of pharmacological compounds with ecological and evolutionary significance, as well as with therapeutic and biotechnological potentials. Among the most promising venomous animals, cone snails produce potent neurotoxic venom to facilitate prey capture and defend against aggressors. Conus striatus, one of the largest piscivorous species, is widely distributed, from east African coasts to remote Polynesian Islands. In this study, we investigated potential intraspecific differences in venom composition between distinct geographical populations from Mayotte Island (Indian Ocean) and Australia (Pacific Ocean). Significant variations were noted among the most abundant components, namely the κA-conotoxins, which contain three disulfide bridges and complex glycosylations. The amino acid sequence of a novel κA-conotoxin SIVC, including its N-terminal acetylated variant, was deciphered using tandem mass spectrometry (MS/MS). In addition, the glycosylation pattern was found to be consisting of two HexNAc and four Hex for the Mayotte population, which erge from the previously characterized two HexNAc and three Hex combinations for this species, collected elsewhere. Whereas the biological and ecological roles of these modifications remain to be investigated, population-specific glycosylation patterns provide, for the first time, a new level of intraspecific variations in cone snail venoms.
Glycine Transporter Dimers
Publisher: Elsevier BV
Date: 04-2008
Evolution of separate predation-and defence-evoked venoms in carnivorous cone snails
Publisher: Springer Science and Business Media LLC
Date: 24-03-2014
DOI: 10.1038/NCOMMS4521
Inhibitory Glycine Receptors: An Update
Publisher: Elsevier BV
Date: 11-2012
Dramatic intraspecimen variations within the injected venom of Conus consors: An unsuspected contribution to venom diversity
Publisher: Elsevier BV
Date: 07-2010
DOI: 10.1016/J.TOXICON.2010.02.025
Abstract: With the advent of highly sensitive mass spectrometry techniques, the minute amount of various secretions produced by living animals can be studied to a level of details never attained before. In this study, we used LC-ESI-MS to analyse the injected venom of an indo-pacific piscivorous cone snail, Conus consors. While long-term follow up of several captive specimens have revealed a typical "venom fingerprint" for this species, dramatic variations were also observed. In the most extreme case, a single cone snail unexpectedly produced two very distinct venom profiles containing completely different sets of peptides with no overlap of detected masses. Surprisingly, there was no correlation between the peptides produced in the venom duct and those obtained after milking live cone snails, implying yet unknown mechanisms of selection and regulation. Our study defines the notion of intraspecimen variation and demonstrates how this phenomenon contributes to the overall venom ersity.
A Combined Transcriptomics and Proteomics Approach Reveals the Differences in the Predatory and Defensive Venoms of the Molluscivorous Cone Snail Cylinder ammiralis (Caenogastropoda: Conidae)
Publisher: MDPI AG
Date: 10-09-2021
Abstract: Venoms are complex mixtures of proteins that have evolved repeatedly in the animal kingdom. Cone snail venoms represent one of the best studied venom systems. In nature, this venom can be dynamically adjusted depending on its final purpose, whether to deter predators or hunt prey. Here, the transcriptome of the venom gland and the proteomes of the predation-evoked and defensive venoms of the molluscivorous cone snail Cylinder ammiralis were catalogued. A total of 242 venom-related transcripts were annotated. The conotoxin superfamilies presenting more different peptides were O1, O2, T, and M, which also showed high expression levels (except T). The three precursors of the J superfamily were also highly expressed. The predation-evoked and defensive venoms showed a markedly distinct profile. A total of 217 different peptides were identified, with half of them being unique to one venom. A total of 59 peptides ascribed to 23 different protein families were found to be exclusive to the predatory venom, including the cono-insulin, which was, for the first time, identified in an injected venom. A total of 43 peptides from 20 protein families were exclusive to the defensive venom. Finally, comparisons of the relative abundance (in terms of number of peptides) of the different conotoxin precursor superfamilies showed that most of them present similar abundance regardless of the diet.
Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose
Publisher: Elsevier BV
Date: 12-2014
DOI: 10.1016/J.TOXICON.2014.09.011
Abstract: Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of in idual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, μ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.
Chemical Synthesis of MT1 and MT7 Muscarinic Toxins: Critical Role of Arg-34 in Their Interaction with M1 Muscarinic Receptor
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 2003
DOI: 10.1124/MOL.63.1.26
Abstract: Two muscarinic toxins, MT1 and MT7, were obtained by one-step solid-phase synthesis using the 9-fluorenylmethoxycarbonyl-based method. The synthetic and natural toxins, isolated from the snake venom or recombinantly expressed, display identical physicochemical properties and pharmacological profiles. High protein recovery allowed us to specify the selectivity of these toxins for various muscarinic receptor subtypes. Thus, sMT7 has a selectivity for the M1 receptor that is at least 20,000 times that for the other subtypes. The stability of the toxin-receptor complexes indicates that sMT1 interacts reversibly with the M1 receptor, unlike sMT7, which binds it quasi-irreversibly. The effect of the synthetic toxins on the atropine-induced [3H]N-methylscopolamine (NMS) dissociation confirms that sMT7 targets the allosteric site on the M1 receptor, whereas sMT1 seems interact on the orthosteric one. The great decreases in the binding potencies observed after the R34A modification in sMT1 and sMT7 toxins highlight the functional role of this conserved residue in their interactions with the M1 receptor. Interestingly, after the R34A modification, the sMT7 toxin binds reversibly on the M1 receptor. Furthermore, the potency of sMT7-R34A for the NMS-occupied receptor is lower compared with unmodified toxin, supporting the role of this residue in the allosteric interaction of sMT7. All these results and the different charge distributions observed at the two toxin surfaces of their structure models support the hypothesis that the two toxins recognize the M1 receptor differently.
Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor
Publisher: Proceedings of the National Academy of Sciences
Date: 27-06-2012
Abstract: P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage cl fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120–I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2′,3′- O -(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP–induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.
Venomics of the asp viper Vipera aspis aspis from France
Publisher: Elsevier BV
Date: 04-2020
Computational approaches to understand α‐conotoxin interactions at neuronal nicotinic receptors
Publisher: Wiley
Date: 06-2004
Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity
Publisher: Wiley
Date: 24-10-2017
Abstract: Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC
Conus venom peptide pharmacology
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-03-2012
Abstract: Conopeptides are a erse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a erse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the ersity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.
Transcriptome and proteome of Conus planorbis identify the nicotinic receptors as primary target for the defensive venom
Publisher: Wiley
Date: 17-11-2015
Abstract: Most venomous predators have evolved complex venom primarily to immobilize their prey and secondarily to defend against predators. In a new paradigm, carnivorous marine gastropods of the genus Conus were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, suggesting that the defensive use of venom may have a more important role in venom evolution and specialization than previously thought. To further investigate this phenomenon, the defensive repertoire of a vermivorous species, Conus planorbis, was deciphered using second-generation sequencing coupled to high-throughput proteomics. The venom gland transcriptome of C. planorbis revealed 182 unique conotoxin precursors from 25 gene superfamilies, with superfamily T dominating in terms of read and paralog numbers. Analysis of the defense-evoked venom revealed that this vermivorous species uses a similarly complex arsenal to deter aggressors as more recently evolved fish- and mollusk-hunting species, with MS/MS validating 23 conotoxin sequences from six superfamilies. Pharmacological characterization of the defensive venom on human receptors identified the nicotinic acetylcholine receptors as a primary target. This work provides the first insights into the composition and biological activity of specifically evolved defensive venoms in vermivorous cone snails.
Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors
Publisher: Elsevier BV
Date: 03-2015
Discovery, synthesis, and structure-activity relationships of conotoxins
Publisher: American Chemical Society (ACS)
Date: 10-04-2014
DOI: 10.1021/CR400401E
Transcriptomic messiness in the venom duct of conus miles contributes to conotoxin diversity
Publisher: Elsevier BV
Date: 12-2013
Comparative proteomic study of the venom of the piscivorous cone snail Conus consors
Publisher: Elsevier BV
Date: 03-2009
DOI: 10.1016/J.JPROT.2009.01.019
Abstract: In the context of an exhaustive study of the piscivorous cone snail Conus consors, we performed an in-depth analysis of the intact molecular masses that can be detected in the animal's venom, using MALDI and ESI mass spectrometry. We clearly demonstrated that, for the venom of this species at least, it is essential to use both techniques in order to obtain the broadest data set of molecular masses. Only 20% of the total number of molecules detected were found in both mass lists. The two data sets were also compared in terms of mass range and relative hydrophobicity of the components detected in each. With a view to an extensive analysis of this venom's proteome, we further performed a comparative study by ESI-MS between venom obtained after classical dissection of the venom duct versus venom obtained by milking live animals. Surprisingly, although many fewer components were found in the milked venom than in the dissected venom, approximately 50% of those found had not been seen in the dissected venom. Several questions raised by these observations are discussed. With regards to the current knowledge of the cone snail venom composition, our results emphasize the complementary nature of the mass spectrometry methods and of the two techniques used in venom collection.
ConoServer: Updated content, knowledge, and discovery tools in the conopeptide database
Publisher: Oxford University Press (OUP)
Date: 03-11-2012
DOI: 10.1093/NAR/GKR886
Venomics in medicinal chemistry
Publisher: Future Science Ltd
Date: 10-2014
DOI: 10.4155/FMC.14.117
AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivity
Publisher: Wiley
Date: 26-07-2007
High-resolution picture of a venom gland transcriptome: Case study with the marine snail Conus consors
Publisher: Elsevier BV
Date: 2012
DOI: 10.1016/J.TOXICON.2011.10.001
Abstract: Although cone snail venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole venom duct. Additionally, rare transcripts were usually not identified due to s ling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using venom ducts of Conus consors, an Indo-Pacific living piscivorous cone snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform ersity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most erse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in venom function could be identified for the first time in cone snail venom duct. This work provides one of the first large-scale EST project for a cone snail venom duct using next-generation sequencing, allowing a detailed overview of the venom duct transcripts. This leads to an expanded definition of the overall cone snail venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides ersity and the underlying mechanisms involved in conopeptide evolution.
Proteomic insight into the venom composition of the largest European rear-fanged snake, Malpolon monspessulanus monspessulanus
Publisher: Elsevier BV
Date: 09-2022
Cone snail venomics: From novel biology to novel therapeutics
Publisher: Future Science Ltd
Date: 10-2014
DOI: 10.4155/FMC.14.99
Abstract: Peptide neurotoxins from cone snails called conotoxins are renowned for their therapeutic potential to treat pain and several neurodegenerative diseases. Inefficient assay-guided discovery methods have been replaced by high-throughput bioassays integrated with advanced MS and next-generation sequencing, ushering in the era of ‘venomics’. In this review, we focus on the impact of venomics on the understanding of cone snail biology as well as the application of venomics to accelerate the discovery of new conotoxins. We also discuss the continued importance of medicinal chemistry approaches to optimize conotoxins for clinical use, with a descriptive case study of MrIA featured.
Preferential use of unobstructed lateral portals as the access route to the pore of human ATP-gated ion channels (P2X receptors)
Publisher: Proceedings of the National Academy of Sciences
Date: 08-2011
Abstract: P2X receptors are trimeric cation channels with widespread roles in health and disease. The recent crystal structure of a P2X4 receptor provides a 3D view of their topology and architecture. A key unresolved issue is how ions gain access to the pore, because the structure reveals two different pathways within the extracellular domain. One of these is the central pathway spanning the entire length of the extracellular domain and covering a distance of ≈70 Å. The second consists of three lateral portals, adjacent to the membrane and connected to the transmembrane pore by short tunnels. Here, we demonstrate the preferential use of the lateral portals. Owing to their favorable diameters and equivalent spacing, the lateral portals split the task of ion supply threefold and minimize an ion's diffusive path before it succumbs to transmembrane electrochemical gradients.
Miniaturized Bioaffinity Assessment Coupled to Mass Spectrometry for Guided Purification of Bioactives from Toad and Cone Snail
Publisher: MDPI AG
Date: 13-02-2014
Deep venomics reveals the mechanism for expanded peptide diversity in cone snail venom
Publisher: Elsevier BV
Date: 02-2013
Combined Proteotranscriptomic-Based Strategy to Discover Novel Antimicrobial Peptides from Cone Snails
Publisher: MDPI AG
Date: 29-03-2021
DOI: 10.3390/BIOMEDICINES9040344
Abstract: Despite their impressive ersity and already broad therapeutic applications, cone snail venoms have received less attention as a natural source in the investigation of antimicrobial peptides than other venomous animals such as scorpions, spiders, or snakes. Cone snails are among the largest genera (Conus sp.) of marine invertebrates, with more than seven hundred species described to date. These predatory mollusks use their sophisticated venom apparatus to capture prey or defend themselves. In-depth studies of these venoms have unraveled many biologically active peptides with pharmacological properties of interest in the field of pain management, the treatment of epilepsy, neurodegenerative diseases, and cardiac ischemia. Considering sequencing efficiency and affordability, cone snail venom gland transcriptome analyses could allow the discovery of new, promising antimicrobial peptides. We first present here the need for novel compounds like antimicrobial peptides as a viable alternative to conventional antibiotics. Secondly, we review the current knowledge on cone snails as a source of antimicrobial peptides. Then, we present the current state of the art in analytical methods applied to crude or milked venom followed by how antibacterial activity assay can be implemented for fostering cone snail antimicrobial peptides studies. We also propose a new innovative profile Hidden Markov model-based approach to annotate full venom gland transcriptomes and speed up the discovery of potentially active peptides from cone snails.
Use of Venom Peptides to Probe Ion Channel Structure and Function
Publisher: Elsevier BV
Date: 04-2010
The Deadly Toxin Arsenal of the Tree-Dwelling Australian Funnel-Web Spiders
Publisher: MDPI AG
Date: 28-10-2022
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.
Venomics Reveals Venom Complexity of the Piscivorous Cone Snail, Conus tulipa
Publisher: MDPI AG
Date: 21-01-2019
DOI: 10.3390/MD17010071
Abstract: The piscivorous cone snail Conus tulipa has evolved a net-hunting strategy, akin to the deadly Conus geographus, and is considered the second most dangerous cone snail to humans. Here, we present the first venomics study of C. tulipa venom using integrated transcriptomic and proteomic approaches. Parallel transcriptomic analysis of two C. tulipa specimens revealed striking differences in conopeptide expression levels (2.5-fold) between in iduals, identifying 522 and 328 conotoxin precursors from 18 known gene superfamilies. Despite broad overlap at the superfamily level, only 86 precursors (11%) were common to both specimens. Conantokins (NMDA antagonists) from the superfamily B1 dominated the transcriptome and proteome of C. tulipa venom, along with superfamilies B2, A, O1, O3, con-ikot-ikot and conopressins, plus novel putative conotoxins precursors T1.3, T6.2, T6.3, T6.4 and T8.1. Thus, C. tulipa venom comprised both paralytic (putative ion channel modulating α-, ω-, μ-, δ-) and non-paralytic (conantokins, con-ikot-ikots, conopressins) conotoxins. This venomic study confirms the potential for non-paralytic conotoxins to contribute to the net-hunting strategy of C. tulipa.
Backbone Cyclization Turns a Venom Peptide into a Stable and Equipotent Ligand at Both Muscle and Neuronal Nicotinic Receptors
Publisher: American Chemical Society (ACS)
Date: 16-10-2020
Recruitment of Glycosyl Hydrolase Proteins in a Cone Snail Venomous Arsenal: Further Insights into Biomolecular Features of Conus Venoms
Publisher: MDPI AG
Date: 31-01-2012
DOI: 10.3390/MD10020258
Synthesis, Pharmacological and Structural Characterization of Novel Conopressins from Conus miliaris
Publisher: MDPI AG
Date: 06-03-2020
DOI: 10.3390/MD18030150
Abstract: Cone snails produce a fast-acting and often paralyzing venom, largely dominated by disulfide-rich conotoxins targeting ion channels. Although disulfide-poor conopeptides are usually minor components of cone snail venoms, their ability to target key membrane receptors such as GPCRs make them highly valuable as drug lead compounds. From the venom gland transcriptome of Conus miliaris, we report here on the discovery and characterization of two conopressins, which are nonapeptide ligands of the vasopressin/oxytocin receptor family. These novel sequence variants show unusual features, including a charge inversion at the critical position 8, with an aspartate instead of a highly conserved lysine or arginine residue. Both the amidated and acid C-terminal analogues were synthesized, followed by pharmacological characterization on human and zebrafish receptors and structural investigation by NMR. Whereas conopressin-M1 showed weak and only partial agonist activity at hV1bR (amidated form only) and ZFV1a1R (both amidated and acid form), both conopressin-M2 analogues acted as full agonists at the ZFV2 receptor with low micromolar affinity. Together with the NMR structures of amidated conopressins-M1, -M2 and -G, this study provides novel structure-activity relationship information that may help in the design of more selective ligands.
Probing the pharmacological properties of distinct subunit interfaces within heteromeric glycine receptors reveals a functional ββ agonist‐binding site
Publisher: Wiley
Date: 27-04-2012
DOI: 10.1111/J.1471-4159.2012.07755.X
Abstract: Synaptic glycine receptors (GlyRs) are hetero-pentameric chloride channels composed of α and β subunits, which are activated by agonist binding at subunit interfaces. To examine the pharmacological properties of each potential agonist-binding site, we substituted residues of the GlyR α(1) subunit by the corresponding residues of the β subunit, as deduced from sequence alignment and homology modeling based on the recently published crystal structure of the glutamate-gated chloride channel GluCl. These exchange substitutions allowed us to reproduce the βα, αβ and ββ subunit interfaces present in synaptic heteromeric GlyRs by generating recombinant homomeric receptors. When the engineered α(1) GlyR mutants were expressed in Xenopus oocytes, all subunit interface combinations were found to form functional agonist-binding sites as revealed by voltage cl recording. The ββ-binding site displayed the most distinct pharmacological profile towards a range of agonists and modulators tested, indicating that it might be selectively targeted to modulate the activity of synaptic GlyRs. The mutational approach described here should be generally applicable to heteromeric ligand-gated ion channels composed of homologous subunits and facilitate screening efforts aimed at targeting inter-subunit specific binding sites.
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.
Intraspecific variations in Conus purpurascens injected venom using LC/MALDI-TOF-MS and LC-ESI-TripleTOF-MS
Publisher: Springer Science and Business Media LLC
Date: 06-06-2015
DOI: 10.1007/S00216-015-8787-Y
Abstract: The venom of cone snails is composed of highly modified peptides (conopeptides) that target a variety of ion channels and receptors. The venom of these marine gastropods represents a largely untapped resource of bioactive compounds of potential pharmaceutical value. Here, we use a combination of bioanalytical techniques to uncover the extent of venom expression variability in Conus purpurascens, a fish-hunting cone snail species. The injected venom of nine specimens of C. purpurascens was separated by reversed-phase high-performance liquid chromatography (RP-HPLC), and fractions were analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) in parallel with liquid chromatography-electrospray ionization (LC-ESI)-TripleTOF-MS to compare standard analytical protocols used in preparative bioassay-guided fractionations with a deeper peptidomic analysis. Here, we show that C. purpurascens exhibits pronounced intraspecific venom variability. RP-HPLC fractionation followed by MALDI-TOF-MS analysis of the injected venom of these nine specimens identified 463 distinct masses, with none common to all specimens. Using LC-ESI-TripleTOF-MS, the injected venom of these nine specimens yielded a total of 5517 unique masses. We also compare the injected venom of two specimens with their corresponding dissected venom. We found 2566 and 1990 unique masses for the dissected venom compared to 941 and 1959 masses in their corresponding injected venom. Of these, 742 and 1004 masses overlapped between the dissected and injected venom, respectively. The results indicate that larger conopeptide libraries can be assessed by studying multiple in iduals of a given cone snail species. This expanded library of conopeptides enhances the opportunities for discovery of molecular modulators with direct relevance to human therapeutics. Graphical Abstract The venom of cone snails are extraordinarily complex mixtures of highly modified peptides. Venom analysis requires separation through RP-HPLC followed by MALDI-TOF mass spectrometry or direct analysis using LC-ESI-TripleTOF-MS. Using these techniques, venom intraspecific variability and comparison between injected and dissected were assessed.
Towards an integrated venomics approach for accelerated conopeptide discovery
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.TOXICON.2012.04.340
Abstract: Conopeptides and conotoxins are small peptides produced by cone snails as a part of their predatory/defense strategies that target key ion channels and receptors in the nervous system. Some of these peptides also potently target mammalian ion channels involved in pain pathways. As a result, these venoms are a source of valuable pharmacological and therapeutic agents. The traditional approach towards conopeptide discovery relied on activity-guided fractionation, which is time consuming and resource-intensive. In this review, we discuss the advances in the fields of transcriptomics, proteomics and bioinformatics that now allow researchers to integrate these three platforms towards a more efficient discovery strategy. In this review, we also highlight the challenges associated with the wealth of data generated with this integrated approach and briefly discuss the impact these methods could have on the field of toxinology.
Toxin insights into nicotinic acetylcholine receptors
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.BCP.2006.03.027
Abstract: Venomous species have evolved cocktails of bioactive peptides to facilitate prey capture. Given their often exquisite potency and target selectivity, venom peptides provide unique biochemical tools for probing the function of membrane proteins at the molecular level. In the field of the nicotinic acetylcholine receptors (nAChRs), the subtype specific snake alpha-neurotoxins and cone snail alpha-conotoxins have been widely used to probe receptor structure and function in native tissues and recombinant systems. However, only recently has it been possible to generate an accurate molecular view of these nAChR-toxin interactions. Crystal structures of AChBP, a homologue of the nAChR ligand binding domain, have now been solved in complex with alpha-cobratoxin, alpha-conotoxin PnIA and alpha-conotoxin ImI. The orientation of all three toxins in the ACh binding site confirms many of the predictions obtained from mutagenesis and docking simulations on homology models of mammalian nAChR. The precise understanding of the molecular determinants of these complexes is expected to contribute to the development of more selective nAChR modulators. In this commentary, we review the structural data on nAChR-toxin interactions and discuss their implications for the design of novel ligands acting at the nAChR.
A rare P2X7 variant Arg307Gln with absent pore formation function protects against neuroinflammation in multiple sclerosis
Publisher: Oxford University Press (OUP)
Date: 17-07-2015
DOI: 10.1093/HMG/DDV278
Abstract: Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.
Characterisation of a Novel A-Superfamily Conotoxin
Publisher: MDPI AG
Date: 20-05-2020
DOI: 10.3390/BIOMEDICINES8050128
Abstract: Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins.
Pmu1a, a novel spider toxin with dual inhibitory activity at pain targets hNaV1.7 and hCaV3 voltage‐gated channels
Publisher: Wiley
Date: 23-03-2023
DOI: 10.1111/FEBS.16773
Abstract: Venom‐derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage‐gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNa V 1.7 and hCa V 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay‐guided HPLC fractionation revealed a 36‐amino acid peptide with three disulfide bridges named μ/ω‐theraphotoxin‐Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNa V 1.7 and hCa V 3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCa V 3.2 and hNa V 1.7 voltage‐gated channels.
Transcriptomic-Proteomic Correlation in the Predation-Evoked Venom of the Cone Snail, Conus imperialis
Publisher: MDPI AG
Date: 19-03-2019
DOI: 10.3390/MD17030177
Abstract: In idual variation in animal venom has been linked to geographical location, feeding habit, season, size, and gender. Uniquely, cone snails possess the remarkable ability to change venom composition in response to predatory or defensive stimuli. To date, correlations between the venom gland transcriptome and proteome within and between in idual cone snails have not been reported. In this study, we use 454 pyrosequencing and mass spectrometry to decipher the transcriptomes and proteomes of the venom gland and corresponding predation-evoked venom of two specimens of Conus imperialis. Transcriptomic analyses revealed 17 conotoxin gene superfamilies common to both animals, including 5 novel superfamilies and two novel cysteine frameworks. While highly expressed transcripts were common to both specimens, variation of moderately and weakly expressed precursor sequences was surprisingly erse, with one specimen expressing two unique gene superfamilies and consistently producing more paralogs within each conotoxin gene superfamily. Using a quantitative labelling method, conotoxin variability was compared quantitatively, with highly expressed peptides showing a strong correlation between transcription and translation, whereas peptides expressed at lower levels showed a poor correlation. These results suggest that major transcripts are subject to stabilizing selection, while minor transcripts are subject to ersifying selection.
Efficient Binding of 4/7 α-Conotoxins to Nicotinic α4β2 Receptors Is Prevented by Arg185 and Pro195 in the α4 Subunit
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 16-07-2012
Abstract: α-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent α3β2 nAChR-selective α-conotoxins have been identified, currently characterized α-conotoxins show no or only weak affinity for α4β2 nAChRs, which are, besides α7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the α4 subunit by the corresponding residues in the α3 subunit. Two-electrode voltage cl analysis of these mutants revealed increased affinity of α-conotoxins MII, TxIA, and [A10L]TxIA at the α4(R185I)β2 receptor. Conversely, α-conotoxin potency was reduced at the reverse α3(I186R)β2 mutant. Replacement of α4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents α-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high α-conotoxin potency to the α4β2 receptor. Molecular dynamics simulations of homology models with docked α-conotoxin indicate that these residues control access to the α-conotoxin binding site.
Comparative Venomics Reveals the Complex Prey Capture Strategy of the Piscivorous Cone Snail Conus catus
Publisher: American Chemical Society (ACS)
Date: 10-09-2015
DOI: 10.1021/ACS.JPROTEOME.5B00630
Abstract: Venomous marine cone snails produce a unique and remarkably erse range of venom peptides (conotoxins and conopeptides) that have proven to be invaluable as pharmacological probes and leads to new therapies. Conus catus is a hook-and-line fish hunter from clade I, with ∼20 conotoxins identified, including the analgesic ω-conotoxin CVID (AM336). The current study unravels the venom composition of C. catus with tandem mass spectrometry and 454 sequencing data. From the venom gland transcriptome, 104 precursors were recovered from 11 superfamilies, with superfamily A (especially κA-) conotoxins dominating (77%) their venom. Proteomic analysis confirmed that κA-conotoxins dominated the predation-evoked milked venom of each of six C. catus analyzed and revealed remarkable intraspecific variation in both the intensity and type of conotoxins. High-throughput FLIPR assays revealed that the predation-evoked venom contained a range of conotoxins targeting the nAChR, Cav, and Nav ion channels, consistent with α- and ω-conotoxins being used for predation by C. catus. However, the κA-conotoxins did not act at these targets but induced potent and rapid immobilization followed by bursts of activity and finally paralysis when injected intramuscularly in zebrafish. Our venomics approach revealed the complexity of the envenomation strategy used by C. catus, which contains a mix of both excitatory and inhibitory venom peptides.
δ-conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails
Publisher: The Royal Society
Date: 22-07-2015
Abstract: Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target K v and Na v channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Na v channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail ( Conus suturatus ). SuVIA is equipotent at hNa V 1.3, hNa V 1.4 and hNa V 1.6 with EC 50 s in the low nanomolar range. SuVIA also increased peak hNa V 1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from –15 mV to –25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.
Diagnosis and treatment of obstructive sleep apnea is key to achieving optimal results after catheter ablation of atrial fibrillation.
Publisher: Elsevier BV
Date: 02-2014
MrIC, a novel α-conotoxin agonist in the presence of PNU at endogenous α7 nicotinic acetylcholine receptors
Publisher: American Chemical Society (ACS)
Date: 30-12-2014
DOI: 10.1021/BI400882S
Abstract: α-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Their high selectivity and affinity for the various subtypes of nAChRs have led to significant advances in our understanding of the structure and function of these key ion channels. Here we report the discovery of a novel 4/7 α-conotoxin, MrIC from the venom duct of Conus marmoreus, which acts as an agonist at the endogenous human α7 nAChR in SH-SY5Y cells pretreated with PNU120596 (PNU). This unique agonist activity of MrIC at α7 nAChRs may guide the development of novel α7 nAChR modulators.
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
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.
A Recurrent Motif: Diversity and Evolution of ShKT Domain Containing Proteins in the Vampire Snail Cumia reticulata
Publisher: MDPI AG
Date: 12-02-2019
Abstract: Proteins of the ShK superfamily are characterized by a small conserved domain (ShKT), first discovered in small venom peptides produced by sea anemones, and acting as specific inhibitors of voltage-dependent and calcium-activated K+ channels. The ShK superfamily includes both small toxic peptides and larger multifunctional proteins with various functions. ShK toxins are often important components of animal venoms, where they perform different biological functions including neurotoxic and immunosuppressive effects. Given their high specificity and effectiveness, they are currently regarded as promising pharmacological lead compounds for the treatment of autoimmune diseases. Here, we report on the molecular analysis of ShKT domain containing proteins produced by the Mediterranean v ire snail Cumia reticulata, an ectoparasitic gastropod that feeds on benthic fishes. The high specificity of expression of most ShK transcripts in salivary glands identifies them as relevant components of C. reticulata venom. These ShK proteins display various structural architectures, being produced either as single-domain secretory peptides, or as larger proteins combining the ShKT with M12 or CAP domains. Both ShKT-containing genes and their internal ShKT domains undergo frequent duplication events in C. reticulata, ensuring a high level of variability that is likely to play a role in increasing the range of their potential molecular targets.
Systematic interrogation of the Conus marmoreus venom duct transcriptome with ConoSorter reveals 158 novel conotoxins and 13 new gene superfamilies
Publisher: Springer Science and Business Media LLC
Date: 16-10-2013
Abstract: Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining. Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies. Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.
Synthesis and Biological Activity of Novel α-Conotoxins Derived from Endemic Polynesian Cone Snails
Publisher: MDPI AG
Date: 09-06-2023
DOI: 10.3390/MD21060356
Abstract: α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools.
Molecular engineering of conotoxins: The importance of loop size to α-conotoxin structure and function
Publisher: American Chemical Society (ACS)
Date: 28-08-2008
DOI: 10.1021/JM800278K
Abstract: Alpha-conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of alpha-conotoxins. This involved the systematic truncation of the second loop in the alpha-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha7 nAChRs, whereas 4/3[A10L]PnIA lost both alpha7 nAChR activity and helicity. In contrast, all truncated analogues lost approximately 100-fold affinity at the AChBP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha7 nAChR and AChBP.
Venomics Reveals a Non-Compartmentalised Venom Gland in the Early Diverged Vermivorous Conus distans
Publisher: MDPI AG
Date: 19-03-2022
Abstract: The defensive use of cone snail venom is hypothesised to have first arisen in ancestral worm-hunting snails and later repurposed in a compartmentalised venom duct to facilitate the dietary shift to molluscivory and piscivory. Consistent with its placement in a basal lineage, we demonstrate that the C. distans venom gland lacked distinct compartmentalisation. Transcriptomics revealed C. distans expressed a wide range of structural classes, with inhibitory cysteine knot (ICK)-containing peptides dominating. To better understand the evolution of the venom gland compartmentalisation, we compared C. distans to C. planorbis, the earliest erging species from which a defence-evoked venom has been obtained, and fish-hunting C. geographus from the Gastridium subgenus that injects distinct defensive and predatory venoms. These comparisons support the hypothesis that venom gland compartmentalisation arose in worm-hunting species and enabled repurposing of venom peptides to facilitate the dietary shift from vermivory to molluscivory and piscivory in more recently erged cone snail lineages.
β2 Subunit Contribution to 4/7 α-Conotoxin Binding to the Nicotinic Acetylcholine Receptor
Publisher: Elsevier BV
Date: 08-2005
Proteotranscriptomic Insights into the Venom Composition of the Wolf Spider Lycosa tarantula
Publisher: MDPI AG
Date: 05-08-2020
Abstract: Spider venoms represent an original source of novel compounds with therapeutic and agrochemical potential. Whereas most of the research efforts have focused on large mygalomorph spiders, araneomorph spiders are equally promising but require more sensitive and sophisticated approaches given their limited size and reduced venom yield. Belonging to the latter group, the genus Lycosa (“wolf spiders”) contains many species widely distributed throughout the world. These spiders are ambush predators that do not build webs but instead rely strongly on their venom for prey capture. Lycosa tarantula is one of the largest species of wolf spider, but its venom composition is unknown. Using a combination of RNA sequencing of the venom glands and venom proteomics, we provide the first overview of the peptides and proteins produced by this iconic Mediterranean spider. Beside the typical small disulfide rich neurotoxins, several families of proteins were also identified, including cysteine-rich secretory proteins (CRISP) and Hyaluronidases. Proteomic analysis of the electrically stimulated venom validated 30 of these transcriptomic sequences, including nine putative neurotoxins and eight venom proteins. Interestingly, LC-MS venom profiles of manual versus electric stimulation, as well as female versus male, showed some marked differences in mass distribution. Finally, we also present some preliminary data on the biological activity of L. tarantula crude venom.
Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides
Publisher: Elsevier BV
Date: 03-2008
Isolation and characterisation of conomap-Vt, a d-amino acid containing excitatory peptide from the venom of a vermivorous cone snail
Publisher: Wiley
Date: 16-06-2006
DOI: 10.1016/J.FEBSLET.2006.06.011
Abstract: Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a d-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture.
Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A
Publisher: Springer Science and Business Media LLC
Date: 15-09-2013
DOI: 10.1038/NM.3270
Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.JPROT.2012.06.001
Abstract: Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.
Determination ofα-conotoxin binding modes on neuronal nicotinic acetylcholine receptors
Publisher: Wiley
Date: 24-06-2004
DOI: 10.1002/JMR.683
Abstract: alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that ImI and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists.
Xenopus Oocytes: A Tool to Decipher Molecular Specificity of Insecticides towards Mammalian and Insect GABA—A Receptors
Publisher: MDPI AG
Date: 19-04-2022
DOI: 10.3390/MEMBRANES12050440
Abstract: The number of insect GABA receptors (GABAr) available for expression studies has been recently increased by the cloning of the Acyrthosiphon pisum (pea aphid) RDL subunits. This large number of cloned RDL subunits from pest and beneficial insects opens the door to parallel pharmacological studies on the sensitivity of these different insect GABAr to various agonists or antagonists. The resulting analysis of the molecular basis of the species-specific GABAr responses to insecticides is necessary not only to depict and understand species toxicity, but also to help at the early identification of unacceptable toxicity of insecticides toward beneficial insects such as Apis mellifera (honeybees). Using heterologous expression in Xenopus laevis oocytes, and two-electrode voltage-cl recording to assess the properties of the GABAr, we performed a comparative analysis of the pharmacological sensitivity of RDL subunits from A. pisum, A. mellifera and Varroa destructor GABAr to three pesticides (fipronil, picrotoxin and dieldrin). These data were compared to similar characterizations performed on two Homo sapiens GABA-A receptors (α2β2γ2 and α2β2γ2). Our results underline a global conservation of the pharmacological profiles of these receptors, with some interesting species specificities, nonetheless, and suggest that this approach can be useful for the early identification of poorly specific molecules.
χ-Conotoxin and Tricyclic Antidepressant Interactions at the Norepinephrine Transporter Define a New Transporter Model
Publisher: Elsevier BV
Date: 06-2007
Related Organisations
Université Paris VII Denis Diderot UFR Sciences Du Vivant
Location: France
Related Funding Activities
Discovery Projects - Grant ID: DP190101667
Start Date: 07-2019
End Date: 12-2022
Amount: $490,000.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP160103826
Start Date: 2016
End Date: 12-2019
Amount: $469,986.00
Funder: Australian Research Council
View Funded ActivityDiscovery Projects - Grant ID: DP170104792
Start Date: 2017
End Date: 12-2020
Amount: $390,000.00
Funder: Australian Research Council
View Funded Activity