Jacqueline Wilce

The TRIM-NHL protein NHL-2 is a co-factor in the nuclear and somatic RNAi pathways in C. elegans

Publisher: eLife Sciences Publications, Ltd

Date: 21-12-2018

DOI: 10.7554/ELIFE.35478

Abstract: Proper regulation of germline gene expression is essential for fertility and maintaining species integrity. In the C. elegans germline, a erse repertoire of regulatory pathways promote the expression of endogenous germline genes and limit the expression of deleterious transcripts to maintain genome homeostasis. Here we show that the conserved TRIM-NHL protein, NHL-2, plays an essential role in the C. elegans germline, modulating germline chromatin and meiotic chromosome organization. We uncover a role for NHL-2 as a co-factor in both positively (CSR-1) and negatively (HRDE-1) acting germline 22G-small RNA pathways and the somatic nuclear RNAi pathway. Furthermore, we demonstrate that NHL-2 is a bona fide RNA binding protein and, along with RNA-seq data point to a small RNA independent role for NHL-2 in regulating transcripts at the level of RNA stability. Collectively, our data implicate NHL-2 as an essential hub of gene regulatory activity in both the germline and soma.

RNA Recognition and Stress Granule Formation by TIA Proteins

Publisher: MDPI AG

Date: 16-12-2014

DOI: 10.3390/IJMS151223377

The androgen receptor mRNA

Publisher: Wiley

Date: 24-05-2004

DOI: 10.1002/BIES.20051

Abstract: Androgens (testosterone), acting via the androgen receptor (AR) a nuclear transcription factor, regulate male sexual development and body composition. In addition, AR expression plays an important role in the proliferation of human prostate cancer and confers a better prognosis in breast cancer. AR mRNA stability is central to the regulation of AR expression in prostate and breast cancer cells, and recent studies have demonstrated binding by members of the ELAV/Hu and poly(C) RNA-binding protein families to a highly conserved UC-rich element in the 3'-untranslated region of AR mRNA, with functional impact on AR protein expression. Remarkably, a CAG trinucleotide repeat in exon 1 of the AR, the length of which has been linked to prostate cancer survival, is also a target for multiple RNA-binding proteins from a variety of human and murine tissues. In this review, we will detail the current knowledge of the mechanisms involved in regulating AR mRNA stability, the nature, potential role and structural biology of several novel AR mRNA-protein interactions, and the implications for novel therapeutics in human prostate cancer.

Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes

Publisher: Wiley

Date: 19-11-2003

DOI: 10.1016/S0014-5793(03)01309-7

Abstract: The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.

Zipping up transcription factors: Rational design of anti-Jun and anti-Fos peptides

Publisher: Springer Science and Business Media LLC

Date: 04-1997

DOI: 10.1007/BF02443517

Oligonucleotide binding proteins: The occurrence of dimer and multimer formation

Publisher: Springer New York

Date: 2012

DOI: 10.1007/978-1-4614-3229-6_6

Abstract: Protein dimers and multimers are often employed by nature for DNA and RNA handling and formation of specific, high-affinity protein-oligonucleotide complexes. The repeating structure of dsDNA lends itself to recognition by multimeric protein complexes that can assemble about the helical structure. In the cases of both DNA and RNA, specific recognition of nucleotide sequences can be achieved by multidomain proteins or protein multimers. Furthermore large multimeric assemblies are utilised for the stable formation of structures such as rings and filaments. Also, the assembly of multimeric structures by interchangeable subunits can add layers of regulation and increase functional complexity. Thus there appear to be many advantages to oligonucleotide interactions that are conferred by dimerisation or multimerisation.

Partners of wild type Grb7 and a mutant lacking its calmodulin-binding domain

Publisher: Elsevier BV

Date: 07-2020

DOI: 10.1016/J.ABB.2020.108386

Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

Publisher: Elsevier BV

Date: 09-2005

DOI: 10.1016/J.BBRC.2005.07.082

Abstract: Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in 1H-15N correlation spectra of a 15N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the 1H-15N correlations was achieved using a suite of triple resonance NMR experiments with 15N,13C,70% 2H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to 1H-15N spectra revealed that the N-termini, alpha3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.

Tandem RNA binding sites induce self-association of the stress granule marker protein TIA-1

Publisher: Oxford University Press (OUP)

Date: 24-02-2021

DOI: 10.1093/NAR/GKAB080

Abstract: TIA-1 is an RNA-binding protein that sequesters target RNA into stress granules under conditions of cellular stress. Promotion of stress granule formation by TIA-1 depends upon self-association of its prion-like domain that facilitates liquid-liquid phase separation and is thought to be enhanced via RNA binding. However, the mechanisms underlying the influence of RNA on TIA-1 self-association have not been previously demonstrated. Here we have investigated the self-associating properties of full-length TIA-1 in the presence of designed and native TIA-1 nucleic acid binding sites in vitro, monitoring phase separation, fibril formation and shape. We show that single stranded RNA and DNA induce liquid-liquid phase separation of TIA-1 in a multisite, sequence-specific manner and also efficiently promote formation of amyloid-like fibrils. Although RNA binding to a single site induces a small conformational change in TIA-1, this alone does not enhance phase separation of TIA-1. Tandem binding sites are required to enhance phase separation of TIA-1 and this is finely tuned by the protein:binding site stoichiometry rather than nucleic acid length. Native tandem TIA-1 binding sites within the 3′ UTR of p53 mRNA also efficiently enhance phase separation of TIA-1 and thus may potentially act as potent nucleation sites for stress granule assembly.

Shortened Penetratin Cell-Penetrating Peptide Is Insufficient for Cytosolic Delivery of a Grb7 Targeting Peptide

Publisher: American Chemical Society (ACS)

Date: 23-02-2017

DOI: 10.1021/ACSOMEGA.6B00561

Controlling leucine zipper specificity with interfacial hydrophobic residues

Publisher: Springer Science and Business Media LLC

Date: 09-1999

DOI: 10.1007/BF02443435

pH-dependent changes in the in vitro ligand-binding properties and structure of human clusterin.

Publisher: American Chemical Society (ACS)

Date: 21-01-2000

DOI: 10.1021/BI991581B

Abstract: Clusterin is a glycoprotein which is locally overexpressed at sites of tissue damage or stress, leading to the proposal that it may be a cytoprotective protein. It has been shown that clusterin has chaperone-like activity, being able to protect proteins against precipitation under stress conditions. It has also been shown that local acidosis is common at sites of tissue damage or stress. We asked whether acidic pH induces structural changes in clusterin and enhances its ability to bind to other proteins. We found by affinity chromatography and ELISA that the binding of clusterin to glutathione-S-transferase, IgG, apolipoprotein A-I, and complement protein C9 was enhanced at mildly acidic compared to physiological pH. Analytical ultracentrifugation and gel filtration studies revealed that clusterin exists in different polymerization states with monomer occurring preferentially at pH 5.5 and multimeric species at pH 7.5. Although circular dichroism showed little difference in the alpha-helical and beta-sheet contents of clusterin at pH 5 compared to pH 7.5, evidence for pH-dependent structural changes in clusterin was obtained from fluorescence experiments. pH titrations showed reversible changes in the fluorescence of tryptophan residues in clusterin. There was a reversible 2-fold increase in the fluorescence of the extrinsic probe 4, 4'-bis(1-anilinonaphthalene-8-sulfonate) bound to clusterin at pH 5. 5 compared to pH 7.5. There was also a 3.5-fold increase in fluorescence resonance energy transfer from tryptophan residues in clusterin to 4,4'-bis(1-anilinonaphthalene-8-sulfonate) at pH 5.5 compared to pH 7.5. These data suggest that pH-induced changes in the structure of clusterin are responsible for its enhanced ability to bind protein ligands at mildly acidic pH.

Context-dependent role of Grb7 in HER2+ve and triple-negative breast cancer cell lines

Publisher: Springer Science and Business Media LLC

Date: 25-01-2014

DOI: 10.1007/S10549-014-2838-5

Abstract: Grb7 is an adapter protein, aberrantly co-overexpressed with HER2 and identified as an independent prognostic marker in breast cancer. It has been established that Grb7 exacerbates the cellular growth and migratory behaviour of HER2+ve breast cancer cells. Less is known about Grb7's role in the context of HER2-ve cells. Here we directly compare the effect of stable Grb7 knockdown in oestrogen sensitive (T47D), HER2+ve (SKBR3) and triple-negative (MDA-MB-468 and MDA-MB-231) breast cancer cell lines on anchorage dependent and independent cell growth, wound healing and chemotaxis. All cell lines showed reduced ability to migrate upon Grb7 knockdown, despite their greatly varied endogenous levels of Grb7. Decreased cell proliferation was not observed in any of the cell lines upon Grb7 knockdown however, decreased ability to form colonies was observed for all but the oestrogen sensitive cell line, depending upon the stringency of the growth conditions. The data reveal that Grb7 plays an important role in breast cancer progression, beyond the context of HER2+ve cell types.

Enhancing the Bioactivity of Bicyclic Peptides Targeted to Grb7-SH2 by Restoring Cell Permeability

Publisher: MDPI AG

Date: 16-05-2022

DOI: 10.3390/BIOMEDICINES10051145

Abstract: The development of peptide inhibitors against intracellular targets depends upon the dual challenge of achieving a high affinity and specificity for the target and maintaining cellular permeability for biological activity. Previous efforts to develop bicyclic peptides targeted to the Grb7 signalling protein implicated in HER2+ve cancer progression have resulted in improved affinity. However, these same peptides demonstrated a lowered activity due to their decreased ability to penetrate cell membranes. Here, we report the testing of a new series of bicyclic G7 peptides designed to possess improved bioactivity. We discovered that the incorporation of two amino acids (Phe-Pro, Phe-Trp or Phe-Arg) within the bicyclic peptide framework maintains an enhanced binding affinity for the Grb7-SH2 domain compared to that of the first-generation monocyclic peptide G7-18NATE. Structure determination using X-ray crystallography revealed that the mode of binding by the expanded bicyclic G7 peptide is analogous to that of G7-18NATE. Interestingly, while the bicyclic peptide containing Phe-Trp did not display the highest affinity for Grb7-SH2 in the series, it was the most potent inhibitor of HER2+ve SKBR3 breast cancer cell migration when coupled to Penetratin. Together, this demonstrates that peptide flexibility as well as the amino acid tryptophan can play important roles in the uptake of peptides into the cell.

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.CELLSIG.2013.08.003

Abstract: The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target.

Publisher: Frontiers Media SA

Date: 26-09-2017

DOI: 10.3389/FMOLB.2017.00064

A functional comparison of the venom of three Australian jellyfish - Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana - on cytosolic Ca2+, haemolysis and Artemia sp. lethality

Publisher: Elsevier BV

Date: 02-2005

DOI: 10.1016/J.TOXICON.2004.10.013

Abstract: Cnidarian venoms produce a wide spectrum of envenoming syndromes in humans ranging from minor local irritation to death. Here, the effects of Chironex fleckeri, Chiropsalmus sp., and Carybdea xaymacana venoms on ventricular myocyte cytosolic Ca2+, haemolysis and Artemia sp. lethality are compared for the first time. All three venoms caused a large, irreversible elevation of cytosolic Ca2+ in myocytes as measured using the Ca2+ sensitive fluorescent probe Indo-1. The L-type Ca2+ channel antagonist verapamil had no effect on Ca2+ influx whilst La3+, a non-specific channel and pore blocker, inhibited the effect. Haemolytic activity was observed for all venoms, with C. xaymacana venom displaying the greatest activity. These activities are consistent with the presence of a pore-forming toxin existing in the venoms which has been demonstrated by transmission electron microscopy in the case of C. fleckeri. The venom of C. fleckeri was found to be more lethal against Artemia sp. than the venom of the other species, consistent with the order of known human toxicities. This suggests that the observed lytic effects may not underlie the lethal effects of the venom, and raises the question of how such potent activities are dealt with by envenomed humans.

Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

Publisher: International Union of Crystallography (IUCr)

Date: 30-09-2011

DOI: 10.1107/S1744309111028004

Supramolecular self-assembly of N-acetyl-capped β-peptides leads to nano- to macroscale fiber formation

Publisher: Wiley

Date: 19-06-2013

DOI: 10.1002/ANIE.201303175

The impact of single cysteine residue mutations on the replication terminator protein

Publisher: Elsevier BV

Date: 10-2003

DOI: 10.1016/J.BBRC.2003.09.126

Abstract: We report the structural and biophysical consequences of cysteine substitutions in the DNA-binding replication terminator protein (RTP) of Bacillus subtilis, that resulted in an optimised RTP mutant suitable for structural studies. The cysteine residue 110 was replaced with alanine, valine or serine. Protein secondary structure and stability (using circular dichroism spectropolarimetry), self-association (using analytical ultracentrifugation), and DNA-binding measurements revealed RTP.C110S to be the most similar mutant to wild-type RTP. The C110A and C110V.RTP mutants were less soluble, less stable and showed lower DNA-binding affinity. The structure of RTP.C110S, solved to 2.5A resolution using crystallographic methods, showed no major structural perturbation due to the mutation. Heteronuclear NMR spectroscopic studies revealed subtle differences in the electronic environment about the site of mutation. The study demonstrates the suitability of serine as a substitute for cysteine in RTP and the high sensitivity of protein behaviour to single amino acid substitutions.

Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist

Publisher: International Union of Crystallography (IUCr)

Date: 21-01-2014

DOI: 10.1107/S2053230X13033414

Abstract: Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1–Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P 6 3 , whereas the G7-B1–Grb7 SH2 domain complex crystallized in the monoclinic space group P 2 1 . The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

Different modes of interaction by TIAR and HuR with target RNA and DNA

Publisher: Oxford University Press (OUP)

Date: 13-01-2011

DOI: 10.1093/NAR/GKQ837

Conformational stability studies of a stapled hexa-β ³- peptide library

Publisher: Royal Society of Chemistry (RSC)

Date: 2012

DOI: 10.1039/C2OB06617C

Abstract: A library of 14-helical hexa β(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-β(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.

Formation of an αcP1-KH3 complex with UC-rich RNA

Publisher: Springer Science and Business Media LLC

Date: 09-03-2005

DOI: 10.1007/S00249-005-0467-Y

Abstract: The alphaCP family of proteins [also known as poly(C)-binding or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alphaCP1 and alphaCP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.

Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Publisher: Springer Science and Business Media LLC

Date: 28-07-2010

DOI: 10.1007/S10989-010-9222-Z

A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

Publisher: Elsevier BV

Date: 05-2013

DOI: 10.1074/JBC.M113.464545

Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26

Publisher: Informa UK Limited

Date: 2020

DOI: 10.1080/22221751.2020.1739565

Toxins not neutralized by brown snake antivenom

Publisher: Elsevier BV

Date: 06-2006

DOI: 10.1016/J.TAAP.2005.09.010

Abstract: The Australian snakes of the genus Pseudonaja (dugite, gwardar and common brown) account for the majority of snake bite related deaths in Australia. Without antivenom treatment, the risk of mortality is significant. There is an accumulating body of evidence to suggest that the efficacy of the antivenom is limited. The current study investigates the protein constituents recognized by the antivenom using 2-DE, immuno-blot techniques and rat tracheal organ bath assays. The 2-DE profiles for all three snake venoms were similar, with major species visualized at 78-132 kDa, 32-45 kDa and 6-15 kDa. Proteins characterized by LC-MS/MS revealed a coagulant toxin ( approximately 42 kDa) and coagulant peptide ( approximately 6 kDa), as well as two PLA(2) ( approximately 14 kDa). Peptides isolated from approximately 78 kDa and 15-32 kDa protein components showed no similarity to known protein sequences. Protein recognition by the antivenom occurred predominantly for the higher molecular weight components with little recognition of 6-32 kDa MW species. The ability of antivenom to neutralize venom activity was also investigated using rat tracheal organ bath assays. The venoms of Pseudonaja affinis affinis and Pseudonaja nuchalis incited a sustained, significant contraction of the trachea. These contractions were attributed to PLA(2) enzymatic activity as pre-treatment with the PLA(2) inhibitor 4-BPB attenuated the venom-induced contractions. The venom of Pseudonaja textilis incited tracheal contractility through a non-PLA(2) enzymatic activity. Neither activity was attenuated by the antivenom treatment. These results represent the first proteomic investigation of the venoms from the snakes of the genus Pseudonaja, revealing a possible limitation of the brown snake antivenom in binding to the low MW protein components.

Structure and mechanism of a proline-specific aminopeptidase from Escherichia coli

Publisher: Proceedings of the National Academy of Sciences

Date: 31-03-1998

DOI: 10.1073/PNAS.95.7.3472

Abstract: The structure of the proline-specific aminopeptidase (EC 3.4.11.9 ) from Escherichia coli has been solved and refined for crystals of the native enzyme at a 2.0-Å resolution, for a dipeptide-inhibited complex at 2.3-Å resolution, and for a low-pH inactive form at 2.7-Å resolution. The protein crystallizes as a tetramer, more correctly a dimer of dimers, at both high and low pH, consistent with observations from analytical ultracentrifuge studies that show that the protein is a tetramer under physiological conditions. The monomer folds into two domains. The active site, in the larger C-terminal domain, contains a dinuclear manganese center in which a bridging water molecule or hydroxide ion appears poised to act as the nucleophile in the attack on the scissile peptide bond of Xaa-Pro. The metal-binding residues are located in a single subunit, but the residues surrounding the active site are contributed by three subunits. The fold of the protein resembles that of creatine amidinohydrolase (creatinase, not a metalloenzyme). The C-terminal catalytic domain is also similar to the single-domain enzyme methionine aminopeptidase that has a dinuclear cobalt center.

Assessment of the robustness of a serendipitous zinc binding fold: Mutagenesis and protein grafting

Publisher: Elsevier BV

Date: 02-2005

DOI: 10.1016/J.STR.2004.12.007

Abstract: Zinc binding motifs have received much attention in the area of protein design. Here, we have tested the suitability of a recently discovered nonnative zinc binding structure as a protein design scaffold. A series of multiple alanine mutants was created to investigate the minimal requirements for folding, and solution structures of these mutants showed that the original fold was maintained, despite changes in approximately 50% of the sequence. We next attempted to transplant binding faces from chosen bimolecular interactions onto one of these mutants, and many of the resulting "chimeras" were shown to adopt a native-like fold. These results both highlight the robust nature of small zinc binding domains and underscore the complexity of designing functional proteins, even using such small, highly ordered scaffolds as templates.

Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type i IRES

Publisher: Oxford University Press (OUP)

Date: 18-06-2020

DOI: 10.1093/NAR/GKAA519

Abstract: The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem–loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem–loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.

Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

Publisher: Oxford University Press (OUP)

Date: 16-02-2012

DOI: 10.1093/NAR/GKS058

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.

An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA

Publisher: Elsevier BV

Date: 07-2007

DOI: 10.1016/J.JMB.2007.02.067

Abstract: In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists

Publisher: Elsevier BV

Date: 2011

DOI: 10.1016/J.BMC.2010.10.030

Abstract: Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC(50) of 86μM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1

Publisher: Oxford University Press (OUP)

Date: 23-02-2005

DOI: 10.1093/NAR/GKI265

Crystallization and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site

Publisher: International Union of Crystallography (IUCr)

Date: 20-10-2006

DOI: 10.1107/S1744309106039108

Detailed protocol for optimised expression and purification of functional monomeric human Heat Shock Factor 1

Publisher: Elsevier BV

Date: 12-2020

DOI: 10.1016/J.PEP.2020.105722

Crystal Structures of the Substrate Free-enzyme, and Reaction Intermediate of the HAD Superfamily Member, Haloacid Dehalogenase DehIVa from Burkholderia cepacia MBA4

Publisher: Elsevier BV

Date: 05-2007

DOI: 10.1016/J.JMB.2007.02.015

Abstract: DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.

¹H‐NMR structural studies of a cystine‐linked peptide containing residues 71–93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

Publisher: Wiley

Date: 06-1999

DOI: 10.1046/J.1432-1327.1999.00423.X

Abstract: The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using 1H-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

Uptake of a cell permeable G7‐18NATE construct into cells and binding with the Grb7‐SH2 domain

Publisher: Wiley

Date: 2011

DOI: 10.1002/BIP.21403

Abstract: Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

Publisher: Wiley

Date: 25-05-2013

DOI: 10.1002/PRO.2262

Conformational rearrangements of RIG-I receptor on formation of a multiprotein:dsRNA assembly.

Publisher: Oxford University Press (OUP)

Date: 15-01-2013

DOI: 10.1093/NAR/GKS1477

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation

Publisher: Springer Science and Business Media LLC

Date: 25-09-2007

DOI: 10.1186/1472-6807-7-58

Abstract: Human g rowth factor r eceptor b ound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 Å resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the μM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of K d = ~35.7 μM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.

Cyclic Peptides Incorporating Phosphotyrosine Mimetics as Potent and Specific Inhibitors of the Grb7 Breast Cancer Target

Publisher: American Chemical Society (ACS)

Date: 23-09-2015

DOI: 10.1021/ACS.JMEDCHEM.5B00609

Abstract: The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 μM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target

Publisher: American Chemical Society (ACS)

Date: 10-11-2017

DOI: 10.1021/ACS.JMEDCHEM.7B01320

Abstract: Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (K

Crystallization of theBacillus subtilisRTP–DNA complex prepared using NMR spectroscopy

Publisher: International Union of Crystallography (IUCr)

Date: 03-2001

DOI: 10.1107/S0907444900019508

Abstract: The replication terminator protein (RTP)-DNA complex of Bacillus subtilis is responsible for the arrest of DNA replication at terminator sites in the B. subtilis chromosome. The crystallization and preliminary diffraction data analysis for the complex of an (15)N-labelled mutant form of RTP and a symmetrical form of its DNA-binding site is reported. NMR spectroscopy was used to assess the stoichiometry of complex formation, with the s le containing the most homogeneous solution of complex giving rise to diffracting crystals. Synchrotron-radiation data to 2.5 A were collected from a crystal of space group P3(2)21, unit-cell parameters a = b = 44.780, c = 395.582 A, containing an RTP dimer within the asymmetric unit.

Identification of PLA2 and α-Neurotoxin Proteins in the Venom of Pseudonaja affinis (Dugite)

Publisher: Elsevier BV

Date: 06-2002

DOI: 10.1006/TAAP.2002.9416

Abstract: The Western brown snake Pseudonaja affinis (dugite), common to the Perth area of Western Australia, possesses one of the most lethal venoms in the world. Little is known, however, about the toxic protein constituents of the venom, other than those causing coagulopathic and procoagulant effects. The current study was therefore undertaken in order to identify other protein constituents and activities present. Crude venom induced a contraction in rat tracheal preparations through phospholipase A(2) (PLA(2)) activity, as shown by the complete and partial inhibition of contraction by PLA(2) inhibitors 4-bromophenacyl bromide and quinacrine. Further, a reduced degree of smooth muscle contraction in the presence of the leukotriene receptor antagonist SKF104353 suggested that this effect was mediated by leukotriene metabolites. The venom-induced contraction did not reoccur upon a second administration of the venom, despite the muscle retaining its contractile function and appearing histologically undamaged. Chromatographic separation of the protein constituents of the venom showed that PLA(2) activity was associated with all protein fractions. A low-molecular-weight component of the venom was further investigated through N-terminal sequencing and found to possess high identity to the short-chain alpha-neurotoxin family of toxins. Venom activity on cultured rat cardiac myocytes and cultured cortical neurons was also examined. The crude venom was found to temporarily inhibit the beating of the cardiac myocytes, after which the beating resumed erratically. Cortical neurons, however, were irreversibly affected, showing concentration-dependent cell death.

The Crystal Structure of DehI Reveals a New α-Haloacid Dehalogenase Fold and Active-Site Mechanism

Publisher: Elsevier BV

Date: 04-2008

DOI: 10.1016/J.JMB.2008.02.035

Abstract: Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I alpha-haloacid dehalogenase that can process both L- and D-substrates. The structure shows that the DehI monomer consists of two domains of approximately 130 amino acids that have approximately 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both L- and D-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to D-only enzymes. These details will assist with future bioengineering of dehalogenases.

The impact of cell-penetrating peptides on membrane bilayer structure during binding and insertion

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.BBAMEM.2016.05.004

Abstract: We have studied the effect of penetratin and a truncated analogue on the bilayer structure using dual polarisation interferometry, to simultaneously measure changes in mass per unit area and birefringence (an optical parameter representing bilayer order) with high sensitivity during the binding and dissociation from the membrane. Specifically, we studied penetratin (RQIKIWFQNRRMKWKK), along with a shortened and biotinylated version known as R8K-biotin (RRMKWKKK(Biotin)-NH2). Overall both peptides bound only weakly to the neutral DMPC and POPC bilayers, while much higher binding was observed for the anionic DMPC/DMPG and POPC/POPG. The binding of penetratin to gel-phase DMPC/DMPG was adequately represented by a two-state model, whereas on the fluid-phase POPC/POPG it exhibited a distinctly different binding pattern, best represented by a three-state kinetic model. However, R8K-biotin did not bind well to DMPC/DMPG and showed a more transitory and superficial binding to POPC/POPG. Comparing the modelling results for both peptides binding to POPC/POPG suggests an important role for a securely bound intermediate prior to penetratin insertion and translocation. Overall these results further elucidate the mechanism of penetratin, and provide another ex le of the significance of the ability of DPI to measure structural changes and the use of kinetic analysis to investigate the stages of peptide-membrane interactions.

RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Publisher: Public Library of Science (PLoS)

Date: 25-07-2013

DOI: 10.1371/JOURNAL.PGEN.1003628

Cooperative interplay of let-7 mimic and HuR with MYC RNA

Publisher: Informa UK Limited

Date: 30-07-2015

DOI: 10.1080/15384101.2015.1069930

Comparison between clickable cyclic TAT and penetratin for delivery of cyclic and bicyclic-peptide cargos

Publisher: Wiley

Date: 31-03-2020

DOI: 10.1002/PEP2.24163

NMR analysis of G7‐18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein

Publisher: Wiley

Date: 2007

DOI: 10.1002/BIP.20667

Abstract: G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.

The intracellular and nuclear‐targeted delivery of an antiandrogen drug by carrier peptides

Publisher: Wiley

Date: 2008

DOI: 10.1002/BIP.20986

Abstract: Cell permeable carrier peptides are currently of interest for their potential to improve the delivery of bioactive molecules into cells and to specific cellular compartments. We have investigated the activity of a derivative of the antiandrogen drug, bicalutamide, attached to the cell-permeable carrier peptide penetratin(R). We have used both disulfide (labile) and thioether (nonlabile) linkages to attach the bicalutamide derivative to the peptide in order to assess whether one type of chemistry has advantages over the other. In addition we have added a nuclear localization sequence (NLS) to the carrier peptide to investigate whether localization of the drug to the nucleus of the cell affects the activity of the drug. Biotin-labeled peptides were used to demonstrate that the carrier peptide is rapidly accumulated inside cultured cells, and that the incorporation of an NLS in the sequence results in its nuclear targeting. The bicalutamide derivative linked to carrier peptides via a disulfide-linkage exerted no greater antiproliferative effect in LNCaP cells, than the bicalutamide derivative alone. The bicalutamide derivative linked to the carrier peptide by a non-labile thioether linkage showed a similar activity profile. When the construct includes a nuclear targeting sequence, however, a markedly increased antiproliferative effect was observed. This study has thus shown that the activity of bicalutamide may be enhanced by the nonlabile attachment of a cell-permeable and nuclear-targeted peptide, which has implications for the development of novel antiandrogens for the treatment of prostate cancer.

The Discovery of Phenylbenzamide Derivatives as Grb7‐Based Antitumor Agents

Publisher: Wiley

Date: 17-12-2012

DOI: 10.1002/CMDC.201200400

Abstract: Grb7 is a non-catalytic protein, the overexpression of which has been associated with the proliferative and migratory potentials of cancer cells. Virtual screening strategies involving a shape-based similarity search, molecular docking, and 2D-similarity searches complemented by experimental binding studies (Thermofluor and isothermal titration calorimetry) resulted in the identification of nine novel phenylbenzamide-based antagonists of the Grb7 SH2 domain. Moderate binding affinities were observed, ranging from K(d)=32.3 μM for lead phenylbenzamide NSC 104999 (1) to K(d)=1.1 μM for a structurally related compound, NSC 57148 (2). Deconvolution of the affinity data into its components revealed differences in lead binding, from being entropy based (lead 1) to enthalpically driven (NSC 100874 (3), NSC 55158 (4), and compound 2). Finally, the lead compound 1 was found to decrease the growth of MDA-MB-468 breast cancer cells, with an IC(50) value of 39.9 μM. It is expected that these structures will serve as novel leads in the development of Grb7-based anticancer therapeutics.

Synthesis of stapled beta3-peptides through ring-closing metathesis.

Publisher: American Chemical Society (ACS)

Date: 31-08-2009

DOI: 10.1021/OL901803D

Abstract: The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease

Publisher: Oxford University Press (OUP)

Date: 05-2001

DOI: 10.1093/NAR/29.9.1852

Abstract: The selective manipulation of mitochondrial DNA (mtDNA) replication and expression within mammalian cells has proven difficult. One promising approach is to use peptide nucleic acid (PNA) oligomers, nucleic acid analogues that bind selectively to complementary DNA or RNA sequences inhibiting replication and translation. However, the potential of PNAs is restricted by the difficulties of delivering them to mitochondria within cells. To overcome this problem we conjugated a PNA 11mer to a lipophilic phosphonium cation. Such cations are taken up by mitochondria through the lipid bilayer driven by the membrane potential across the inner membrane. As anticipated, phosphonium-PNA (ph-PNA) conjugates of 3.4-4 kDa were imported into both isolated mitochondria and mitochondria within human cells in culture. This was confirmed by using an ion-selective electrode to measure uptake of the ph-PNA conjugates by cell fractionation in conjunction with immunoblotting by confocal microscopy by immunogold-electron microscopy and by crosslinking ph-PNA conjugates to mitochondrial matrix proteins. In all cases dissipating the mitochondrial membrane potential with an uncoupler prevented ph-PNA uptake. The ph-PNA conjugate selectively inhibited the in vitro replication of DNA containing the A8344G point mutation that causes the human mtDNA disease 'myoclonic epilepsy and ragged red fibres' (MERRF) but not the wild-type sequence that differs at a single nucleotide position. Therefore these modified PNA oligomers retain their selective binding to DNA and the lipophilic cation delivers them to mitochondria within cells. When MERRF cells were incubated with the ph-PNA conjugate the ratio of MERRF to wild-type mtDNA was unaffected, even though the ph-PNA content of the mitochondria was sufficient to inhibit MERRF mtDNA replication in a cell-free system. This unexpected finding suggests that nucleic acid derivatives cannot bind their complementary sequences during mtDNA replication. In summary, we have developed a new strategy for targeting PNA oligomers to mitochondria and used it to determine the effects of PNA on mutated mtDNA replication in cells. This work presents new approaches for the manipulation of mtDNA replication and expression, and will assist in the development of therapies for mtDNA diseases.

Novel Binding of HuR and Poly(C)-binding Protein to a Conserved UC-rich Motif within the 3′-Untranslated Region of the Androgen Receptor Messenger RNA

Publisher: Elsevier BV

Date: 07-2002

DOI: 10.1074/JBC.M202883200

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

Sequence requirements for RNA binding by HuR and AUF1

Publisher: Oxford University Press (OUP)

Date: 25-02-2012

DOI: 10.1093/JB/MVS010

Abstract: The stability of RNAs bearing AU-rich elements in their 3'-UTRs, and thus the level of expression of their protein products, is regulated by interactions with cytoplasmic RNA-binding proteins. Binding by HuR generally leads to mRNA stabilization and increased protein production, whereas binding by AUF1 isoforms generally lead to rapid degradation of the mRNA and reduced protein production. The exact nature of the interplay between these and other RNA-binding proteins remains unclear, although recent studies have shown close interactions between them and even suggested competition between the two for binding to their cognate recognition sequences. Other recent reports have suggested that the sequences recognized by the two proteins are different. We therefore performed a detailed in vitro analysis of the binding site(s) for HuR and AUF1 present in androgen receptor mRNA to define their exact target sequences, and show that the same sequence is contacted by both proteins. Furthermore, we analysed a proposed HuR target within the 3'-UTR of MTA1 mRNA, and show that the contacted bases lie outside of the postulated motif and are a better match to a classical ARE than the postulated motif. The defining features of these HuR binding sites are their U-richness and single strandedness.

Synthesis and Structural Analysis of the N-Terminal Domain of the Thyroid Hormone-Binding Protein Transthyretin

Publisher: Walter de Gruyter GmbH

Date: 10-01-2002

DOI: 10.1515/CCLM.2002.212

Grb7-SH2 domain dimerisation is affected by a single point mutation

Publisher: Springer Science and Business Media LLC

Date: 20-04-2005

DOI: 10.1007/S00249-005-0480-1

Abstract: Growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is co-overexpressed and forms a tight complex with the ErbB2 receptor in a number of breast tumours and breast cancer cell lines. The interaction of Grb7 with the ErbB2 receptor is mediated via its Src homology 2 (SH2) domain. Whilst most SH2 domains exist as monomers, recently reported studies have suggested that the Grb7-SH2 domain exists as a homodimer. The self-association properties of the Grb7-SH2 domain were therefore studied using sedimentation equilibrium ultracentrifugation. Analysis of the data demonstrated that the Grb7-SH2 domain is dimeric with a dissociation constant of approximately 11 muM. We also demonstrate, using size-exclusion chromatography, that mutation of phenylalanine 511 to an arginine produces a monomeric form of the Grb7-SH2 domain. This mutation represents the first step in the engineering of a Grb7-SH2 domain with good solution properties for further biophysical and structural investigation.

Constraints within major histocompatibility complex class I restricted peptides: presentation and consequences for T-cell recognition

Publisher: Proceedings of the National Academy of Sciences

Date: 08-03-2010

DOI: 10.1073/PNAS.1000032107

Abstract: Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of “independent pegs” that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of αβ-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many self- and viral peptides show extensive and direct interresidue interactions, an unexpected finding that has led us to the idea of “constrained” peptides. Mutational analysis of two constrained peptides (the HLA B44 restricted self-peptide (B44DPα–EEFGRAFSF) and an H2-D b restricted influenza peptide (D b PA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLEN F RAYV → SSLEN A RAYV) within the constrained PA peptide resulted in a diminished cytotoxic T lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between in idual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, whereas the perturbation of such constraints can lead to a previously unappreciated mechanism of viral escape.

Elucidation of a C-Rich Signature Motif in Target mRNAs of RNA-Binding Protein TIAR

Publisher: Informa UK Limited

Date: 10-2007

DOI: 10.1128/MCB.01036-07

Erratum: Structural characterisation of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target (Protein Science (2013) 22 (762-773))

Publisher: Wiley

Date: 19-12-2014

DOI: 10.1002/PRO.2399

Direct Interaction between Calmodulin and the Grb7 RA-PH Domain

Publisher: MDPI AG

Date: 17-02-2020

DOI: 10.3390/IJMS21041336

Abstract: Grb7 is a signalling adapter protein that engages activated receptor tyrosine kinases at cellular membranes to effect downstream pathways of cell migration, proliferation and survival. Grb7’s cellular location was shown to be regulated by the small calcium binding protein calmodulin (CaM). While evidence for a Grb7/CaM interaction is compelling, a direct interaction between CaM and purified Grb7 has not been demonstrated and quantitated. In this study we sought to determine this, and prepared pure full-length Grb7, as well as its RA-PH and SH2 subdomains, and tested for CaM binding using surface plasmon resonance. We report a direct interaction between full-length Grb7 and CaM that occurs in a calcium dependent manner. While no binding was observed to the SH2 domain alone, we observed a high micromolar affinity interaction between the Grb7 RA-PH domain and CaM, suggesting that the Grb7/CaM interaction is mediated through this region of Grb7. Together, our data support the model of a CaM interaction with Grb7 via its RA-PH domain.

Engineering the ovarian hormones inhibin A and inhibin B to enhance synthesis and activity

Publisher: The Endocrine Society

Date: 22-06-2020

DOI: 10.1210/ENDOCR/BQAA099

Abstract: Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing β-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of β-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin β A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this β A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin β B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.

Distinct binding properties of TIAR RRMs and linker region

Publisher: Informa UK Limited

Date: 04-2013

DOI: 10.4161/RNA.24341

Combined roles of ATP and small hairpin RNA in the activation of RIG-I revealed by solution-based analysis

Publisher: Oxford University Press (OUP)

Date: 13-01-2018

DOI: 10.1093/NAR/GKX1307

Targeting large molecules to mitochondria

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0169-409X(01)00134-X

Abstract: Mitochondrial function is central to a range of cell processes and mitochondrial dysfunction contributes to a number of human diseases. Consequently there is growing interest in delivering large molecules such as nucleic acids, proteins, enzyme mimetics, drugs and probes to mitochondria within cells. The reasons for doing this are to understand how mitochondria function in the cell and to develop therapies for diseases involving mitochondrial damage. Here we review the methods that have been used to target large molecules to mitochondria and discuss some approaches under development.

Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase fromPseudomonas putidastrain PP3

Publisher: International Union of Crystallography (IUCr)

Date: 07-06-2008

DOI: 10.1107/S1744309108008865

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.CELREP.2017.04.029

Abstract: Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7

Publisher: Springer Science and Business Media LLC

Date: 03-06-2016

DOI: 10.1038/SREP27060

Abstract: The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain ( K D = 0.83 μM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains ( K D 100 μM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical ex le of structure aided inhibitor development.

Venom as a source of useful biologically active molecules

Publisher: Wiley

Date: 03-2001

DOI: 10.1046/J.1442-2026.2001.00174.X

Abstract: In the specialty area of venomology, emergency physicians traditionally have been most interested in the description of a variety of envenomation syndromes and, subsequent to this, the most appropriate investigative and therapeutic strategies to employ when envenomation is present. Taking an alternative viewpoint, in this paper we have reviewed a selection of interesting areas of biomedical research in which venom components are being investigated for their potential as novel therapeutic agents, pesticides and ion-channel probes. In addition, we describe the molecular imaging tools of X-ray crystallography and nuclear magnetic resonance spectroscopy, key techniques in the development of rationally designed therapeutic agents.

Preparation and crystallization of the Grb7 SH2 domain in complex with the G7-18NATE nonphosphorylated cyclic inhibitor peptide

Publisher: International Union of Crystallography (IUCr)

Date: 26-11-2010

DOI: 10.1107/S1744309110041850

RNA-binding proteins that target the androgen receptor mRNA

Publisher: Wiley

Date: 12-2002

DOI: 10.1080/15216540216033

Abstract: Recent efforts to elucidate mechanisms of posttranscriptional gene regulation have resulted in the description of both cis- and trans-acting factors which affect the stability and translational efficiency of specific mRNAs. Here we summarise some of the concepts in this field, with particular attention to recently discovered protein/RNA interactions involving the human androgen receptor mRNA. Such systems are of interest for their potential as targets for the modulation of protein production.

Preparation and cellular uptake of bicyclic-peptide cargo clicked to cell penetrating peptides

Publisher: Wiley

Date: 09-03-2018

DOI: 10.1002/PEP2.24037

Synthesis of an Analog of the Thyroid Hormone-binding Protein Transthyretin via Regioselective Chemical Ligation

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1074/JBC.M101228200

Design and testing of bicyclic inhibitors of Grb7—are two cycles better than one?

Publisher: Wiley

Date: 09-2013

DOI: 10.1002/BIP.22237

Abstract: Grb7 is an adapter protein involved in the propagation of signals in cancer cell migration and proliferation, and is thus a target for the development of novel anti‐cancer agents. An 11‐residue thioether‐cyclized peptide known as G7‐18NATE has previously been developed, that inhibits Grb7 via specific interactions with its SH2 domain with micromolar affinity. Here we explore whether the peptide binding is enhanced by the addition of a second linkage designed to restrain the peptide in its bound conformation and thus reduce the entropic loss upon binding. The use of an O‐ally ser covalent linkage between residue positions 1 and 8 successfully enhanced the affinity, and ITC showed that the entropic loss was reduced. A peptide with thioether‐cyclization exchanged for an amide linkage showed reduce affinity, though the formation of a disulfide bond between positions 1 and 8 in this peptide enhanced its binding. This study paves the way for improving the G7‐18NATE scaffold for second generation inhibitors of Grb7. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 543–549, 2013.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

Publisher: Springer Science and Business Media LLC

Date: 03-2001

DOI: 10.1038/84934

Interaction of the non‐phosphorylated peptide G7‐18NATE with Grb7‐SH2 domain requires phosphate for enhanced affinity and specificity

Publisher: Wiley

Date: 29-12-2011

DOI: 10.1002/JMR.2148

Abstract: Src-homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7-18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7-18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7-18NATE is specific for the Grb7-SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7-18NATE binds with micromolar binding affinity to Grb7-SH2 domain (K(D) = 4-6 μm) compared with 50-200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2-(N-Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7-18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7-18NATE binding to the Grb7-SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition.

Structural Basis of Binding by Cyclic Nonphosphorylated Peptide Antagonists of Grb7 Implicated in Breast Cancer Progression

Publisher: Elsevier BV

Date: 09-2011

DOI: 10.1016/J.JMB.2011.07.030

Abstract: Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Monash University

Location: Australia

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Monash Institute Of Pharmaceutical Sciences

Location: Australia

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Structural Basis For Ribosomal Docking On A Picornavirus Type I IRES

Start Date: 2019

End Date: 2022

Funder: National Health and Medical Research Council

Discovery Projects - Grant ID: DP110102056

Start Date: 2011

End Date: 12-2013

Amount: $350,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0987761

Start Date: 01-2009

End Date: 12-2011

Amount: $420,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200102737

Start Date: 2020

End Date: 11-2023

Amount: $420,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989504

Start Date: 07-2009

End Date: 07-2010

Amount: $1,400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0560712

Start Date: 04-2005

End Date: 06-2005

Amount: $630,837.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0879279

Start Date: 01-2008

End Date: 12-2010

Amount: $719,500.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0882977

Start Date: 09-2008

End Date: 12-2009

Amount: $600,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0346892

Start Date: 03-2003

End Date: 06-2005

Amount: $689,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0345376

Start Date: 03-2003

End Date: 12-2006

Amount: $340,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE130100024

Start Date: 2013

End Date: 12-2013

Amount: $520,000.00

Funder: Australian Research Council