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Structural and dynamic studies of macromolecular interactionsDr Jackie WilceSenior Lectureremail: Jackie.Wilce@med.monash.edu.au Research interestsMy research involves the study of protein/protein and protein/oligonucleotide (RNA and DNA) interactions that occur in human cells in pathways that control gene expression, cell growth and cell proliferation. We seek to better understand the molecular recognition that takes place by determining precise three-dimensional structures of the macromolecular complexes using x-ray crystallography and NMR spectroscopy as well as using other biophysical techniques. Structure and RNA binding of poly(C)-binding protein aCP1 is a member of the poly(C)-binding protein family of proteins which include aCP1, aCP2, aCP3,aCP4 (also known as hnRNP E or PCBP) and the earliest member to be characterized, the heterologous ribonucleoprotein K, hnRNP K. These proteins contain a triplet K homology (KH) RNA-binding motif, as first identified in hnRNP K, which confer specificity for single-stranded poly(C) tracts of both RNA and DNA. Upon binding to RNA, they are involved in a diverse range of functions affecting post-transcriptional regulation of specific genes. These include the shuttling of mRNA between the nucleus and the cytoplasm, the stabilization of specific mRNAs, translational silencing and translational enhancement. Our interest follows on from the finding that androgen receptor mRNA is bound by aCP1 in a specific region of its 3’UTR which affects translation.
We have shown that the third KH domain of aCP1 binds pyrimidine-rich RNA using surface plasmon resonance (using BIACORE) and RNA electrophoretic mobility-shift assays (REMSA). In addition we have solved its structure to 2.1 Å resolution using x-ray crystallographic techniques. This has allowed us to model the interaction between this domain and poly(C)-RNA using NAMD molecular dynamics simulation software (www.ks.uiuc.edu/Research/namd/). Future work will involve the pursuit of the structure of the protein:RNA complex and other KH domains using NMR spectroscopy and x-ray crystallography. We also will measure how tightly the complexes form with RNA in the presence of other proteins which also bind mRNA. The structural and affinity measurements will help us understand the role aCP1 plays in affecting the translation of mRNA. Structure and peptide binding of the Grb7-SH2 Domain Growth factor receptor bound protein-7 (Grb7) is a member of a family of SH2 domain containing adaptor proteins. SH2 domains are present in a diverse group of proteins which are implicated in tyrosine kinase signalling. The SH2 domain of Grb7 binds to phosphorylated tyrosine residues located in the cytoplasmic domain of several growth factor receptors including the epidermal growth factor receptor-2, erbB2. Grb7 is over-expressed with erbB2 in a subset of human breast cancer cell lines and breast tumours, suggesting erbB2 signaling via Grb7 may be increased in these cancers. There is also a strong correlation between erbB-2 and Grb7 over-expression in oesophageal and gastric carcinoma. As it has been shown that breast tumours that over-express the erbB2 receptor are generally estrogen-receptor negative and have a poor prognosis, the Grb7-erbB2 complex provides an attractive target for the development of novel therapeutics in the treatment of breast cancer.
We are investigating the structure of the Grb7-SH2 domain. This will provide insight into the specificity of the Grb7-SH2 domain for binding phosphotyrosines contained within the pYXN sequence motif. We have also synthesized a cyclic, non-phosphorylated peptide reported to have binding specificity for the Grb7-SH2 domain. We are currently examining the interaction between the peptide and the Grb7-SH2 domain using biophysical techniques. We hope this will reveal how tightly the peptide binds and why it is specific for Grb7 and not, for example, the similar Grb14 molecule. It is anticipated these studies will serve as a starting point in the design of therapeutics to target the erbB2-Grb7 interaction. Characterisation of mRNA binding by RNA-binding proteins We are investigating the molecular basis of protein-RNA recognition which underlies the regulation of messenger RNA (mRNA) stability and translational efficiency. A significant subset of mRNA transcripts are regulated by Adenylate/uridylate-Rich Elements (AREs) in their 3’-untranslated regions. AREs are the binding targets of a number of RNA-binding proteins that either up-regulate or down-regulate the mRNA transcript’s stability and translational efficiency. Binding by the T-cell restricted antigen 1 (TIA-1) protein and its related homologue (TIAR) results in translational silencing of mRNA transcripts encoding inflammatory proteins. The AU binding Factor (AUF1, also known as hnRNP D) also binds to AREs leading to the rapid degradation of the mRNA. Alternatively, the ARE-binding protein HuR has a protective effect. We are examining the interactions of TIA-1/TIAR (in comparison with AUF-1 and HuR) with ARE motifs to discover the factors which determine the preferential binding of these proteins in the cell, which dictates the amount of gene product produced in the cell. Characterisation of mRNA binding by RNA-binding proteins The innate immune response is invoked by cellular stresses such as microbial invasion and other immune stimuli resulting in the production of key inflammatory mediators. We are interested in the effect of double stranded (ds)RNA in initiating the innate immune response pathway through its recognition by the retinoic acid inducible gene I (RIG-I). RIG-I recognises double-stranded RNA (dsRNA) that enters the cell as a result of viral infection, resulting in the induction of interferon and proinflammatory cytokines as part of the innate immune response. This has important implications for the application of RNA interference (RNAi) which leads to the specific destruction of a targeted mRNA. RNAi provides a spectacularly successful method of studying the functional outcome of a gene, and there is enormous potential for its application in medicine. It is limited, however, when it also triggers the innate immune response of the cell. We are exploring the structural and biophysical basis of RIG-I activation in the development of optimised siRNA that do not induce the innate immune response. CareerI completed a B. Sc (Hons) degree at Monash University, specialising in novel peptide chemistries at honours level. In 1994 I graduated with a Ph.D. in Medicinal Chemistry from the Victorian College of Pharmacy in the field of NMR structural and dynamics studies of peptides and proteins. After a year off, sailing along the East coast of Australia, I took up a position at Queensland University in the Centre for Drug Design and Development with Professor David Craik, synthesising proteins using peptide and novel linkage chemistries. I then spent several years at the University of Sydney with Professor Glenn King, extending my expertise to include molecular biological and protein expression and purification techniques as well as 3D heteronuclear NMR spectroscopy and analytical ultracentrifugation. Here I began to develop an interest in structural and dynamic studies of novel cancer targets as well as the use of peptides for transporting bioactive compounds across cell walls. I also embarked on structural studies of a protein:DNA complex involved in replication fork arrest for which I was awarded an ARC Postdoctoral Fellowship. In 1999 I established my own laboratory as a joint member of Biochemistry and Chemistry at the Department at the University of Western Australia, where I extended my research into the field of snake and jellyfish venom (about which little was known for the local WA species) as well as structural and dynamic studies of protein-RNA interactions involved in mRNA stabilization in collaboration with partner (and crystallographer guru) A/Prof Matthew Wilce and Professor Peter Leedman. In 2001 I was awarded an ARC Fellowship to pursue the molecular interactions studies, and have added surface plasmon resonance using BIACORE to the repertoire of biophysical techniques that underpins the work. In 2005 I moved with Matthew Wilce to Monash University to establish labs in the dynamic Department of Biochemistry and Molecular Biology. Here I look forward to developing my research interests and forging new collaborative efforts. Postgraduate OpportunitiesMy work has very much been a joint effort with talented students along the way. I have enjoyed training them at Honours and PhD level and believe this is an important role of the researcher. I have successfully supervised 7 honours and 3 PhD students to completion, who have taken up post-doctoral positions, industry or furthered their training in medicine or post-graduate studies. There are currently positions vacant to study at Honours and PhD level under my supervision at Monash University, including a PhD Scholarship available for a suitably qualified candidate with a excellent background in biochemistry, molecular biology or chemistry, and an interest in studies of macromolecular interactions (see projects above). Please email me at Jackie.Wilce@med.monash.edu.au. if you are interested in studying in my laboratory. Wilce lab AlumniHonours levelAdam Hastings – 1998 Honours in Biochemistry, Sydney University (1st Class) PhD completions2004 Roopwant Judge (co-supervisor Matthew Wilce) – Biochemistry/ Pharmacology University of Western Australia PhD currentMahjooba Sidiqi – (co-supervised with Matthew Wilce and Peter Leedman) Biochemistry/Pharmacology University of Western Australia Recent PublicationsJ.A. Wilce, R.G Wake, G.F. King (2001) Termination of replication in bacteria. In: Encyclopedia of Life Sciences, Nature Publishing Group: London, http://www.els.net. J.P.Vivian, J.A.Wilce, A.F. Hastings and M.C.J. Wilce (2001) Crystallization of the B. subtilis RTP/DNA complex. Acta Crystallographica D57 421-424. J. A. Wilce, J. P. Vivian, A.F. Hastings, G. Otting, R. Folmer, I.G. Duggin, R.G. Wake and M.C.J Wilce, (2001) Structure of the RTP/DNA complex and the mechanism of polar replication fork arrest. Nature Structural Biology 8, 206-210. P. Bailey and J.A. Wilce (2001) Venom as a source of useful biologically active molecules. Emergency Medicine 13, 28-36. A. Muratovska, R.H. Lightowlers, R.W. Taylor, R.W., D.M. Turnbull, R.A.J. Smith, J.A. Wilce, S.W. Martin and M. Murphy (2001) Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations. Nucleic Acid Research 29, 1852-1863. A. Muratovska, R.N. Lightowlers, R.W. Taylor, J.A.Wilce and M. Murphy (2001) Targeting large molecules to mitochondria. Advanced Drug Delivery Reviews 49, 189-198. J.Wilce, S.Love, S. Richardson, P. Alewood and D.Craik (2001) Synthesis of an analog of the thyroid-hormone-binding protein transthyretin via regioselective chemical ligation. J. Biol. Chem 276, 25997-26003. Reinhard I. Boysen, Agnes J.O. Jong, Jackie A. Wilce, Glenn F. King and Milton T. W. Hearn (2002) Role of interfacial hydrophobic residues in the stabilisation of the leucine zipper structures of the transcription factors c-Fos and c-Jun. J. Biol. Chem. 277, 23-31. R K. Judge, P.J. Henry, A. d’Aprile, D. Lynch, G.A. Jelinek, M.C.J. Wilce and J.A. Wilce (2002) Identification of PLA2 and a-neurotoxin proteins in the venom of Pseudonaja affinis (dugite). Toxicology and Applied Pharmacology 181,184-191. B.B. Yeap, D. Voon, J.Vivian, R. McCulloch, A.M. Thomson, M.F. Czyzyk-Krzeska, H. Furneaux, M.C.J Wilce, J.A.Wilce and P.J. Leedman (2002). 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. J. Biol. Chem. 277, 27183-27192 J.A. Wilce, N.L. Daly and D.J. Craik (2002) Synthesis and structural analysis of the N-terminal domain of the thyroid-hormone binding protein transthyretin. Clin. Chem. Lab. Med. 40, 1221-1228. J.A. Wilce, Peter J Leedman and Matthew CJ Wilce (2002) RNA-binding proteins that target the androgen receptor mRNA IUBMB Life 54, 345-349. P.M. Bailey, M. Little, G.A Jelinek and J.A. Wilce (2003) Jellyfish envenoming syndromes: Unknown toxic mechanisms and unproven therapies. Med. Journal of Australia 178, 34-37. J.P. Vivian, A.F. Hastings, I.G. Duggin, R.G. Wake, M.C.J. Wilce and J.A. Wilce, (2003) The impact of single cysteine residue mutations on the replication terminator protein. Biochem. Biophys. Res. Comm. 310, 1096-1103. P.M. Finnegan, A.L. Umbach and J.A. Wilce (2003) Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes. FEBS Lett. 555, 425-430. B.B. Yeap, J.A. Wilce and P.J. Leedman (2004) Our Favourite Molecule: The Androgen Receptor mRNA. BioEssays 26, 672-682. P.M. Bailey, A. Bakker, A, J. Seymour and J.A. Wilce (2005) 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. Toxicon 45, 233-242. B.K. Sharpe, C. K. Liew, J.A. Wilce, M. Crossley, J.M. Matthews and J.P. Mackay (2005) Assessment of the robustness of a serendipitous zinc binding fold: mutagenesis and protein grafting. Structure 13, 1-10. M. Sidiqi, J.A Wilce, C.J., Porter, A. Barker, P.J. Leedman and M.C.J.Wilce (2005) Formation of an alphaCP1-KH3 complexed with UC-rich RNA. Eur Biophys J. 34, 423-429. Porter CJ, Wilce MCJ, MacKay JP, Leedman PJ, Wilce JA (2005) Grb7-SH2 domain dimerisation is affected by a single point mutation. Eur Biophys J. 34, 454-460. M. Sidiqi, J.A. Wilce, J.P.Vivian, C.J. Porter, P.J. Leedman and M.C.J. Wilce (2005) Structure and RNA binding of the third domain of poly(C)-binding protein. Nucleic Acids Research 33,1213-1221. Hastings, A.F., Otting, G., Folmer, R.H.A., Duggin I.G.,Wake, R.G. Wilce, M.C.J.and Wilce, J.A., (2005) Interaction of the Replication Terminator Protein of Bacillus subtilis with DNA probed by NMR spectroscopy. Biochem. Biophys. Res. Comm 335, 361-366. Judge R.K., Henry, P.J., Mirtschin P., Jelinek G. and Wilce J.A. (2006) Toxins not neutralized by brown snake antivenom. Toxicol Appl Pharmacol. 213, 117-125. Hatchell EC, Colley SM, Beveridge DJ, Epis MR, Stuart LM, Giles KM, Redfern, AD, Miles LE, Barker A, MacDonald LM, Arthur PG, Lui JC, Golding JL, McCulloch RK, Metcalf CB, Wilce JA, Wilce MC, Lanz RB, O'Malley BW, Leedman PJ. (2006) SLIRP, a small SRA binding protein, is a nuclear receptor corepressor. Mol Cell. 22, 657-668. Vivian J.P., Porter, C.J, Wilce, J.A. and Wilce M.C.J. (2006) Crystallisation and preliminary X-ray diffraction analysis of the Bacillus subtilis replication termination protein in complex with the 37-base-pair TerI-binding site. Acta Crys Section F 62, 1104-1107. Porter C.J. and Wice, J.A. (2007) NMR analysis of G7-18NATE, a non-phosphorylated cyclic peptide inhibitor of the Grb7 adapter protein. Biopolymers. 2007 Jan 5; [Epub ahead of print]. Vivian, J.P., Porter, C.J., Wilce, J.A., Wilce, M.C.J. (2007) An asymmetric structure of replication terminator protein in complex with DNA (accepted Feb 17 - Journal of Molecular Biology ). Schmidberber, J.W., Wilce, J.A., Tsang, J.S.H. and Wilce, M.C.J. (2007) Crystal Structures of the substrate free-enzyme, and reaction intermediate of the HAD Superfamily member, Haloacid Dehalogenase DehIVa from Burkholderia cepacia MBA4 (accepted Feb 8 - Journal of Molecular Biology. ContactDr Jackie Wilce |
