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Postgraduate and honours research projects - protein X-ray crystallographyProjects in the Rossjohn laboratoryProtein X-ray crystallography is the major technique used to determine the precise three-dimensional shape of proteins. Our laboratory is a major user of synchrotron radiation, where the intense X-rays enable us to acquire more accurate structures of the protein molecules we are investigating. The research within our laboratory is aimed at understanding processes central to infection and immunity and using this information to rationally design therapeutics. ImmunityHosts defend themselves from microbial attack by mounting responses that are controlled by the innate and adaptive arms of the cellular immune system. We are investigating the structural basis of innate and adaptive immunity; how pathogenic peptides and lipids are presented by the immune system; T-cell mediated transplant rejection and T-cell mediated autoimmune disorders. InfectionBacteria and viruses have developed a suite of strategies to overcome the defenses of the immune system. Within the ARC Centre of Excellence for Structural and Functional Microbial Genomics, we are investigating virulence factors that control invasion, components that assist immune evasion and essential cellular machinery that is unique to certain pathogenic bacteria. Rational drug designProtein crystallography is the main tool we use in rationally designing therapeutics to combat disease. We aim to design therapeutics that augment immunity and control infection. In addition, in collaboration with an Australian based biotechnology company, we are designing drugs against protein tyrosine kinases that cause cancer and inflammation. For further information, please contact:
Prof. Jamie Rossjohn, ARC Federation Fellow Projects in the Wilce laboratoryStructural biology is a powerful means by which to understand the molecular basis of disease. We examine the way in which RNA-binding proteins recognise mRNA in the regulation of inflammatory genes and also the way in which dsRNA triggers the innate immune response in cells. We are also characterising potential drug targets in fighting cancer and infection. We use X-ray crystallography (in-house as well as synchrotron radiation), NMR spectroscopy and molecular dynamics simulations to obtain and analyse molecular structural information. Surface plasmon resonance (using BIACORE), isothermal titration calorimetry and analytical ultracentrifugation are used to determine the affinities of interaction and self-association. These data reveal the basis for molecular recognition and facilitate the design of novel therapeutics. Regulating the expression of inflammatory genesin collaboration with Dr Myriam Gorospe (NIH) and Dr Paul Anderson (Harvard) We are investigating the molecular basis of protein-RNA recognition which underlies the regulation of messenger RNA (mRNA) translation. Many mRNA transcripts are regulated by specific sequences (known as AREs) in their 3’-untranslated regions. These are recognised by several RNA-binding proteins that either up-regulate or down-regulate the mRNA transcript’s translation. 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 binds to these sequences leading to the rapid degradation of the mRNA. Alternatively, the HuR protein has a protective effect. Therefore the fate of the mRNA, and the amount of gene product produced in the cell, depends on which of these proteins preferentially binds to it. We are examining the interactions of TIA-1/TIAR (in comparison with AUF-1 and HuR) with target mRNA sequences to discover the factors which determine the preferential binding of these proteins. With a better understanding of this regulatory system we may be able to design agents which regulate gene expression at the level of mRNA.
Avoiding the innate immune response in gene therapyin collaboration with Dr Bryan Williams and Dr Tony Sadler (Monash Institute for Medical Research) 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 application of RNAi that do not induce the innate immune response. Regulating androgen receptor expression in prostate cancerin collaboration with A/Prof Prof Peter Leedman (UWA) Poly(C)-binding (also known as αCP) proteins bind to C-rich RNA and 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, an important cancer target, is bound by αCP1 in a specific region of its 3'UTR which affects translation. We hypothesise that this interaction could be targeted to destabilise the androgen receptor mRNA and thereby prevent prostate cancer progression.
We have studied separate domains of αCP1 (αCP1 is comprised of three KH domains) binding to C-rich RNA in order to better understand the molecular interactions. We have solved the structure of the third KH domain and the first KH domain in complex with ssDNA using x-ray crystallographic techniques. This has allowed us examine the basis for poly-C binding specificity. We've also measured the way in which the affinity of interaction is affected by changing the RNA sequence, and have deduced which RNA bases underlie the specific interaction. Future work will involve the investigation of the second KH domain, whose role in RNA binding remains elusive. We will discover whether it plays a role in RNA binding or has another function - such as interacting with other RNA-binding proteins. In this way we will build up the picture of the way in which mRNA is bound by multiple proteins to regulate the expression of mRNA. Targeting the Grb7 protein involved in cancer cell migrationin collaboration with Prof David Krag and Dr Stephanie Pero (University of Vermont, USA), Jacqui Matthews and Joel Mackay (Sydney University) 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. Understanding antibiotic resistancein collaboration with Professor Rood, Department of Microbiology, Monash Antibiotic resistance is an increasing problem worldwide. Inappropriate use of antibiotics both in humans and animals has led to the development of resistance to all commercially available antibiotics by micro-organisms. The spread of resistance genes within mixed bacterial populations does not occur randomly but involves mobile genetic elements such as transposons, other integrative elements and integrons. For further information, please contact:
Dr Jackie Wilce
Associate Professor Matthew Wilce Projects in the Harley laboratoryStructural characterisation of clinical mutations in the chromatin-remodelling protein, ATRXAnthony Argentaro and Vincent Harley, Prince Henrys Institute, Monash Medical Centre in conjunction with Matthew Wilce, Dept Biochemistry, Monash University The ATRX syndrome is a severe form of mental retardation associated with alpha thalassaemia, facial, skeletal and urogenital abnormalities. Point mutations leading to single amino acid substitions within ATRX cluster in the ADD domain. We have solved the 0.48A ADD structure by NMR (Argentaro et al., 2007, PNAS). It comprises a GATA-like zinc-finger, a PHD finger and a long C-terminal alpha helix. Several clinical ATRX mutants are predicted to be solvent exposed on a putative protein-protein interaction surface. The project involves crystallisation and structure solution of of wildtype and mutant ATRX ADD domains. For further information, please contact:
Associate Professor Vincent R Harley
Associate Professor Matthew Wilce |
