Structural studies of two cancer targets involved in protein-protein interactions
AuthorTang, Wai Kin Julian
AffiliationMedicine (St Vincent's)
MetadataShow full item record
Document TypePhD thesis
Access StatusOpen Access
© 2017 Dr. Wai Kin Julian Tang
Protein-protein interactions play a key role in biological processes. With experimental data showing an increasing number of protein-protein interactions being important in tumorigenesis and progression, structure-guided design of small molecules as potential inhibitors of such interactions offer attractive and exciting opportunities for therapeutic intervention. In this work, structural investigations were focused on two key proteins involved in cancer: the tetraspanin CD151 and the ubiquitin ligase SIAH, which are validated targets in prostate and breast cancers, respectively. In both cases, the proteins utilise protein-protein interactions for signalling. The tetraspanin CD151 has been associated with the regulation of cancer invasion and metastasis by initiating signalling events across the cell surface. Higher levels of CD151 are associated with poor prognosis in lung and prostate cancer, and overexpression of CD151 promotes metastasis in colon carcinoma and fibrosarcoma cells. In this study, CD151 EC2 (extracellular domain 2) has been successfully expressed and purified to facilitate structural and functional studies by X-ray crystallography and nuclear magnetic resonance. The SIAH (seven-in-absentia homologue) family of proteins functions as ubiquitin ligases for specific intracellular targets. SIAH proteins have been implicated in the ubiquitination and degradation of a range of proteins, including the PHD (prolyl- hydroxylase) family of proteins, β-catenin and netrin receptor. Under hypoxia, SIAH proteins are up-regulated and target PHD for degradation, leading to an increased expression of HIF-1α, the major transcription factor controlling hypoxic and angiogenic responses. We are collaborating with other laboratories to discover small molecule inhibitors of SIAH signalling that might be developed into anti-cancer drugs. The crystal structures of the substrate-binding domain of SIAH, in apo and peptide-bound states, have been previously determined to moderate resolution. In this work, a high-resolution structure was achieved, enabling a fragment-based approach for the discovery of SIAH inhibitors. In addition, the improved structure is very valuable for guiding development of small molecules through high-throughput screening approaches.
Keywordscrystallography; protein purification; biochemistry; molecular biology
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