Abstract/Summary

The human ROCO protein family consists of four proteins, including Leucine-Rich Repeat Kinase 2 (LRRK2), a widely studied protein for its role in familial Parkinson's disease; Leucine-Rich Repeat Kinase 1 (LRRK1), which is involved in immunity and bone resorption; Death Associated Protein Kinase 1 (DAPK1), which is linked to cancer; and Malignant Fibrous Histiocytoma-Amplified Sequence 1 (MFHAS1), a protein of unknown function implicated in cancer and immunity. The presence of a ROC-COR supradomain distinguishes this family of proteins. The Ras Of Complex protein (ROC) domain has been described as a small GTPase and is universally followed by a 300-400 amino-acid C-terminal domain of unknown function, termed C terminal Of ROC (COR). Focusing on MFHAS1, the smallest of the ROCO proteins, this project aims to develop a structural and functional understanding of the ROCO proteins. In this thesis, MFHAS1 was studied with three approaches: structural analysis of MFHAS1 at the Research Complex at Harwell and Diamond Light Source, use of bioinformatic tools, and investigation of the cell biology and pathways surrounding MFHAS1. For the former, MFHAS1 was over-expressed and purified attempted for structural and biophysical characterisation. Bioinformatic analysis was utilised to define a modelled structure of MFHAS1, analyse the post-translational modification presents in MFHAS1 and build a protein-protein interaction network. Finally, cellular studies were used to understand the physiological role of MFHAS1, with the steady state of MFHAS1 and the expression level of surrounding proteins were analysed following artificial mutation of residues subject to putative post translational modification. Immunoprecipitation was employed to determine proteins interacting with MFHAS1, providing evidence that it interacts directly with MAP3K4 in a cellular context. Endogenous MAP3K4 is found to interact with overexpressed wild-type and mutants MFHAS1. Together, these analyses and experiments give an insight into MFHAS1 protein, illuminating the function of this least studied of the human ROCO proteins.