Structural and biochemical analysis of yeast ubiquitin-specific protease 15

点击翻译按钮获取中文摘要

The attachment of ubiquitin to target proteins plays important regulatory tasks in eukaryotic cell processes. One of the effects of protein ubiquitination is degradation by the 26S proteasome. Protein ubiquitination is both dynamic and reversible. The ubiquitination of target proteins can be reversed by the action of deubiquitinating enzymes. The roles of the enzymes involved in deubiquitination are not well understood. One class of these enzymes is known as ubiquitin specific proteases (UBPs or USPs) which are large multidomain proteins ranging in size from 50 to 300 kDa. Ubiquitin specific proteases are involved in the removal of ubiquitin molecules from specific target proteins therefore sequence variation in the non-catalytic domains can provide target protein specificity to individual ubiquitin specific proteases. There are several examples in the literature that demonstrate the importance of ubiquitin specific proteases in cellular functions. One of the most studied is USP7. USP7 regulates the levels of p53 and mdm2 proteins by deubiquitination once they have been targeted for degradation by ubiquitination. The USP7 protein is found in a large number of eukaryotes ranging from yeast to humans. I will focus most of my research on UBP15, the yeast homolog of USP7. I would like to identify and biochemically characterize proteins that interact with UBP15 in order to provide insight into the functional role of UBP15. A combination of binding, mutagenesis and structural studies will follow the initial identification of interacting proteins. One of the goals of this proposal is to determine the structure of the C-terminal domain of UBP15. Another goal of this project is to study deubiquitination in greater detail in yeast especially the structural basis of ubiquitin-specific proteases-substrate interactions. As a starting point, I propose to determine crystal structures of the domains of the other yeast ubiquitin specific proteases. Since the ubiquitin specific proteases are all predicted to contain one or more specificity domains, insight into the nature of substrate recognition will be beneficial to the overall characterization of these proteins. The significance of this research will be to enhance our understanding of deubiquitination in cellular function.