Structural and biochemical analysis of yeast ubiquitin-specific protease 15

泛素与靶蛋白的结合在真核细胞过程中起着重要的调控作用。蛋白质泛素化的作用之一是26S蛋白酶体的降解。蛋白质泛素化既是动态的，也是可逆的。靶蛋白的泛素化可以通过脱泛素化酶的作用而逆转。参与脱泛素化的酶的作用还不是很清楚。其中一类酶被称为泛素特异性蛋白水解酶(UBPs或USPS)，它是一种大的多域蛋白质，大小从50 kDa到300 kDa不等。泛素特异性蛋白水解酶参与从特定的靶蛋白中去除泛素分子，因此非催化区的序列变异可以为个别泛素特异性蛋白水解酶提供靶蛋白专一性。文献中有几个例子证明了泛素特定的蛋白水解酶在细胞功能中的重要性。其中研究最多的是USP7。一旦P53和MDM2蛋白被泛素化降解，USP7就通过去泛素化来调节它们的水平。从酵母到人类的大量真核生物中都发现了USP7蛋白。我将把我的大部分研究集中在UBP15上，它是USP7的酵母同源物。我想要鉴定和生化描述与UBP15相互作用的蛋白质，以便深入了解UBP15的功能作用。在初步鉴定相互作用的蛋白质之后，将进行结合、突变和结构研究。该提案的目标之一是确定UBP15的C-末端结构域的结构。这个项目的另一个目标是更详细地研究酵母中的去泛素化作用，特别是泛素特定的蛋白酶-底物相互作用的结构基础。作为起点，我建议确定其他酵母泛素特异性蛋白水解酶结构域的晶体结构。由于泛素特异性蛋白水解酶都被预测含有一个或多个特异性结构域，深入了解底物识别的性质将有助于对这些蛋白质的全面表征。这项研究的意义将是加深我们对细胞功能中去泛素化的理解。

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.