Structural and biochemical analysis of yeast ubiquitin-specific protease 15

泛素与靶蛋白的附着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被26S蛋白酶体降解。蛋白质泛素化是动态可逆的。靶蛋白的泛素化可以通过去泛素化酶的作用逆转。在去泛素化过程中所涉及的酶的作用尚不清楚。其中一类酶被称为泛素特异性蛋白酶（ubp或USPs），它们是大的多结构域蛋白，大小从50到300 kDa不等。泛素特异性蛋白酶参与从特定靶蛋白中去除泛素分子，因此非催化结构域的序列变化可以为单个泛素特异性蛋白酶提供靶蛋白特异性。文献中有几个例子证明了泛素特异性蛋白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通过去泛素化来调节它们的水平。USP7蛋白存在于从酵母到人类的大量真核生物中。我将把我的大部分研究重点放在UBP15上，它是USP7的酵母同源物。我想鉴定和生化表征与UBP15相互作用的蛋白质，以便深入了解UBP15的功能作用。结合、诱变和结构研究将在相互作用蛋白的初步鉴定之后进行。本提案的目标之一是确定UBP15的c端结构域的结构。本项目的另一个目标是更详细地研究酵母中的去泛素化，特别是泛素特异性蛋白酶-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酶的结构域的晶体结构。由于泛素特异性蛋白酶都被预测含有一个或多个特异性结构域，因此深入了解底物识别的本质将有利于这些蛋白质的整体表征。本研究的意义在于增强我们对去泛素化在细胞功能中的认识。

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.