Structure-Based Investigation of the Activation Mechanism of Deubiquitinase USP7 by X-ray Crystallography

• Main Authors: Chi-Wen Wu, 吳啟聞
• Other Authors: 詹迺立
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/62847812827510070380

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 103 === Ubiquitylation is one of the most important forms of post translational modifications (PTMs) that is involved in numerous physiological processes, including cell cycle regulation, DNA repair responses, protein sorting and proteasomal degradation. Consistent with its highly diversified cellular functions, different types of covalently linked polyubiquitin chains can be generated through the formation of isopeptide bond catalyzed by a cascade of enzymes, including the activating (E1), conjugating (E2) and ligating (E3) enzymes. Similar to most other PTMs, ubiquitylation is reversible. A group of proteins, called deubiquitinases (DUBs), are capable of reversing ubiquitylation by cleaving the isopeptide bond. The human genome encodes more than 90 DUBs, each one displays its own unique specificity for substrates. One of the best characterized deubiquitinases is USP7 (also known as the herpes virus-associated ubiquitin-specific protease, or HAUSP), which plays key roles in a number of cellular processes, such as tumor suppression, DNA repair and virus infection. Previous studies have led to a model that USP7 may be subjected to self-activation through its C-terminal domain-mediated regulation of the core domain, with the flexible C-terminal tail and switching loop being essential for activation. However, no three-dimensional structure of full-length hUSP7 is currently available to confirm this hypothesis. In addition, mounting evidences show that down-regulation of USP7 can inhibit the growth of several types of cancer cells by inducing anti-proliferation signaling events and apoptosis in these cells, making USP7 a potential therapeutic target for treating tumor progression. The specific aim of this project is to determine the crystal structure of hUSP7 using X-ray crystallography to understand the mechanism of self-activation, as well as investigate how a small-molecule inhibitor may associate with hUSP7 to interfere with its catalytic activity. Here, we observed that hUSP7 mainly exists in dimeric form in vitro based on size exclusion chromatography. This initial finding was further confirmed by using native non-reducing gel electrophoresis, glutaraldehyde crosslinking and analytic ultracentrifugation (AUC). Yet, due to the lack of structural information on full-length hUSP7, it has remained unknown how the dimerization of hUSP7 is achieved and whether the formation of hUSP7 dimer is required for its catalytic activity. To facilitate the development of USP7-targeting anticancer drugs, we also examined the effects of the inhibitor hbx41108 on hUSP7. Unexpectedly, we found that hbx41108 could associate with core domain of hUSP7 and induce protein aggregation. Although the assembly mechanism and functional significance of the hUSP7 aggregation are yet to be characterized, the effect of hbx41108 nevertheless infers the difficulty associated with the cocrystallization of hUSP7 with this inhibitor. Recently, we have successfully crystallized an N-terminal domain-truncated hUSP7 ( covering residues 208-1102) in complex with ubiquitin and a native X-ray diffraction data set to 2.3 Å has been collected, and structure determination is currently underway. This structure is expected to reveal new insights regarding the activation mechanism of hUSP7.