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研究生(外文):Chi-Wen Wu
論文名稱(外文):Structure-Based Investigation of the Activation Mechanism of Deubiquitinase USP7 by X-ray Crystallography
外文關鍵詞:ubiquitin specific protease 7 (USP7)deubiquitinaseprotein crystal structuresmall-molecule anticancer drug
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泛素化 (Ubiquitylation)是真核生物中最常見的後轉譯修飾 (Post translational modification, PTM)之一,並在細胞內各式反應中扮演關鍵的角色,包含細胞週期調控、去氧核醣核酸修復機制、蛋白質胞內運輸以及蛋白酶體(Proteasome)之蛋白質降解機制。鑒於其生物功能的多元性,因此不令人意外地,泛素可經由不同位置之離胺酸所形成的類胜肽鍵(isopeptide bond)在目標蛋白上組成結構及長度不同的修飾。如同其他後轉錄修飾,泛素化亦可經由去泛素酶 (deubiquitinases, DUBs)將類胜肽鍵水解而去除。在人類的基因體上,已經發現超過90種去泛素酶,每一種去泛素酶都有其獨特的受質專一性。
第七型泛素特異性蛋白酶 (USP7)或稱為疱疹病毒相關泛素特異性蛋白酶 (Herpes virus-associated ubiquitin-specific protease, or HAUSP),是最被廣為研究的去泛素酶之一。 USP7被證實在腫瘤抑制調控、DNA修復以及病毒感染等過程中扮演重要角色。根據目前已知的研究結果,此去泛素酶可能的活化機制如下: USP7會透過C端結構域與核心結構的交互作用而自我活化。科學家進一步發現其C末端區以及一段核心結構域的表面環狀結構對此活性提升極為重要,突變此區域中的關鍵胺基酸,將導致其活性完全喪失。然而,因為缺少USP7全長蛋白質的結構資訊,科學家仍無法判斷此活化機制的模型是否正確。
本研究的主要目的之一是希望利用X光結晶學解析人類USP7的整體結構,並進而了解其C端結構域誘發其活化的機制。同時,我們也希望進一步了解目前已知的小分子抑制劑 Hbx41108如何與USP7結合並抑制其活性。本研究首先利用凝膠過濾色譜法(gel filtration chromatography)發現 USP7主要是以同質二聚體的形式存在於溶液中。我們進一步以非變性凝膠 (native gel)、戊二醛交聯反應 (glutaraldehyde crosslinking)、分析超速離心 (analytic ultracentrifuge, AUC )確認在我們的實驗條件下USP7的確以二聚體形式存在。但是因為缺少此蛋白質的全長結構,我們尚無法回答二聚體如何形成,同時,二聚體形成與USP7活化之間的關聯亦待探討。此外,為了發展以USP7為標靶的抗癌藥物,我們也嘗試探討小分子抑制物 Hbx41108與USP7的交互作用。我們意外發現hbx41108會與核心結構域產生交互作用,並且引發蛋白質聚集反應。雖然此蛋白質聚集反應發生的原因及生理意義仍屬未知,但是這個現象暗示USP7-hbx41108複合體的晶體培養有其困難性。最近,我們成功得到USP7 N-端刪除蛋白與泛素形成之複合體的晶體,並以收集一組解析度達到2.3Å的繞射數據,目前正在進行結構解析。預期此結構將可加深我們對於USP7活化機制的了解。

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.

口試委員會審定書 #
誌謝 i
中文摘要 ii
Chapter 1 Introduction 1
1.1 Deubiquitinases: A Group of Regulators that Control Protein Homeostasis through Reversing Ubiquitylation 1
1.1.1 Ubiquitylation: A Biological Label 1
1.1.2 Deubiquitylation: A Reverse Reaction of Ubiquitylation 2
1.2 Structure of the USP Subfamily: From Conserved Structural Features to the Molecular Mechanism of Isopeptide Cleavage 3
1.3 Allosteric Regulation for Activation of USP Subfamily 4
1.4 The Specificity of USPs to Chain Linkage 6
1.5 Structure and Function of Herpes-Virus-Associated Ubiquitin-Specific Protease_ (HAUSP) 7
1.5.1 Regulation of Several Tumor Suppressors 8
1.5.2 Modulation of Cell Cycle 8
1.6 Targeting ubiquitin specific protease 7 (USP7) In Cancer Therapeutic Treatment 9
1.7 Specific Aims 10
Chapter 2 Materials and Methods 11
2.1 Construction of Different Truncated USP7 11
2.1.1 Human USP7core (hUSP7core, residues 208-564) 11
2.1.2 Human USP7* (hUSP7*; residues 208-1102) 11
2.1.3 Human Full-Length Ubiquitin (hUb; residues 1-76) 11
2.2 Site-directed Mutagenesis 12
2.3 Protein Expression 13
2.3.1 Expression of hUSP7core and hUSP7* 13
2.3.2 Expression of hUb 13
2.4 Protein Purification 13
2.4.1 Purification of Wild-Type hUSP7core and mutant hUSP7core 13
2.4.2 Purification of hUSP7* and all type of mutant hUSP7* 14
2.4.3 Purification of ubiquitin 15
2.5 Protein Crystallization 15
2.5.1 Crystallization and Data Collection for hUSP7core 15
2.5.2 Crystallization and Data Collection for hUSP7* 15
2.6 Post-Crystallization Drug Soaking 16
2.7 Protein Complex Analyzed by Size Exclusion Chromatography 16
2.8 Oligomerization Analyzed by Native Gel and Gel Filtration 16
2.9 Limited Proteolytic Digestion 17
2.10 Differential Scanning Calorimeter (DSC) 17
2.11 Glutaraldehyde Cross-linking 18
2.12 Analytical Ultracentrifuge (AUC) 18
Chapter 3 Results 19
3.1 Construction of wild-type and mutant forms of hUSP7 19
3.2 Protein Expression by IPTG Induction 19
3.3 Protein Purification 20
3.4 C-terminal Domain Is Required for hUSP7 Oligomerization 22
3.5 Crystallization of the hUSP7* (C223S) mutant -Ubiquitin Binary Complex 23
3.6 Hbx41108-induced aggregation of hUSP7 24
Chapter 4 Conclusion and Discussion 26
Chapter 5 Figures 28
Chapter 6 References 47

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