本文選題：去泛素水解酶 + NMR��； 參考：《中國(guó)科學(xué)院上海藥物研究所》2016年博士論文

【摘要】：泛素化是蛋白質(zhì)翻譯后修飾方式之一,其修飾和去修飾過程分別由泛素化酶組(E1、E2和E3)和去泛素化水解酶(DUB)催化完成。去泛素化蛋白水解酶分為六個(gè)亞家族,共有近100個(gè)家族成員,他們保證了體內(nèi)蛋白質(zhì)泛素化和去泛素化的動(dòng)態(tài)平衡,以實(shí)現(xiàn)對(duì)細(xì)胞生命活動(dòng)如細(xì)胞的代謝、分化、增殖等過程的精確調(diào)控。其中USP亞家族最為龐大,成員數(shù)量占去泛素化水解酶總量50%以上。近年來(lái)的研究報(bào)道發(fā)現(xiàn),部分USP家族成員的表達(dá)異常和功能紊亂與癌癥的發(fā)生發(fā)展密切相關(guān)。由上可知,從結(jié)構(gòu)生物學(xué)角度闡明USP的功能調(diào)控機(jī)制具有重要的科學(xué)意義。此外,USP相關(guān)的結(jié)構(gòu)生物學(xué)研究成果,還可以進(jìn)一步指導(dǎo)我們理性的設(shè)計(jì)具有抗癌活性的USP抑制劑,從而為癌癥的治療做出貢獻(xiàn)。本論文的研究目標(biāo)Usp25和Usp28均為USP家族成員,且具有重要的生理功能。Usp25參與肌生成、內(nèi)質(zhì)網(wǎng)蛋白質(zhì)降解、免疫信號(hào)調(diào)節(jié)等多種重要的生理過程,而Usp28的異常表達(dá)與癌癥的發(fā)生、發(fā)展密切相關(guān)。Usp25與Usp28具有較高的序列同源性,它們都是由一個(gè)N端泛素結(jié)合結(jié)構(gòu)區(qū)域UBR(包含UBA、UIM結(jié)構(gòu)域)和一個(gè)核心的催化結(jié)構(gòu)域USP構(gòu)成。泛素結(jié)合結(jié)構(gòu)區(qū)域UBR通過參與對(duì)泛素底物的識(shí)別而在Usp25和Usp28的催化功能展示過程中發(fā)揮重要調(diào)控作用。此外,需要指出的是,在Usp25和Usp28的泛素結(jié)合結(jié)構(gòu)區(qū)域中還包含一個(gè)和類泛素蛋白SUMO相互作用的區(qū)域(SIM),這一事實(shí)提示我們SUMO分子有可能參與對(duì)Usp25和Usp28酶活功能展示的調(diào)控。在本論文中,我們主要采用液體核磁共振以及生化實(shí)驗(yàn)等手段研究Usp25和Usp28的N端UBR結(jié)構(gòu)區(qū)域在它們的酶活展示過程中所發(fā)揮的作用。研究結(jié)果顯示,UBR結(jié)構(gòu)區(qū)域能夠特異性的結(jié)合泛素以及SUMO2。SUMO2與UBR結(jié)構(gòu)域SIM區(qū)的非共價(jià)結(jié)合,會(huì)競(jìng)爭(zhēng)性的屏蔽UBR結(jié)構(gòu)域與泛素底物之間的相互作用,從而下調(diào)Usp25和Usp28的去泛素水解能力。最終,基于已獲得的實(shí)驗(yàn)結(jié)果,我們提出了UBR結(jié)構(gòu)區(qū)域調(diào)控Usp25和Usp28酶活功能的分子機(jī)制模型。
[Abstract]:Ubiquidization is one of the post-translational modification methods of proteins. The process of modification and de-modification is catalyzed by Ubiquitin group E _ (1) E _ 2 and E _ 3) and desuginization hydrolase (DUB) respectively. The diubiquitin hydrolase is divided into six subfamilies of nearly 100 family members. They maintain a dynamic balance between ubiquitization and deubiquification in the body, in order to achieve cell life activities such as cell metabolism and differentiation. Precise regulation of processes such as proliferation. The USP subfamily is the largest, and the number of members accounts for more than 50% of the total ubiquitin hydrolase. Recent studies have found that abnormal expression and dysfunction of some members of USP family are closely related to the occurrence and development of cancer. From the above, it is of great scientific significance to elucidate the functional regulation mechanism of USP from the point of view of structural biology. In addition, the research results of USP-related structural biology can further guide us in the rational design of USP inhibitors with anticancer activity, thus contributing to the treatment of cancer. The objective of this study is that Usp25 and Usp28 are members of USP family, and have important physiological functions. Usp25 is involved in many important physiological processes, such as myogenesis, endoplasmic reticulum protein degradation, immune signal regulation, and the abnormal expression of Usp28 and carcinogenesis. The development of Usp25 is closely related to the sequence homology of Usp28, which consists of a N-terminal ubiquitin binding domain (UBR) and a core catalytic domain (USP). Ubiquitin binding structure region (UBR) plays an important regulatory role in the display of catalytic functions of Usp25 and Usp28 by participating in the recognition of ubiquitin substrates. In addition, it should be pointed out that the region of ubiquitin binding structure of Usp25 and Usp28 also contains a region interacting with ubiquitin protein SUMO, which suggests that the SUMO molecule may be involved in the regulation of Usp25 and Usp28 enzyme activity. In this thesis, we mainly use liquid nuclear magnetic resonance (LNMR) and biochemical experiments to study the role of N-terminal UBR region of Usp25 and Usp28 in the process of enzyme activity display. The results showed that the UBR domain could specifically bind ubiquitin and the noncovalent binding of SIM region of SUMO2.SUMO2 and UBR domain, which would be able to screen the interaction between UBR domain and ubiquitin substrate competitively. Thus, the deoxyuridine hydrolysis ability of Usp25 and Usp28 was down-regulated. Finally, based on the obtained experimental results, we proposed a molecular model for the regulation of Usp25 and Usp28 activity by UBR structural regions.
【學(xué)位授予單位】：中國(guó)科學(xué)院上海藥物研究所
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R914

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