本文選題：泛素連接酶 + LNX��； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：泛素化是真核生物中最重要的翻譯后修飾之一。泛素化過程除了參與蛋白酶體降解之外,還參與調(diào)節(jié)許多生物學(xué)過程,包括細胞內(nèi)轉(zhuǎn)運,DNA修復(fù),信號傳導(dǎo)和蛋白質(zhì)蛋白質(zhì)相互作用。泛素化過程通過多步酶促級聯(lián)反應(yīng)實現(xiàn)。在這一過程中,泛素連接酶(E3)決定了泛素化底物識別的特異性。然而多數(shù)E3的底物尚不清楚。鑒定這些底物是當前泛素化研究的主要難點。目前,多數(shù)底物的是在不同實驗室用不同的方法逐一鑒定出來的,需要更好,更快,更經(jīng)濟的高通量方法鑒定E3底物。 目前已經(jīng)有幾種高通量研究E3底物的方法,包括,1.利用蛋白質(zhì)芯片作為底物高效篩選E3底物；2.體內(nèi)(in vivo)非標記定量或SILAC (Stable Isotope Labeling by Amino Acid)標記定量質(zhì)譜的方法；3.體內(nèi)GPSP (Global Proteins Stability profiling)的方法。在本研究中,我們建立了與上述方法不同的兩種較高通量鑒定泛素連接酶底物的新策略。 許多E3特異性識別底物是通過他們的蛋白質(zhì)相互作用結(jié)構(gòu)域?qū)崿F(xiàn)的,在本論文的前一部分,我們建立了一套在蛋白質(zhì)組水平上系統(tǒng)鑒定含蛋白質(zhì)相互作用結(jié)構(gòu)域的泛素連接酶底物的策略(見圖1)。本策略通過篩選隨機多肽文庫鑒定蛋白質(zhì)相互作用結(jié)構(gòu)域的配體結(jié)合特性。通過構(gòu)建了一系列人工底物(artificial degron),包括一個可以被泛素化的序列和一系列識別底物的序列用于體外泛素化實驗。通過這一策略,除了底物之外還能鑒定到非底物調(diào)節(jié)蛋白。該策略還能鑒定到底物識別的詳細機制,這對于藥物的研發(fā)很有幫助。本課題中以LNX (Ligand of Numb protein X)家族E3(一組含有PDZ結(jié)構(gòu)域的RING-type泛素連接酶)為例,來闡述和驗證這一策略。我們鑒定到大量LNX1的潛在底物；在選出的9個候選底物中,8個在體外泛素化實驗中被LNX1泛素化。在進一步的細胞內(nèi)驗證中,本研究驗證出兩個LNX1內(nèi)源底物—-PBK和BCR。進一步實驗證明LNX1催化PBK的泛素化和降解,抑制細胞增殖,促進阿霉素誘導(dǎo)的細胞死亡。我們還描述了LNX1識別底物的詳細機制,即LNX1通過哪個PDZ結(jié)合底物。這一策略作為一個在蛋白質(zhì)組水平上系統(tǒng)鑒定E3底物的有力工具,能夠擴展到其他含有蛋白質(zhì)相互作用結(jié)構(gòu)域的泛素連接酶的研究。 在本文的后一部分,我們建立了另一套較高通量鑒定E3底物的新策略(見圖2)。該策略利用活噬菌體展示文庫做為E3底物進行篩選。本研究以MDM2為例闡述和驗證該策略。通過4次不同方法的篩選,本策略鑒定到MDM2的16個天然潛在底物和許多非天然潛在底物。有些底物可以在不同的實驗中重復(fù)篩選到。在選出的12個候選底物中,10個在體外泛素化體系中被MDM2泛素化。在進一步的細胞內(nèi)驗證中,本實驗驗證出三個MDM2的新底物蛋白——DX42,TP53RK和RPL36a。進一步的研究發(fā)現(xiàn)了MDM2促進TP53RK泛素化導(dǎo)致其被蛋白酶體的降解。在本策略中,除了多聚底物之外,還能鑒定到更多的MDM2單泛素化或寡聚泛素化底物。只要某一個E3適合體外泛素化系統(tǒng),且不泛素化空噬菌體,這一策略就能夠推廣到該泛素連接酶底物的篩選中。
[Abstract]:Ubiquitination is one of the most important post-translational modifications in eukaryotes. In addition to proteasome degradation, ubiquitination is involved in regulating many biological processes, including intracellular transport, DNA repair, signal transduction and protein protein interaction. The process of ubiquitination through multistep enzyme catalyzed cascade reaction. Ubiquitin ligase (E3) determines the specificity of ubiquitination substrate identification. However, most E3 substrates are not yet clear. Identification of these substrates is a major difficulty for current ubiquitination. Most substrates are identified in different laboratories in different laboratories and need to be better, faster, and more economical to identify the bottom of E3. Things.
At present, there are several high throughput methods for the study of E3 substrates, including 1. using protein chips as substrates to efficiently screen E3 substrates; 2. in vivo (in vivo) non labeled quantitative or SILAC (Stable Isotope Labeling by Amino Acid) method of labeling quantitative mass spectrometry; 3. in the body. In this study, we established two new strategies to identify ubiquitin ligase substrates, which are different from the above methods.
Many E3 specific substrates are realized through their protein interaction domains. In the first part of this paper, we established a set of strategies for systematically identifying ubiquitin ligase substrates containing protein interaction domains at proteome level (see Figure 1). This strategy is based on screening random peptide library identification proteins. The ligand binding properties of a mass interaction domain. A series of artificial substrates (artificial degron), including a sequence that can be ubiquitous and a series of identification substrates, are used for in vitro ubiquitination experiments. Through this strategy, a non substrate regulatory protein can be identified except the substrate. The strategy can also be identified. The detailed mechanism of the identification is helpful for the development of drugs. In this topic, the LNX (Ligand of Numb protein X) family E3 (a group of RING-type ubiquitin ligase containing PDZ domains) is used as an example to illustrate and verify this strategy. We identified the potential of a large number of LNX1 in the substrate; of the selected 9 candidate substrates, 8 are in body. In the external ubiquitination experiment, LNX1 is ubiquitination. In further intracellular validation, two LNX1 endogenous substrates, -PBK and BCR., have been verified by further experiments that LNX1 catalyzes the ubiquitination and degradation of PBK, inhibits cell proliferation and promotes adriamycin induced cell death. We also describe the detailed mechanism of LNX1 recognition of the substrate, that is, LNX1 pass. Which PDZ combines substrates. This strategy, as a powerful tool for systematic identification of E3 substrates at the proteome level, can be extended to other ubiquitin ligase containing protein interaction domains.
In the latter part of this article, we have established a new set of new strategies for identifying E3 substrates at high flux (see Figure 2). This strategy uses a live phage display library as a substrate for E3 screening. This study describes and validates the strategy with MDM2 as an example. By screening 4 different methods, this strategy identifies 16 natural potential substrates and many of the MDM2's natural substrates. Non natural potential substrates. Some substrates can be repeated in different experiments. Of the selected 12 candidate substrates, 10 are ubiquitin by MDM2 in the extracorporeal ubiquitination system. In further intracellular validation, this experiment verified that three new MDM2 substrate proteins, DX42, TP53RK and RPL36a., found MDM2 The promotion of ubiquitination of TP53RK leads to the degradation of proteasome. In this strategy, more MDM2 ubiquitin or oligoubiquitin substrates can be identified in addition to polysubstrates. As long as a single E3 is suitable for an in vitro ubiquitination system and without ubiquitin phage, this strategy can be extended to the screening of the ubiquitin ligase substrate. Middle.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R3411

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相關(guān)期刊論文 前1條

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本文編號：2057872