两种高效研究泛素连接酶底物的新策略

发布时间：2018-06-23 17:35

本文选题：泛素连接酶 + LNX　；参考：《北京协和医学院》2012年博士论文

【摘要】：泛素化是真核生物中最重要的翻译后修饰之一。泛素化过程除了参与蛋白酶体降解之外,还参与调节许多生物学过程,包括细胞内转运,DNA修复,信号传导和蛋白质蛋白质相互作用。泛素化过程通过多步酶促级联反应实现。在这一过程中,泛素连接酶(E3)决定了泛素化底物识别的特异性。然而多数E3的底物尚不清楚。鉴定这些底物是当前泛素化研究的主要难点。目前,多数底物的是在不同实验室用不同的方法逐一鉴定出来的,需要更好,更快,更经济的高通量方法鉴定E3底物。目前已经有几种高通量研究E3底物的方法,包括,1.利用蛋白质芯片作为底物高效筛选E3底物；2.体内(in vivo)非标记定量或SILAC (Stable Isotope Labeling by Amino Acid)标记定量质谱的方法；3.体内GPSP (Global Proteins Stability profiling)的方法。在本研究中,我们建立了与上述方法不同的两种较高通量鉴定泛素连接酶底物的新策略。许多E3特异性识别底物是通过他们的蛋白质相互作用结构域实现的,在本论文的前一部分,我们建立了一套在蛋白质组水平上系统鉴定含蛋白质相互作用结构域的泛素连接酶底物的策略(见图1)。本策略通过筛选随机多肽文库鉴定蛋白质相互作用结构域的配体结合特性。通过构建了一系列人工底物(artificial degron),包括一个可以被泛素化的序列和一系列识别底物的序列用于体外泛素化实验。通过这一策略,除了底物之外还能鉴定到非底物调节蛋白。该策略还能鉴定到底物识别的详细机制,这对于药物的研发很有帮助。本课题中以LNX (Ligand of Numb protein X)家族E3(一组含有PDZ结构域的RING-type泛素连接酶)为例,来阐述和验证这一策略。我们鉴定到大量LNX1的潜在底物；在选出的9个候选底物中,8个在体外泛素化实验中被LNX1泛素化。在进一步的细胞内验证中,本研究验证出两个LNX1内源底物—-PBK和BCR。进一步实验证明LNX1催化PBK的泛素化和降解,抑制细胞增殖,促进阿霉素诱导的细胞死亡。我们还描述了LNX1识别底物的详细机制,即LNX1通过哪个PDZ结合底物。这一策略作为一个在蛋白质组水平上系统鉴定E3底物的有力工具,能够扩展到其他含有蛋白质相互作用结构域的泛素连接酶的研究。在本文的后一部分,我们建立了另一套较高通量鉴定E3底物的新策略(见图2)。该策略利用活噬菌体展示文库做为E3底物进行筛选。本研究以MDM2为例阐述和验证该策略。通过4次不同方法的筛选,本策略鉴定到MDM2的16个天然潜在底物和许多非天然潜在底物。有些底物可以在不同的实验中重复筛选到。在选出的12个候选底物中,10个在体外泛素化体系中被MDM2泛素化。在进一步的细胞内验证中,本实验验证出三个MDM2的新底物蛋白——DX42,TP53RK和RPL36a。进一步的研究发现了MDM2促进TP53RK泛素化导致其被蛋白酶体的降解。在本策略中,除了多聚底物之外,还能鉴定到更多的MDM2单泛素化或寡聚泛素化底物。只要某一个E3适合体外泛素化系统,且不泛素化空噬菌体,这一策略就能够推广到该泛素连接酶底物的筛选中。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R3411

【参考文献】