Proteomics of Ubiquitin-Dependent N-End Rule Pathway

描述(由申请人提供)：泛素依赖的N-末端规则通路将蛋白质在体内的半衰期与其N-末端残基的同一性联系起来。我们已经报道了UBR1和UBR2在功能上重叠的E3，并用小鼠基因敲除的方法阐明了它们在体内的作用。出乎意料的是，尽管UBR1和UBR2都与N-降解物强烈结合，但UBR1-/-UBR2-/-细胞仍然保持着N-末端规则E3的活性，这表明还存在未知的N-识别素(N-降解子识别E3)。本研究的目的是鉴定和鉴定N-识别素及其底物，并阐明其E3-底物相互作用的生理意义。为了鉴定哺乳动物的N-识别素，我们开发了一种新的基于亲和力的蛋白质组学方法，利用含有N-降解素的合成肽，产生了一个新的570 kDa的蛋白质UBR4和一个300 kDa的E3连接酶EDO(称为UBR5)。UBr1、-2、-4和-5共享一个锌指状结构域，命名为UBR box。哺乳动物基因组似乎编码七种UBR蛋白，命名为UBR1到UBR7。此外，通过使用功能蛋白质组学方法，我们已经从兔网织红细胞裂解物中表达的-14,000种不同蛋白质中获得了-35个候选N-末端规则底物。对候选底物的初步表征揭示了几种新的体内N-末端规则底物(RGS4、RGS5、RGS16和CDC6)，这是首次在哺乳动物中发现的。为了进一步扩大我们目前对N-端规则途径的理解，我们提出了以下目标。目的1.鉴定UBR盒蛋白为候选N-识别素。我们将检测：1)UBR突变细胞中模型N-末端规则底物的蛋白分解，2)UBR盒蛋白与N-降解物的相互作用和特异性，以及3)UBR盒蛋白体外泛素化模型底物。目的2.确定UBR盒基序是否是N-降解子的识别结构域。我们将确定N-识别素的UBR盒是否需要以及是否足以与N-降解子直接结合，并确定识别N-降解子所必需的残基。目的3.鉴定生理N-末端规则底物。我们将分析网织红细胞裂解物和UBR突变细胞中候选底物的蛋白分解，确定N-识别素与底物的相互作用，并在体外测试N-识别素是否支持底物泛素化。目的4.剖析已鉴定的N-末端规则底物的生理过程。我们将研究体内出现的N-端规则底物RGS4、-5和-16依赖于N-端规则的蛋白质分解的生理学意义。

DESCRIPTION (provided by applicant): The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have previously reported UBR1 and UBR2 as its functionally overlapping E3s and elucidated their in vivo roles using mouse knockout approach. Unexpectedly, although both UBR1 and UBR2 strongly bound to N-degrons, UBR1-/-UBR2-/- cells still retained the N-end rule E3 activities, indicating the presence of yet unidentified N-recognins (N-degron-Recognizing E3s). The goal of this study is to identify and characterize N-recognins and their substrates and to elucidate the physiological meaning of their E3-substrate interaction. To identify mammalian N-recognins, we developed a novel affinity-based proteomic approach using synthetic peptides bearing N-degron, yielding a novel 570 kDa-protein named UBR4 and a 300 kDa-E3 ligase EDO (termed UBR5). UBR1, -2, -4, and -5 shared a zinc finger-like domain named the UBR box. Mammalian genome appears to encode seven UBR proteins, named UBR1 through UBR7. Further, by using a functional proteomic approach, we have obtained -35 candidate N-end rule substrates from -14,000 different proteins expressed in the rabbit reticulocyte lysates. Preliminary characterization of candidate substrates unveiled several novel in vivo N-end rule substrates (RGS4, RGS5, RGS16, and CDC6), the first to be identified in mammals. To further extend our current understanding of the N-end rule pathway, we propose the following Aims. Aim 1. To characterize UBR box proteins as candidate N-recoqnins. We will examine: 1) proteolysis of model N-end rule substrates in UBR mutant cells, 2) the interaction and specificity of UBR box proteins with N-degrons, and 3) in vitro ubiquitylation of model substrates by UBR box proteins. Aim 2. To determine whether UBR box motif is the recognition domain for N-degron. We will determine whether the UBR boxes of N-recognins are required and sufficient for direct binding to N-degrons and identify essential residues for recognition of N-degron. Aim 3. To identify physiological N-end rule substrates. We will dissect proteolysis of candidate substrates in reticulocyte lysates and UBR mutant cells, determine the N-recognin-substrate interaction, and test whether N-recognins support substrate ubiquitylation in vitro. Aim 4. To dissect physiological processes underlying identified N-end rule substrates. We will examine the physiological significance underlying the N-end rule dependent proteolysis of RGS4, -5, and -16, emerging in vivo N-end rule substrates.