Proteomics of Ubiquitin-Dependent N-End Rule Pathway

描述（由申请人提供）：泛素依赖性N-末端规则途径将蛋白质的体内半衰期与其N-末端残基的身份联系起来。我们以前曾报道过UBR 1和UBR 2作为其功能重叠的E3，并阐明了它们在体内的作用，使用小鼠敲除方法。出乎意料的是，尽管UBR 1和UBR 2都与N-降解决定子强烈结合，但UBR 1-/-UBR 2-/-细胞仍然保留了N-末端规则E3活性，表明存在尚未鉴定的N-识别蛋白（N-degron-Recognizing E3）。本研究的目的是鉴定和表征N-识别及其底物，并阐明其E3-底物相互作用的生理意义。为了鉴定哺乳动物N-识别蛋白，我们开发了一种新的基于亲和力的蛋白质组学方法，使用带有N-降解决定子的合成肽，产生了一种新的570 kDa蛋白质，命名为UBR 4和一种300 kDa-E3连接酶EDO（称为UBR 5）。UBR 1、-2、-4和-5共有一个锌指结构域，称为UBR盒。哺乳动物基因组似乎编码七种UBR蛋白，分别命名为UBR 1至UBR 7。此外，通过使用功能性蛋白质组学方法，我们已经从兔网织红细胞裂解物中表达的约14，000种不同蛋白质中获得了约35种候选N-末端规则底物。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（RGS 4、RGS 5、RGS 16和CDC 6），这是第一种在哺乳动物中鉴定的底物。为了进一步扩展我们目前对N端规则途径的理解，我们提出以下目标。目标1。鉴定UBR盒蛋白作为候选N-重组蛋白。我们将研究：1）UBR突变细胞中模型N-末端规则底物的蛋白水解，2）UBR盒蛋白与N-降解决定子的相互作用和特异性，以及3）UBR盒蛋白对模型底物的体外泛素化。目标二。确定UBR box模体是否为N-降解决定子的识别域。我们将确定N-识别蛋白的UBR盒是否是直接结合N-降解决定子所必需的和足够的，并确定识别N-降解决定子的必需残基。目标3。识别生理N端规则底物。我们将解剖网织红细胞裂解物和UBR突变细胞中候选底物的蛋白水解，确定N-识别蛋白-底物相互作用，并测试N-识别蛋白是否支持体外底物泛素化。目标4。解剖生理过程的基础上确定的N端规则基板。我们将研究RGS 4，-5和-16的N-末端规则依赖性蛋白水解的生理意义，这些蛋白水解在体内出现N-末端规则底物。

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.