Proteomics of Ubiquitin-Dependent N-End Rule Pathway

泛素依赖的N-末端规则途径将蛋白质的体内半衰期与其N-末端的身份联系起来 残余物我们以前曾报道UBR 1和UBR 2是其功能重叠的E3，并阐明了它们在E3中的作用。 使用小鼠敲除方法的体内作用。出乎意料的是，尽管UBR 1和UBR 2都强烈地结合到 UBR 1 ^'、UBR 2 ^'细胞仍然保留N端规则E3活性，表明存在尚未表达的N-degron。 未鉴定的N-识别蛋白（N-降解决定子-识别E3）。本研究的目的是识别和表征 N-识别蛋白及其底物的研究，并阐明其E3-底物相互作用的生理意义。到 为了鉴定大肠杆菌N-识别蛋白，我们开发了一种新的基于亲和性的蛋白质组学方法， 携带N-降解决定子，产生一种新的570 kDa蛋白质，命名为UBR 4和一种300 kDa的E3连接酶EDO（命名为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哺乳动物基因组似乎编码 七种UBR蛋白，命名为UBR 1到UBR 7。此外，通过使用功能蛋白质组学方法，我们 从兔网织红细胞中表达的约14，000种不同蛋白质中获得约35种候选N-末端规则底物 裂解物。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 (RGS4、RGS 5、RGS 16和CDC 6），这是第一个在哺乳动物中被鉴定的。为了进一步扩大我们目前的 为了理解N端规则途径，我们提出以下目标。目标1。表征UBR盒 蛋白作为候选N-重组蛋白。我们将研究：1）UBR突变体中N-末端规则底物的蛋白水解 细胞，2）UBR盒蛋白与N-降解决定子的相互作用和特异性，以及3）模型的体外泛素化 UBR盒蛋白的底物。目标二。为了确定UBR盒基序是否是 N-degron。我们将确定N-识别蛋白的UBR盒是否是直接结合所需的和足够的 并鉴定识别N-降解决定子的必需残基。目标3：识别生理N端 规则衬底。我们将剖析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-识别蛋白是否支持底物泛素化， 体外目标4。解剖生理过程的基础上确定的N端规则基板。我们会研究 RGS 4，-5和-16的N-末端规则依赖性蛋白水解的生理意义，在体内出现 N端规则基板。

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 mammalianN-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 appearsto 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.