Proteomics of Ubiquitin-Dependent N-End Rule Pathway

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-末端的特性联系起来 残留物。我们之前已经报告了UBR1和UBR2是其功能重叠的E3，并在 活体角色采用小鼠基因敲除方法。出乎意料的是，尽管UBR1和UBR2都强烈绑定到 N-退化，UBr1‘^’UBr2^‘细胞仍保留N-末端规则E3活性，表明存在 未知的N-识别蛋白(N-降解素识别E3)。这项研究的目标是确定和表征 N-识别素及其底物，并阐明其E3-底物相互作用的生理意义。至 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，使用合成肽 含有N-降解子，产生一个新的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 7个UBR蛋白，命名为UBR1至UBR7。此外，通过使用功能蛋白质组学方法，我们已经 从兔网织红细胞表达的-14,000种不同蛋白中获得-35个候选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、RGS5、RGS16和CDC6)，首次在哺乳动物中发现。为了进一步扩展我们目前的 对N-端规则途径的理解，我们提出了以下目标。目标1.UBR BOX的特性 作为候选N-识别蛋白的蛋白质。我们将研究：1)UBR突变体中模型N-末端规则底物的蛋白质降解 2)UBR盒蛋白与N-降解物的相互作用和特异性；3)体外泛素化模型的建立 以UBR盒蛋白为底物。目的2.确定UBR盒基序是否是 N-degron。我们将确定是否需要N-识别素的UBR盒，以及是否足以直接结合 识别N-降解子所必需的残基。目的3.识别生理N-端 规则基材。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质分解， 确定N-识别蛋白与底物的相互作用，并检测N-识别蛋白是否支持底物泛素化。 体外培养。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 依赖N-端规则的RGS4、-5和-16体内蛋白降解的生理意义 N端规则衬底。

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.