Proteomics of Ubiquitin-Dependent N-End Rule Pathway

泛素依赖性 N 端规则途径的蛋白质组学

基本信息

批准号：
7350248
负责人：
YONG TAE KWON
金额：
$ 26.55万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7350248
关键词：
Affinity Apoptosis Inhibitor Binding Biochemical Genetics Biological Models Boxing CDC6 gene Cardiovascular system Cells Chromosomal Instability Class Coupled Defect Dissection Embryo Embryonic Heart Family Female infertility Figs - dietary Goals Growth HIV-1 Half-Life Heart Hypertrophy Hormones In Vitro Integrase Knockout Mice Mammals Mediating Modeling Monitor Mus Muscle Proteins Myocardial N Domain N-terminal Names Neural Tube Development Neurotransmitters Oryctolagus cuniculus PTPRJ gene Pathway interactions Phenotype Physiological Physiological Processes Polymerase Process Production Proteins Proteolysis Proteomics RGS Proteins RNA Interference Reporting Reticulocytes Retroviridae Role Saccharomyces cerevisiae Signal Pathway Signal Transduction Sindbis Virus Specificity Substrate Interaction System Techniques Technology Testing Ubiquitin Zinc Fingers base cohesin human CDC6 protein in vivo inhibitor/antagonist lipid metabolism male mammalian genome mutant novel protein degradation recognins small molecule synthetic peptide ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

泛素依赖性 N 端规则途径将蛋白质的体内半衰期与其 N 端的身份联系起来残留物。我们之前曾报道过 UBR1 和 UBR2 作为其功能重叠的 E3，并阐明了它们的作用使用小鼠敲除方法进行体内作用。出乎意料的是，尽管 UBR1 和 UBR2 都与 N端降解决定子，UBR1'^'UBR2^'细胞仍然保留N端规则E3活性，表明还存在未识别的 N-识别蛋白（N-degron-识别 E3）。本研究的目的是识别和表征 N-识别蛋白及其底物，并阐明其 E3-底物相互作用的生理意义。到为了识别哺乳动物 N-识别蛋白，我们使用合成肽开发了一种新的基于亲和力的蛋白质组学方法带有 N-degron，产生一种名为 UBR4 的新型 570 kDa 蛋白质和一种 300 kDa-E3 连接酶 EDO（称为 UBR5）。 UBR1、-2、-4 和 -5 共享一个名为 UBR 盒的锌指状结构域。哺乳动物基因组似乎编码七个 UBR 蛋白，命名为 UBR1 至 UBR7。此外，通过使用功能蛋白质组学方法，我们有从兔网织红细胞中表达的-14,000种不同蛋白质中获得-35种候选N端规则底物裂解物。候选底物的初步表征揭示了几种新型体内 N 端规则底物（RGS4、RGS5、RGS16 和 CDC6），第一个在哺乳动物中被发现。为了进一步扩展我们目前的为了理解 N 端规则路径，我们提出以下目标。目标 1. 表征 UBR 盒作为候选 N-recoqnin 的蛋白质。我们将检查：1) UBR 突变体中模型 N 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 2. 确定 UBR 盒基序是否是 N-德隆。我们将确定 N-识别蛋白的 UBR 盒是否是直接结合所必需的和足够的 N-降解决定子并鉴定识别 N-降解决定子所必需的残基。目标 3. 识别生理 N 端规则基板。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作用，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. 剖析已识别的 N 端规则底物背后的生理过程。我们将检查体内出现的 RGS4、-5 和 -16 N 端规则依赖性蛋白水解的生理意义 N端规则基板。