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

• 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
• 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

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.

泛素依赖的n端规则途径将蛋白质的体内半衰期与其n端身份联系起来