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IP3 Receptor Ubiquitination and Down-regulation

IP3 受体泛素化和下调

基本信息

批准号：
8325019
负责人：
RICHARD J H WOJCIKIEWICZ
金额：
$ 33.47万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 2015-08-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in vertebrate intracellular signaling. They are a family of three very similar proteins that form tetrameric ion channels in endoplasmic reticulum (ER) membranes. For the last 15 years, I have been studying the molecular biology of IP3Rs and for the last 10 years or so, have been examining how they are degraded by the ubiquitin-proteasome pathway (UPP). During these studies, it became obvious that IP3Rs are degraded by the ERAD pathway (the facet of the UPP that accounts for the disposal of aberrant ER proteins) and that given their unique properties, IP3Rs might be particularly useful tools for identifying the proteins and mechanisms that mediate ERAD in mammalian cells. Thus, in the last grant cycle we sought to identify and characterize the proteins and mechanisms that mediate IP3R ERAD, and discovered that the ubiquitin conjugates that become attached to activated IP3Rs are unexpectedly complex, and that a completely novel, and very large (>1MDa) complex composed of two uncharacterized ER membrane proteins, termed SPFH1 and 2, mediates the ERAD of activated IP3Rs and participates in the degradation of model ERAD substrates. Interestingly, we have also very recently found that RNF170, an uncharacterized, putative ubiquitin ligase, interacts strongly with the SPFH1/2 complex. The current proposal is designed to extend this work. As in the past, the bulk of the work will be done with endogenous proteins in mammalian cells, ensuring that the results obtained closely mirror the physiological situation. The Specific Aims of the current proposal are to (i) identify and characterize the proteins that govern the levels of ubiquitin conjugates on activated IP3Rs, and (ii) investigate the structure and function of the SPFH1/2 complex. These goals are significant for three main reasons. First, the studies in Aim 1 on the fine details of IP3R ubiquitination, should help solve the recently revealed mystery of how and why UPP substrates are modified with diverse ubiquitin conjugates. Second, Aim 2 should generate a clearer understanding of the structure and function of the recently discovered, but poorly understood, SPFH1/2 complex. Third, our focus on endogenous proteins in mammalian cells means that the results obtained will accurately model the in vivo situation, and will provide information on the ERAD pathway under near- physiological conditions. Results from this work should have a significant impact on our understanding of both ERAD and the UPP in general, and will hopefully facilitate efforts aimed at developing drugs targeting these pathways. PUBLIC HEALTH RELEVANCE: The degradation of proteins via the proteasome, which is governed by another protein called ubiquitin, is a fundamental cellular process that controls the levels of many key proteins, and has enormous potential as a therapeutic target in diseases such as cystic fibrosis, neuropathies, diabetes and cancer. The studies I propose will investigate in mammalian cells some of the mechanisms that underpin this process with the aim of obtaining a clearer understanding of this area.

描述（由申请人提供）：1，4，5-三磷酸肌醇（IP 3）受体（IP 3R）在脊椎动物细胞内信号传导中起关键作用。它们是一个由三种非常相似的蛋白质组成的家族，在内质网（ER）膜中形成四聚体离子通道。在过去的15年里，我一直在研究IP 3R的分子生物学，在过去的10年左右，我一直在研究它们是如何被泛素-蛋白酶体途径（UPP）降解的。在这些研究中，很明显，IP 3Rs是由ERAD途径（UPP的一个方面，负责处理异常ER蛋白）降解的，并且鉴于其独特的性质，IP 3Rs可能是鉴定哺乳动物细胞中介导ERAD的蛋白质和机制的特别有用的工具。因此，在最后一个研究周期中，我们试图鉴定和表征介导IP 3R ERAD的蛋白质和机制，并发现与活化的IP 3R连接的泛素缀合物是出乎意料的复杂的，并且发现了一种完全新颖的，非常大（> 1 MDa）的复合物，由两种未表征的ER膜蛋白组成，称为SPFH 1和2，介导激活的IP 3R的ERAD并参与模型ERAD底物的降解。有趣的是，我们最近还发现RNF 170，一种未表征的，假定的泛素连接酶，与SPFH 1/2复合物强烈相互作用。目前的建议旨在扩大这项工作。与过去一样，大部分工作将在哺乳动物细胞中的内源性蛋白质上进行，以确保获得的结果密切反映生理情况。本提案的具体目的是（i）鉴定和表征控制活化IP 3R上泛素缀合物水平的蛋白质，以及（ii）研究SPFH 1/2复合物的结构和功能。这些目标之所以重要，主要有三个原因。首先，目标1中关于IP 3R泛素化的细节的研究，应该有助于解决最近揭示的UPP底物如何以及为什么被不同的泛素缀合物修饰的谜团。其次，目标2应该对最近发现但知之甚少的SPFH 1/2复合物的结构和功能有更清楚的了解。第三，我们对哺乳动物细胞中内源性蛋白的关注意味着所获得的结果将准确地模拟体内情况，并将提供关于近生理条件下ERAD途径的信息。这项工作的结果应该对我们理解ERAD和UPP产生重大影响，并有望促进旨在开发针对这些途径的药物的努力。公共卫生关系：通过蛋白酶体降解蛋白质，这是由另一种蛋白质称为泛素，是一个基本的细胞过程，控制许多关键蛋白质的水平，并有巨大的潜力作为疾病的治疗目标，如囊性纤维化，神经病，糖尿病和癌症。我提出的研究将在哺乳动物细胞中调查一些支持这一过程的机制，目的是更清楚地了解这一领域。