Protein quality control at eukaryotic membranes.

内质网相关降解（ERAD）是内质网蛋白降解的过程， 因为它们是错误折叠的，或者因为它们的降解是受生理学调节的。的标志性基质 该过程是HMG-CoA还原酶，一种催化胆固醇合成中限速步骤的酶。在 除了降解代谢调节或错误折叠的蛋白质外，ERAD还作为应激反应系统发挥作用 缓解急诊室压力虽然其正常功能是靶向错误折叠或调节的蛋白质，但ERAD也是 被细菌和病毒病原体利用以进入细胞质。在正常ERAD期间，蛋白质底物 在内质网腔内被选择，穿过膜移动到胞质溶胶（逆向易位），在那里它们 被多泛素化，从膜中提取并被蛋白酶体降解。从生理上讲- 选择受调节的底物进行降解，并在ERAD的控制下选择特定的细胞途径 是模棱两可的。最近的研究表明，一个中心的，保守的泛素连接酶称为Hrd 1，形成一个 泛素门控的蛋白传导通道，足以允许错误折叠的管腔蛋白的反向易位。 和完整的膜蛋白。自动量化门控机制提出了一个难题;在正常情况下， 在某些情况下，泛素化将导致修饰蛋白的降解。然而，Hrd 1相对稳定 这意味着存在未鉴定的机制来保护autoubiquitinated Hrd 1免于降解， 逆转泛素门控激活。沿着Hrd 1的调节，许多相关过程包括 ERAD对底物选择是神秘的。本提案的目标是：1）定义ERAD系统如何 2）确定ERAD系统如何识别其目标，3）定义目标引导的特征 4）理解膜如何有助于ERAD功能，以及5）识别 其他（非ERAD）完整膜蛋白质质量控制系统的细胞功能。我们将使用一个 生物化学，细胞生物学，遗传学和蛋白质组学的多方面方法来解决这些核心问题 膜相关蛋白质质量控制中的问题。我们将利用我们独特的体内和体外试验 (and继续设计创新的检测方法）来剖析这些真核系统的基本机制。一 对系统如何运作（包括ERAD）及其调节过程的机械理解将 将这些系统作为蛋白质错误折叠的医学相关途径中的可行治疗靶点， 蛋白质失调和病原体劫持

Endoplasmic reticulum-associated degradation (ERAD) is a process in which ER proteins are degraded, either because they are misfolded or because their degradation is physiologically regulated. The hallmark substrate of this process is HMG-CoA reductase, an enzyme catalyzing the rate-limiting step in cholesterol synthesis. In addition to degrading metabolically-regulated or misfolded proteins, ERAD functions as stress-response system to alleviate ER stress. While its normal function is to target misfolded or regulated proteins, ERAD is also exploited by bacterial and viral pathogens to gain access to the cytosol. During normal ERAD, protein substrates are selected within the ER lumen, moved across the membrane to the cytosol (retrotranslocation), where they are polyubiquitinated, extracted from the membrane and degraded by the proteasome. How physiologically- regulated substrates are selected for degradation and the specific cellular pathways under the control of ERAD are ambiguous. Recent work has demonstrated that a central, conserved ubiquitin ligase called Hrd1, forms a ubiquitin-gated protein-conducting channel that that is sufficient to allow retrotranslocation of misfolded lumenal and integral membrane proteins. The autoubiquitination-gating mechanism presents a conundrum; under normal circumstances, ubiquitination will result in degradation of the modified protein. However, Hrd1 is relatively stable meaning there are unidentified mechanisms in place to protect autoubiquitinated Hrd1 from degradation and to reverse the ubiquitin-gated activation. Along with the regulation of Hrd1, many associated processes including substrate selection by ERAD are mysterious. The goals of this proposal are to 1) define how the ERAD system is regulated, 2) determine how the ERAD system recognizes its targets, 3) define the features of targets directing them to ERAD (the degrons), 4) understand how the membrane contributes to ERAD function, and 5) identify the cellular function of other (non-ERAD) integral membrane protein quality control systems. We will use a multifaceted approach with biochemistry, cell biology, genetics, and proteomics to address these central questions in membrane-associated protein quality control. We will leverage our unique in vivo and in vitro assays (and continue to design innovative assays) to dissect the basic mechanisms of these eukaryotic systems. A mechanistic understanding of how the systems function (including ERAD) and the processes they regulate will establish these systems as viable therapeutic targets in medically relevant pathways of protein misfolding, protein misregulation, and pathogen hijacking.