Mechanism and Function of Cotranslational Ubiquitylation

项目摘要/摘要 真核细胞中存在蛋白质质量控制(QC)机制来监测和清除错误折叠， QC系统中受损的或聚集的蛋白质和缺陷被认为是几种 疾病状态，特别是神经退行性疾病、癌症、心血管疾病、代谢性疾病， 衰老。许多蛋白质质量控制系统在蛋白质生命的早期阶段起作用，确保 新合成的蛋白质达到成熟和功能状态。而泛素-蛋白酶体系统 (UPS)长期以来一直被认为在蛋白质质控中发挥重要作用，这通常被认为是 在正确折叠新合成的蛋白质的尝试失败后发生。相比之下，我们制作了 新生多肽的泛素化也发生在主动翻译中，这一令人惊讶的发现 复合体，本质上是在蛋白质合成完成之前标记要破坏的蛋白质。共同-- 翻译泛素化(CTU)在人类细胞中是一个令人惊讶的强大过程，大约有10% 新生的链在活跃的翻译复合体中泛素化。氨基的掺入 新生链的酸性类似物，以及Hsp70抑制剂，导致CTU急剧增加，强烈 提示CTU反映了以异常或错误折叠的新生细胞为靶点的蛋白质QC途径 多肽。重要的是，CTU不同于发生在 有缺陷和拆解的核糖体复合体的背景(“RQC”核糖体质量控制 途径，依赖于Ltn1泛素连接酶)。这项提案的目标是描述 CTU途径与泛素连接酶、靶点、影响因素和潜在的 规范CTU，并评估CTU的生物学重要性。具体目标是1)确定 CTU泛素连接酶，在酵母和人类细胞中，通过生化和遗传方法，2) 用蛋白质组学方法鉴定CTU的优先靶点以确定其特征 由该QC系统识别的新生多肽，以及3)决定 影响和规范CTU。这将显著影响我们对两国关系的理解。 蛋白质合成和蛋白质质量控制，与蛋白质平衡所在的疾病状态有关 尤其关键。

Project Summary/Abstract Protein quality control (QC) mechanisms exist in eukaryotic cells to monitor and clear misfolded, damaged, or aggregated proteins, and defects in QC systems are recognized to be causative in several disease states, particularly neurodegenerative disorders, cancer, cardiovascular, metabolic diseases, aging. Many protein QC systems function at the earliest stages of the life of a protein, ensuring that newly synthesized proteins reach a mature and functional state. While the ubiquitin-proteasome system (UPS) has long been recognized to play an important role in protein QC, this was generally assumed to occur after unsuccessful attempts at correctly folding newly synthesized proteins. In contrast, we made the surprising discovery that ubiquitylation of nascent polypeptides also occurs within active translation complexes, essentially marking proteins for destruction before their synthesis is even complete. Co- translation ubiquitylation (CTU) is a surprisingly robust process in human cells, with approximately 10% of nascent chains being ubiquitylated within active translation complexes. The incorporation of amino acid analogs into nascent chains, as well as Hsp70 inhibitors, leads to sharp increases in CTU, strongly suggesting that CTU reflects a protein QC pathway that targets aberrant or misfolded nascent polypeptides. Importantly, CTU is distinct from ubiquitylation of nascent chains that occurs in the context of defective and disassembled ribosome complexes (the “RQC” Ribosome Quality Control pathway, dependent on the Ltn1 ubiquitin ligase). The goals of this proposal are to characterize the CTU pathway with respect to the ubiquitin ligases, the targets, the factors that influence and potentially regulate CTU, and to assess the biological importance of CTU. The specific aims are to 1) identify the CTU ubiquitin ligases, in both yeast and human cells, through biochemical and genetic approaches, 2) characterize the preferential targets of CTU by proteomic approaches in order to determine the features of nascent polypeptides that are recognized by this QC system, and 3) determine the factors that influence and regulate CTU. This will significantly impact our understanding of the relationship between protein synthesis and protein quality control, with implications for disease states where proteostasis is particularly critical.