Mechanism and Function of Cotranslational Ubiquitylation

项目总结/摘要 蛋白质质量控制（QC）机制存在于真核细胞中以监测和清除错误折叠， 受损或聚集的蛋白质，以及质量控制系统的缺陷被认为是几种疾病的病因。 疾病状态，特别是神经变性疾病，癌症，心血管，代谢疾病， 衰老许多蛋白质QC系统在蛋白质生命的最早阶段起作用，确保 新合成的蛋白质达到成熟和功能状态。虽然泛素-蛋白酶体系统 (UPS)长期以来被认为在蛋白质质量控制中起着重要作用，这通常被认为是 在尝试正确折叠新合成的蛋白质失败后发生。相比之下， 令人惊讶的发现是，新生多肽的泛素化也发生在主动翻译中， 复合物，基本上是在蛋白质合成完成之前就将其破坏。共- 翻译泛素化（CTU）是人类细胞中令人惊讶的强大过程，大约10%的细胞在翻译过程中被泛素化。 在活跃的翻译复合物中被泛素化的新生链。氨基的掺入 酸类似物进入新生链，以及Hsp 70抑制剂，导致CTU急剧增加，强烈 这表明CTU反映了一种蛋白质QC途径，其靶向异常或错误折叠的新生 多肽。重要的是，CTU不同于发生在细胞中的新生链的泛素化。 有缺陷的和分解的核糖体复合物的背景（“RQC”核糖体质量控制 途径，依赖于Ltn 1泛素连接酶）。本提案的目标是描述 CTU途径的泛素连接酶，靶点，影响因素和潜在的 调节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，通过生物化学和遗传学方法，2） 通过蛋白质组学方法表征CTU的优先靶点，以确定特征 的新生多肽，这是承认这个QC系统，和3）确定的因素， 影响和管理CTU这将极大地影响我们对 蛋白质合成和蛋白质质量控制，以及蛋白质稳态 特别关键。

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.