Translational Rescue Mechanisms in Eukaryotes

真核生物的转化救援机制

• 批准号: 9176399
• 负责人: Natalia Shcherbik
• 金额: $ 31.4万
• 依托单位: ROWAN UNIVERSITY SCHOOL/OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9176399
• 关键词: ATP phosphohydrolase; Address; Binding; Biochemical; Biochemical Genetics; Cardiovascular Diseases; Cell physiology; Cells; Cleaved cell; Client; Complex; Controlled Study; Disease; Eukaryota; Eukaryotic Cell; Event; Excision; Face; Goals; Human; Knowledge; Light; Longevity; Malignant Neoplasms; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Genetics; Neurodegenerative Disorders; Pathway interactions; Peptides; Physiological; Play; Positioning Attribute; Process; Production; Protein Biosynthesis; Proteins; Proteome; Quality Control; RNA, ribosomal, 25S; Reading Frames; Recovery; Recycling; Reporter; Ribosomal RNA; Ribosomes; Role; Saccharomyces cerevisiae; Sense Codon; Site; Staging; Stress; System; Techniques; Terminator Codon; Testing; Therapeutic Intervention; Time; Translations; Ubiquitin; Ubiquitination; Yeasts; Zinc Fingers; base; genetic approach; insight; multicatalytic endopeptidase complex; mutant; novel; novel therapeutic intervention; particle; polypeptide; prevent; protein aggregate; protein aggregation; public health relevance; release factor; response; ubiquitin-protein ligase

PROJECT SUMMARY Synthesis of the correctly folded and functional proteins is crucial for maintaining the healthy state of the cell. Eukaryotic cells have evolved a variety of quality control (QC) mechanisms that recognize and eliminate abnormal polypeptides at different stages of their lifespan. One of such surveillance mechanisms, the co-translational protein QC, targets stalled ribosomes containing aberrant nascent polypeptides and promotes the release of the 60S subunits with a nascent chain attached. The aberrant polypeptides then undergo ubiquitination by the E3 ubiquitin ligase Ltn1 followed by proteasomal degradation facilitated by the ATPase Cdc48. Although the Ltn1-Cdc48-dependent segment of this QC pathway has been recently investigated, the identity of factors acting upstream in this pathway remains uncertain. We have recently discovered that the canonical release factors eRF1-eRF3 (eRFs) are responsible for generating most of the 60S-associated complexes containing nascent polypeptides that are eliminated through the Ltn1-Cdc48 pathway. Thus, eRFs accomplish two novel functions during co- translational protein QC: they initiate clearance of aberrant polypeptides and resolve translational stalls, thereby allowing translation to continue. We also found that in situations when protein QC malfunctions or is overwhelmed, accumulation of unresolved translational stalls triggers a specific endonucleolytic cleavage in the 25S rRNA of the large ribosomal subunit. Our central hypothesis is that eRF-mediated ribosome recycling and cleavage of stalled ribosomes constitute two key parts of the eukaryotic Translation REscue Mechanism (TREM), devoted to protecting the translational apparatus from terminal ribosomal stalling and preventing the escape of aberrant polypeptides. In this way, TREM likely plays a fundamental role in the defense against translational and proteotoxic stress. The broad objective of the current proposal is to obtain insight into the molecular basis of eukaryotic TREM using biochemical, genetic and molecular biology approaches in the yeast Saccharomyces cerevisiae. In this study, we will (1) define clients of the eRF-mediated translational rescue pathway; (2) elucidate the mechanistic function of eRF1 during translational rescue; and (3) evaluate the potential role of rRNA cleavage in the recovery from translational stalls. In addition to expanding our knowledge of the mechanisms of eRF1-eRF3 functioning during co- translational protein QC, this study will shed light on a crucial system that protects eukaryotic cells during proteotoxic stress. Because a number of pathological consequences are known to result from accumulation of harmful protein aggregates and defective translation when protein QC is dysfunctional or overloaded, this study may open previously unexplored avenues for therapeutic intervention.

项目总结 正确折叠和功能蛋白质的合成对维持健康至关重要 单元的状态。真核细胞已经进化出各种质量控制(QC)机制， 识别和消除生命周期不同阶段的异常多肽。这样的人之一 监控机制，共翻译蛋白QC，针对含有异常的停滞的核糖体 新生多肽，并促进60S亚基的释放，并与新生链连在一起。这个 异常多肽随后被E3泛素连接酶Ltd n1泛素化，随后 由ATPase CDC48促进的蛋白酶体降解。尽管Ltd.-CDC48依赖于 最近对这一QC途径的片段进行了研究，确定了作用于 这条道路仍然不确定。 我们最近发现，标准释放因子eRF1-eRF3(ERF)是 负责产生大多数与60S相关的包含新生多肽的复合体，这些多肽 通过Ltn1-CDC48途径被消除。因此，ERF在协同作用过程中完成了两个新的功能。 翻译蛋白QC：它们启动异常多肽的清除并解决翻译停滞， 从而允许继续进行翻译。我们还发现，在蛋白质质量控制出现故障的情况下 或者是不堪重负，未解决的翻译停滞的积累触发了特定的核酸内切作用 大核糖体亚基的25S rRNA裂解。 我们的中心假设是ERF介导的核糖体循环和切割停滞不前 核糖体构成真核生物翻译救援机制(TREM)的两个关键部分，致力于 保护翻译装置不受核糖体末端停滞的影响，防止 异常多肽。在这种情况下，特雷姆很可能在防御中发挥重要作用。 翻译和蛋白毒性应激。当前提案的广泛目标是深入了解 用生化、遗传学和分子生物学方法研究真核细胞TREM的分子基础 在酿酒酵母中。在这项研究中，我们将(1)定义ERF中介的客户 翻译救援途径；(2)阐明eRF1在翻译过程中的作用 拯救；以及(3)评估rRNA切割在从翻译停滞中恢复的潜在作用。 除了扩大我们对eRF1-eRF3在协同作用过程中的作用机制的了解 翻译蛋白QC，这项研究将阐明保护真核细胞的关键系统 在蛋白毒性应激期间。因为已知的一些病理后果是由 当蛋白质QC功能失调时有害蛋白质聚集物的积累和翻译缺陷 或者超负荷，这项研究可能会打开以前从未探索过的治疗干预途径。