Translational Rescue Mechanisms in Eukaryotes

项目摘要 正确折叠和功能蛋白质的合成对于维持健康至关重要。 细胞的状态。真核细胞已经进化出多种质量控制（QC）机制， 识别并消除生命周期不同阶段的异常多肽。一名该等 监视机制，共翻译蛋白质QC，靶向含有异常的核糖体， 新生多肽，并促进具有新生链连接的60 S亚基的释放。的 然后，异常多肽通过E3泛素连接酶Ltn 1进行泛素化， 由ATP酶Cdc 48促进的蛋白酶体降解。虽然Ltn 1-Cdc 48依赖性 最近研究了这一QC途径的一段，确定了上游作用的因子， 这一途径仍然不确定。 我们最近发现，典型的释放因子eRF 1-eRF 3（eRF）是 负责产生大多数含有新生多肽的60 S-相关复合物， 通过Ltn 1-Cdc 48途径消除。因此，eRF在共-共-期间实现两个新颖功能。 翻译蛋白QC：它们启动异常多肽的清除并解决翻译停滞， 从而允许翻译继续。我们还发现，在蛋白质质控出现故障的情况下， 或不堪重负时，未解决的翻译停滞的积累触发了特异性内切核酸裂解酶， 核糖体大亚基的25 S rRNA中的切割。 我们的中心假设是eRF介导的核糖体再循环和停滞的核糖体的切割， 核糖体是真核生物翻译回收机制（TREM）的两个关键部分， 保护翻译装置免于末端核糖体停滞和防止 异常多肽通过这种方式，TREM可能在防御 翻译和蛋白毒性应激。本提案的总体目标是深入了解 利用生物化学、遗传学和分子生物学方法研究真核TREM的分子基础 在酿酒酵母中。在本研究中，我们将（1）定义eRF介导的客户端 （2）阐明eRF 1在翻译拯救途径中的作用机制 拯救;和（3）评估rRNA切割在从翻译停滞恢复中的潜在作用。 除了扩大我们对eRF 1-eRF 3在共同作用过程中发挥作用的机制的了解外， 翻译蛋白QC，这项研究将揭示一个重要的系统，保护真核细胞 在蛋白毒性应激中。因为我们知道， 当蛋白质QC功能障碍时，有害蛋白质聚集体的积累和翻译缺陷 或超载，这项研究可能会打开以前未探索的治疗干预途径。

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.