Cytotoxicity and function of incomplete proteins

项目摘要/摘要 原理：所有细胞都会产生不完整的蛋白质，这是基因中常见的惊人错误的结果 表情。细胞已经进化出多种机制来抑制不完整蛋白质的表达，包括 含有提前终止密码子(无意义变体)的mRNAs的降解。这种胡说八道的中介 原核生物和真核生物都存在着信使核糖核酸的质量控制。细菌的过程植根于 Rho和随后的RNA降解导致转录提前终止，这是一种依赖于紧密连接的机制 翻译与转录的耦合。无意义中介性质的存在及其表观普遍性 对照研究表明，不完全蛋白质具有高度的细胞毒性。然而，这一命题具有 没有得到系统的调查。此外，李实验室最近的工作揭示了一大片 细菌--特别是枯草杆菌和其他细菌(芽孢杆菌)--完全缺乏无意义的介导性。 调控和转录-翻译耦合。在最近的其他工作中，我开发了一种方法 通过来自以下来源的复杂蛋白质片段文库平行测量细菌生长抑制 阵列合成的寡核苷酸。这一方法创新提供了以前缺失的工具 需要系统地研究不完全蛋白质的细胞毒性。在这份提案中，我将这些 概念和技术突破，以及计算分析和其他补充 方法，以解决关于不完全蛋白质的生理影响和 他们镇压的重要性。我假设大多数不完整的蛋白质形式是无害的，但只是一个子集 是细胞毒性的，枯草杆菌蛋白序列已经进化到限制截断的变异毒性。我也会 研究不完全蛋白质对核糖体抑制抗生素杀菌活性的贡献，以及 由于缺乏对过早终止密码子突变的抑制而导致的进化途径。 目的：为了解决这些问题，我将通过以下方法系统地定义不完全蛋白质的细胞毒性 测量来自枯草杆菌和大肠杆菌蛋白质组的不完全蛋白质的生长速度效应， 确定与细胞毒性相关的蛋白质特性，并进一步研究 使用核糖体图谱、RNA测序和下拉式质谱学选择蛋白质组。我也会 通过以下方法确定不完全蛋白质如何影响核糖体抑制抗生素的杀菌活性 结合药物诱导的不完全蛋白质产生的翻译基因组学和蛋白质组学测量 大量平行的细胞毒性测量。在第三种方法中，我将重新定义功能和进化 利用基因敲除文库研究假基因在早停假基因中的作用 功能，并使用生物信息学分析来确定复制和截断作为 通向新基因的道路。最后，我将使用高通量分析来系统地绘制 可能有益的截断变体。

PROJECT SUMMARY / ABSTRACT Rationale: All cells produce incomplete proteins, a consequence of surprisingly common errors in gene expression. Cells have evolved multiple mechanisms to suppress expression of incomplete proteins, including the degradation of mRNAs containing premature stop codons (nonsense variants). Such nonsense-mediated mRNA quality control occurs in both prokaryotes and eukaryotes. The bacterial process is grounded in premature termination of transcription by Rho and subsequent RNA degradation, a mechanism reliant on tight coupling of transcription and translation. The existence and apparent universality of nonsense-mediated quality control has been taken to suggest that incomplete proteins are highly cytotoxic. However, this proposition has not been systematically investigated. Furthermore, recent work from the Li lab has revealed that a broad swath of bacteria – notably B. subtilis and other Firmicutes (Bacillota) – altogether lack nonsense-mediated quality control and transcription-translation coupling. In other recent work, I developed an approach for massively parallel measurements of bacterial growth inhibition by complex libraries of protein fragments, derived from array-synthesized oligonucleotides. This methodological innovation provides the previously missing tool needed to systematically investigate cytotoxicity of incomplete proteins. In this proposal I bring together these conceptual and technological breakthroughs, along with computational analyses and other complementary methods, to address fundamental questions about the physiological impacts of incomplete proteins and the importance of their suppression. I hypothesize that most incomplete proteoforms are innocuous, but a subset are cytotoxic, and that B. subtilis protein sequences have evolved to limit truncated variant toxicity. I will also investigate the contributions of incomplete proteins to bactericidal activity of ribosome-inhibitory antibiotics, and the evolutionary pathways enabled by a lack of suppression of premature stop codon mutants. Objective: To address these questions, I will systematically define the cytotoxicity of incomplete proteins by measuring growth rate effects of incomplete proteins from across the B. subtilis and E. coli proteomes, determining protein features associated with cytotoxicity, and further investigating physiological effects of select proteoforms using ribosome profiling, RNA sequencing, and pull-down mass spectrometry. I will also determine how incomplete proteins contribute to bactericidal activity of ribosome-inhibiting antibiotics by combining translatomic and proteomic measurements of drug-induced incomplete protein production with massively parallel cytotoxicity measurements. In a third approach, I will redefine the functions and evolutionary roles of pseudogenes in Firmicutes by using a knockout library to demonstrate premature-stop pseudogene function and employing bioinformatic analyses to determine the prevalence of duplication and truncation as a path to novel genes. Finally, I will use a high-throughput assay to systematically map the landscape of potentially beneficial truncation variants.