Cytotoxicity and function of incomplete proteins

项目总结/摘要 原理：所有细胞都产生不完整的蛋白质，这是基因突变中令人惊讶的常见错误的结果。 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（无义变体）的mRNA的降解。这种无意义介导的 mRNA质量控制发生在原核生物和真核生物中。细菌的过程是基于 Rho导致的转录提前终止和随后的RNA降解，这是一种依赖于紧密结合的机制。 转录和翻译的耦合。无意义介导性的存在及其明显的普遍性 已经进行了对照，以表明不完整的蛋白质是高度细胞毒性的。然而，这一主张 没有进行系统的调查。此外，Li实验室最近的工作表明， 尤其是B。枯草杆菌和其他厚壁菌门（芽孢杆菌门）-完全缺乏无义介导的质量 控制和转录-翻译偶联。在最近的其他工作中，我开发了一种方法， 通过蛋白质片段的复杂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少的工具 需要系统地研究不完整蛋白质的细胞毒性。在这个建议中，我把这些 概念和技术突破，沿着计算分析和其他补充 方法，以解决有关不完整蛋白质的生理影响的基本问题， 镇压的重要性。我假设大多数不完整的蛋白质是无害的，但一个子集 是有细胞毒性的，而B.枯草芽孢杆菌蛋白质序列已经进化以限制截短的变体毒性。我也会 研究不完整蛋白质对核糖体抑制抗生素杀菌活性的贡献， 由于缺乏对过早终止密码子突变体的抑制而实现的进化途径。 目的：为了解决这些问题，我将系统地定义不完整蛋白质的细胞毒性， 测量来自整个B的不完整蛋白质的生长速率效应。subtilis和E.大肠杆菌蛋白质组， 确定与细胞毒性相关的蛋白质特征，并进一步研究 使用核糖体分析、RNA测序和下拉质谱法选择蛋白形式。我也会 确定不完全蛋白质如何有助于核糖体抑制抗生素的杀菌活性， 将药物诱导的不完全蛋白质产生的翻译组学和蛋白质组学测量与 大规模平行细胞毒性测量。在第三种方法中，我将重新定义功能和进化 利用敲除文库展示厚壁菌门中的早熟终止假基因 功能，并采用生物信息学分析，以确定复制和截断的流行率， 通往新基因的道路最后，我将使用高通量分析系统地绘制 潜在有益的截短变体。

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.