Cytotoxicity and function of incomplete proteins

不完整蛋白质的细胞毒性和功能

• 批准号: 10570685
• 负责人: Andrew Savinov
• 金额: $ 12.4万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570685
• 关键词: Address; Amino Acid Sequence; Aminoglycosides; Antibiotics; Bacillus subtilis; Bacteria; Bioinformatics; Cells; Code; Combined Antibiotics; Complex; Computer Analysis; Coupling; DNA; Data; Dominant-Negative Mutation; Drug toxicity; Escherichia coli; Eukaryota; Evolution; Exhibits; Firmicutes; Gene Expression; Gene Family; Genes; Genetic Transcription; Growth; Knock-out; Laboratories; Libraries; Link; Macrolides; Maps; Mass Spectrum Analysis; Measurement; Measures; Mediating; Messenger RNA; Methodology; Methods; Mutation; Nonsense Codon; Oligonucleotides; Open Reading Frames; Organism; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Physiological; Prevalence; Process; Production; Prokaryotic Cells; Protein Biosynthesis; Protein Fragment; Protein Truncation; Proteins; Proteome; Proteomics; Pseudogenes; Puromycin; Quality Control; RNA Degradation; Resolution; Ribosomes; Role; Techniques; Technology; Testing; Toxic effect; Translational Repression; Translations; Variant; Work; bactericide; cytotoxic; cytotoxicity; high throughput screening; human disease; innovation; mRNA Transcript Degradation; mutant; novel; premature; rho; ribosome profiling; tool; transcription termination; transcriptome sequencing; translatome

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.

项目概要/摘要 基本原理：所有细胞都会产生不完整的蛋白质，这是基因中令人惊讶的常见错误的结果 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 含有过早终止密码子（无义变体）的 mRNA 的降解。 mRNA 质量控制发生在原核生物和真核生物中。细菌过程基于细菌过程。 Rho 过早终止转录以及随后的 RNA 降解，这是一种依赖于紧密关系的机制 转录和翻译的耦合。无意义介导的质量的存在和明显的普遍性。 已采取控制措施表明不完整的蛋白质具有高度细胞毒性，但这一主张已被证实。 此外，李实验室最近的工作表明，广泛的范围。 细菌——显着的枯草芽孢杆菌和其他厚壁菌门（芽孢杆菌）——完全缺乏无意义介导的质量 在最近的其他工作中，我开发了一种大规模的方法。 通过复杂的蛋白质片段文库平行测量细菌生长抑制，源自 这种方法创新提供了以前缺失的工具。 需要系统地研究不完整蛋白质的细胞毒性。在本提案中，我将这些结合在一起。 概念和技术突破，以及计算分析和其他补充 方法，解决有关不完整蛋白质的生理影响和 我急忙说，大多数不完整的蛋白质形式是无害的，但只是其中的一个子集。 具有细胞毒性，并且枯草芽孢杆菌蛋白质序列已经进化到限制截短变体的毒性。 研究不完整蛋白质对核糖体抑制性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体的抑制而实现的进化途径。 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整蛋白质的细胞毒性： 测量枯草芽孢杆菌和大肠杆菌蛋白质组中不完整蛋白质的生长率影响， 确定与细胞毒性相关的蛋白质特征，并进一步研究其生理效应 我还将使用核糖体分析、RNA 测序和下拉质谱来选择蛋白质形式。 确定不完整蛋白质如何促进核糖体抑制抗生素的细菌活性 将药物诱导的不完全蛋白质生产的翻译原子学和蛋白质组学测量与 在第三种方法中，我将重新定义功能和进化。 通过使用敲除文库来证明假基因在厚壁菌门中的作用，以证明过早停止的假基因 功能并采用生物信息学分析来确定重复和截断的普遍程度 最后，我将使用高通量系统地绘制检测分析图谱。 潜在有益的截断变体。