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.

项目摘要 /摘要 基本原理：所有细胞产生不完全的蛋白质，这是基因中常见误差的结果 表达。细胞已经发展了多种机制，以抑制不完整蛋白的表达，包括 含有过早终止密码子（无义变体）的mRNA降解。这种废话介导的 mRNA质量控制发生在原核生物和真核生物中。细菌过程接地 RHO和随后的RNA降解过早终止转录，这是一种负责紧密的机制 转录和翻译的耦合。废话介导的质量的存在和明显的宇宙 已经采取控制表明不完全的蛋白质是高度细胞毒性的。但是，该提议有 没有系统地研究。此外，李实验室的最新工作揭示了一个广阔的片段 细菌 - 尤其是枯草芽孢杆菌和其他坚硬（杆菌） - 完全缺乏胡说八道介导的质量 控制和转录翻译耦合。在最近的其他工作中，我开发了一种大规模的方法 通过蛋白质片段的复杂文库抑制细菌生长的平行测量，源自 阵列合成的寡核苷酸。这种方法论创新提供了先前缺少的工具 需要系统地研究不完全蛋白质的细胞毒性。在这个建议中，我将这些汇总在一起 概念和技术突破以及计算分析和其他完整性 方法，以解决有关不完整蛋白质的物理影响的基本问题和 我假设大多数不完整的蛋白质成型都是无害的，但是子集 是细胞毒性的，并且枯草芽孢杆菌蛋白序列已演变成限制截短的变异毒性。我也会 研究不完全蛋白质蛋白对核糖体抑制性抗生素的生物学活性的贡献， 由于缺乏过早的终止密码子突变体的抑制，进化途径可以实现。 目的：要解决这些问题，我将系统地定义不完整蛋白质的细胞毒性 测量来自枯草芽孢杆菌和大肠杆菌蛋白的不完全蛋白质的生长速率影响， 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质成型。我也会 确定蛋白质不完全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和蛋白质组学测量与 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 通过使用敲除库展示过早的伪元，伪基因基因生成的作用 功能并采用生物信息学分析来确定重复和截断的流行率 通往新基因的路径。最后，我将使用高通量测定法来系统地绘制景观 潜在有益的截断变体。