Characterization of the Abundant Small Proteome of Mycobacteria

分枝杆菌丰富的小蛋白质组的表征

• 批准号: 9090002
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 19.43万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9090002
• 关键词: 5' Untranslated Regions; Amino Acids; Animal Model; Appearance; Architecture; Bacteria; Bacterial Genes; Biological; Biology; Cell division; Cell physiology; Cells; Characteristics; Chronic; Clinical; Codon Nucleotides; Communities; Coupled; Coupling; Cysteine; Cytoplasm; Data; Disease; Drug resistance; Enzymes; Escherichia coli; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Genus Mycobacterium; Health; Knowledge; Laboratories; Maps; Mass Spectrum Analysis; Measures; Membrane; Methodology; Molecular Genetics; Molecular Profiling; Multi-Drug Resistance; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Open Reading Frames; Operon; Pathway interactions; Pharmaceutical Preparations; Play; Preparation; Proteins; Proteome; Proteomics; Public Health; RNA; Reading; Reading Frames; Regulation; Reporter; Resolution; Resources; Role; Seeds; Signal Transduction; Single Nucleotide Polymorphism Map; Site; Technology; Testing; Transcription Initiation; Transcription Initiation Site; Translating; Translation Initiation; Translations; Transmembrane Transport; Tuberculosis; Vaccines; Work; design; field study; genetic approach; genetic makeup; genome annotation; genome-wide; global health; innovation; insight; intercellular communication; killings; liquid chromatography mass spectrometry; mycobacterial; novel; pathogen; protein folding; public health relevance; research study; resistant strain; ribosome profiling; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): Mycobacterial disease, primarily tuberculosis, kills nearly two million people annually. Ineffective vaccines, as well as multi-drug and extremely-drug resistant strains of Mycobacterium tuberculosis, exacerbate this chronic global crisis. The design of new efficacious drugs necessitates a fundamental understanding of the biology of the bacterium and its genetic make-up. In turn, the successful application of genomic sequence information requires accurate gene annotation and a comprehensive knowledge of gene architecture and expression profiles. We have empirically determined transcription and translation initiation sites on a genome scale, using RNA- seq and ribosomal profiling (ribo-seq). This work has revealed two novel characteristics of mycobacterial gene architecture: one-third of transcription start sites lack a 5' UTR (leaderless genes lacking a Shine- Dalgarno sequence), and the presence of many hundreds of small open-reading-reading frames encoding small proteins of less than 50 amino acids (sproteins). To date, sproteins have been largely overlooked in bacteria, as they are hard to detect by traditional methodologies or annotation pipelines. Moreover, sproteins represent a completely unexplored class of proteins in mycobacteria, and are likely to have a significant impact on cell physiology. Importantly, our preliminary studies indicate that sproteins are present in large numbers in both the fast-growing model-organism, Mycobacterium smegmatis, and the slow- growing pathogen, M. tuberculosis, Here, we propose to generate the first comprehensive, experimentally validated small proteome for both slow- and fast- growing Mycobacteria. This will be achieved by combining state-of-the art sproteome mass spectrometry approaches with ribo-seq and transcription start site mapping. Together, these approaches will provide an empirically defined, high-confidence data resource for the mycobacterial community. In addition, we will define the mechanism of action for a subset of the sproteins that we hypothesize act in cis to regulate downstream genes. This proposal is highly innovative as it focuses on the discovery of an entirely unexpected class of abundant proteins that has escaped scientific scrutiny for any bacterium. The application of high-throughput, cutting- edge tools to facilitate this analysis will provide a comprehensive overview of the mycobacterial sproteome, while also providing mechanistic insight into potential functions of sproteins. Thus, we anticipate both an immediate and long-term impact on the mycobacterial field, providing new biological insights that will seed multiple emerging fields of study, while expanding our knowledge of gene architecture and regulation for all bacteria.

 描述(申请人提供)：分枝杆菌病，主要是结核病，每年导致近200万人死亡。无效的疫苗，以及多种药物和极端耐药的结核分枝杆菌菌株，加剧了这一长期的全球危机。设计新的有效药物需要对细菌的生物学及其基因构成有基本的了解。反过来，基因组序列信息的成功应用需要准确的基因注释和全面的基因结构和表达谱知识。我们使用RNA-SEQ和核糖体图谱(RIBO-SEQ)在基因组水平上经验地确定了转录和翻译起始点。这项工作揭示了分枝杆菌基因结构的两个新特征：三分之一的转录起始点缺乏5‘UTR(无领导基因缺乏Shine-Dalgarno序列)，以及数百个编码小于50个氨基酸的小蛋白(s蛋白)的小开放阅读框架的存在。迄今为止，s蛋白质在细菌中很大程度上被忽视了，因为它们很难通过传统的方法或注释管道检测到。此外，s蛋白质代表了分枝杆菌中一类完全未被探索的蛋白质，并可能对细胞生理学产生重大影响。重要的是，我们的初步研究表明，s蛋白质在快速生长的模式生物--污垢分枝杆菌和生长缓慢的病原体--结核分枝杆菌中都大量存在。在这里，我们建议为慢速和快速生长的分枝杆菌生成第一个全面的、经实验验证的小蛋白质组。这将通过将最先进的蛋白质组质谱方法与RIBO-SEQ和转录起始位点作图相结合来实现。总而言之，这些方法将为分枝杆菌群落提供一个经验性定义的、高度可信的数据资源。此外，我们还将定义我们假设的作用于顺式调节下游基因的s蛋白质子集的作用机制。这一提议具有很高的创新性，因为它专注于发现一类完全出人意料的丰富蛋白质，这种蛋白质逃脱了任何细菌的科学审查。应用高通量、尖端的工具来促进这一分析将提供对分枝杆菌s蛋白质组的全面概述，同时也提供对s蛋白质潜在功能的机械洞察。因此，我们预计对分枝杆菌领域将产生即时和长期的影响，提供新的生物学见解，将为多个新兴研究领域提供种子，同时扩大我们对所有细菌的基因结构和调控的知识。