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: 8949153
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 23.62万
• 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 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8949153
• 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; 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; Ribosomes; 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; 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（由申请人提供）：分枝杆菌疾病，主要是结核病，每年夺去近 200 万人的生命。无效的疫苗以及多重耐药和极度耐药的结核分枝杆菌菌株加剧了这一长期的全球危机。新有效药物的设计需要对细菌的生物学及其基因组成有基本的了解。反过来，基因组序列信息的成功应用需要准确的基因注释以及对基因结构和表达谱的全面了解。我们使用 RNA-seq 和核糖体分析 (ribo-seq) 凭经验确定了基因组规模的转录和翻译起始位点。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位点缺乏 5'UTR（缺乏 Shine-Dalgarno 序列的无前导基因），以及存在数百个编码少于 50 个氨基酸的小蛋白（s 蛋白）的小型开放阅读框。迄今为止，细菌中的 s 蛋白在很大程度上被忽视，因为它们很难通过传统方法或注释管道检测到。此外，s蛋白代表了分枝杆菌中完全未被探索的一类蛋白质，并且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蛋白在快速生长的模式生物（耻垢分枝杆菌）和缓慢生长的病原体（结核分枝杆菌）中大量存在。在这里，我们建议为缓慢和快速生长的分枝杆菌生成第一个全面的、经过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核糖测序和转录起始位点定位相结合来实现。这些方法共同将为分枝杆菌群落提供根据经验定义的高可信度数据资源。此外，我们将定义 s 蛋白子集的作用机制，我们假设这些 s 蛋白以顺式作用调节下游基因。该提案具有高度创新性，因为它的重点是发现一类完全出乎意料的丰富蛋白质，这些蛋白质逃脱了对任何细菌的科学审查。应用高通量、尖端工具来促进这种分析将提供分枝杆菌蛋白质组的全面概述，同时还提供对 s 蛋白潜在功能的机制洞察。因此，我们预计会对分枝杆菌领域产生直接和长期的影响，提供新的生物学见解，为多个新兴研究领域播种，同时扩大我们对所有细菌的基因结构和调控的了解。