Systematic Discovery and Analysis of Small Proteins and Small ORFs in Mycobacteria

分枝杆菌中小蛋白和小 ORF 的系统发现和分析

• 批准号: 10663206
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 54.7万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10663206
• 关键词: 5' Untranslated Regions; Algorithms; Amino Acids; Archaea; Architecture; Bacteria; Biochemical; Bioinformatics; Biology; Birth; Cell Extracts; Codon Nucleotides; Collaborations; Coupling; Cryoelectron Microscopy; Cysteine; Data; Data Set; Detection; Eukaryota; Genes; Genome; Genomics; Genus Mycobacterium; Goals; Infection; Knowledge; Laboratories; Life; Mass Spectrum Analysis; Medical; Messenger RNA; Methods; Molecular Biology; Molecular Genetics; Mutation; Open Reading Frames; Operon; Organism; Pathogenesis; Pathogenicity; Peptides; Preparation; Prevalence; Prokaryotic Cells; Property; Proteins; Proteomics; Regulation; Research Personnel; Ribosomal Proteins; Ribosomes; Role; Selection Bias; Signal Transduction; Structure; Testing; Training; Translating; Translations; Work; attenuation; comparative; experimental study; genetic approach; genome annotation; genome-wide; improved; insight; member; mutant; mycobacterial; novel; protein function; response; ribosome profiling; whole genome

The last few decades have seen the birth and maturation of the field of Molecular Biology. Initially, mutant genes were focal points of genome exploration. Now, entire genomes are routinely sequenced, and the resident genes are automatically identified by annotation algorithms. Alternatively, proteomic approaches prepare proteolytic peptides of whole-cell extracts for analysis by mass spectrometry. Each of these approaches are strongly biased for large genes: large genes are frequent targets for mutation, long-open- reading frames are easily discerned in genomic sequence, and large proteins generate many peptides for mass spectrometry identification. This unintended bias has also created a large gap in our understanding of molecular biology. Recent work in eukaryotes and prokaryotes alike have uncovered multitudes of small genes or their encoded proteins. The numbers of small proteins (considered as 50 aa or less) rival that of traditionally large proteins, yet only a handful have been ascribed a function. The goal of this proposal is to propel this nascent field forward by facilitating both small protein discovery and functional characterization. Our preliminary data identify specific examples that clearly define cis- and trans-classes of function for short- open-reading frames and small proteins. These early leads will be pursued to fruition, providing the framework for the expanded rigorous study needed in any new field. We will test an additional subset of small proteins for function, which we anticipate will reveal functions for each member of this training set, while also establishing general principles for short-open-reading frames and small proteins. We will develop and apply our small protein approaches in mycobacteria. Mycobacteria offer many advantages for small protein study. Foremost is that they express >1000 small proteins in standard conditions. An extensive toolkit for modifying, culturing, and analyzing mycobacteria makes them very tractable. A GC-rich genome provides codon bias selection as one criterion to identify functional small proteins. Moreover, our findings of small gene/protein function in standard laboratory conditions may directly provide insights into the biology and pathogenesis of infection. This proposal integrates the complementary expertise of investigators whose ongoing collaboration has already provided the requisite groundwork leading to this proposal. Through the proposed Aims, we will identify new functional roles of encoded mycobacterial small proteins and develop an optimized, small-proteomics pipeline for efficient application to other bacteria, archaea, and eukaryotes.

过去的几十年见证了分子生物学领域的诞生和成熟。最初，突变体 基因是基因组研究的焦点。现在，整个基因组都被例行测序，而 通过注解算法自动识别驻留基因。或者，蛋白质组学方法 制备全细胞提取液中的蛋白水解肽，用于质谱学分析。这其中的每一个 方法对大基因有很强的偏见：大基因是经常突变的目标，长期开放- 基因组序列中的阅读框很容易辨认，大的蛋白质产生许多多肽 用于质谱学鉴定。这种无意的偏见也在我们的 对分子生物学的理解。 最近在真核生物和原核生物中的工作已经发现了大量的小基因或它们的 编码的蛋白质。小蛋白质的数量(被认为是50aa或更少)可以与传统的 大的蛋白质，但只有少数被认为是一种功能。这项提议的目标是推动 这一新兴领域通过促进小蛋白的发现和功能表征而向前发展。我们的 初步数据确定了明确定义顺式和跨式功能类别的具体例子。 开放阅读框架和小蛋白质。这些早期的线索将被追求到成果，前提是 为任何新领域所需的扩大的严谨研究提供框架。我们将测试另一个子集 小的功能蛋白质，我们预计它将揭示这个训练集的每个成员的功能， 同时还建立了短开放阅读框架和小蛋白的一般原则。 我们将在分枝杆菌中开发和应用我们的小蛋白方法。分枝杆菌提供了许多 小蛋白研究的优势。最重要的是，它们以标准的方式表达&gt；1000小蛋白 条件。用于修改、培养和分析分枝杆菌的广泛工具包使它们非常 很容易驯服。富含GC的基因组提供密码子偏向选择作为识别功能性小分子的一个标准 蛋白质。此外，我们在标准实验室条件下发现的小基因/蛋白质功能可能 直接提供对感染的生物学和发病机制的见解。这项提议整合了 调查人员的互补专业知识，他们正在进行的合作已经提供了必要的 导致这项提议的基础工作。通过建议的目标，我们将确定新的职能角色 编码分枝杆菌小蛋白，并开发优化的小蛋白质组学流水线，以高效 适用于其他细菌、古菌和真核生物。

项目成果

A Practical Guide to Small Protein Discovery and Characterization Using Mass Spectrometry.

• DOI: 10.1128/jb.00353-21
• 发表时间: 2022-01-18
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Ahrens CH;Wade JT;Champion MM;Langer JD
• 通讯作者: Langer JD

Evolution: Mitochondrial Ribosomes Across Species.

进化：跨物种的线粒体核糖体。

• DOI: 10.1007/978-1-0716-3171-3_2
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Agrawal,RajendraK;Majumdar,Soneya
• 通讯作者: Majumdar,Soneya

PrIntMap-R: An Online Application for Intraprotein Intensity and Peptide Visualization from Bottom-Up Proteomics.

• DOI: 10.1021/acs.jproteome.2c00606
• 发表时间: 2023-02-03
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Weaver, Simon D.;DeRosa, Christine M.;Schultz, Sadie R.;Champion, Matthew M.
• 通讯作者: Champion, Matthew M.

Pervasive translation in Mycobacterium tuberculosis.

• DOI: 10.7554/elife.73980
• 发表时间: 2022-03-28
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Smith, Carol;Canestrari, Jill G.;Wang, Archer J.;Champion, Matthew M.;Derbyshire, Keith M.;Gray, Todd A.;Wade, Joseph T.
• 通讯作者: Wade, Joseph T.

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

• DOI: 10.1073/pnas.2302006120
• 发表时间: 2023-05-30
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Li, Yunlong;Majumdar, Soneya;Treen, Ryan;Sharma, Manjuli R.;Corro, Jamie;Gamper, Howard B.;Manjari, Swati R.;Prusa, Jerome;Banavali, Nilesh K.;Stallings, Christina L.;Hou, Ya-Ming;Agrawal, Rajendra K.;Ojha, Anil K.
• 通讯作者: Ojha, Anil K.