DBP-C: ACETYLATION/DEACETYLATION PATHWAYS IN BACTERIA

DBP-C：细菌中的乙酰化/脱乙酰化途径

• 批准号: 7724692
• 负责人: JORGE C ESCALANTE
• 金额: $ 33.84万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724692
• 关键词: Acetylation; Acetyltransferase; Address; Automobile Driving; Bacteria; Biochemical; Biological; Cell physiology; Cells; Chitin; Chromosomes; Coenzyme A; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Condition; Deacetylase; Deacetylation; Enzymes; Eukaryotic Cell; Funding; Gene Expression Regulation; Genes; Genetic; Goals; Grant; Institution; Learning; Ligase; Location; Lysine; Maps; Mass Spectrum Analysis; Measurement; Metabolism; Methods; Molecular Genetics; Monitor; Numbers; Pathway interactions; Personal Satisfaction; Physiological; Plasmids; Prokaryotic Cells; Protein Acetylation; Protein Microchips; Proteins; Proteome; Regulon; Research; Research Personnel; Resources; Role; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Techniques; Technology; United States National Institutes of Health; gene cloning; insight; interest; member; research study; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Driving Biological Project C We seek to answer two central questions about the SDPADS. First, which proteins - other than acetyI-CoA synthetase - comprise the SDPADS regulon? Second, what cell processes are these proteins involved in? We will use powerful technologies developed by other members of the Center to address these questions from a global perspective. MALDI mass spectrometry techniques to will be used to identify new protein members of the SDPADS regulon, to determine the precise number and location of acetylated lysine residues in each protein, and to monitor the abundance of acetylated SDPADS proteins in cells as a function of changing physiological conditions. The genetic system for E. coil is well characterized and their use will greatly facilitate the analysis of the physiological roles of gene products in diverse genetic backgrounds. The availability of such genetic system will be instrumental in validating the results obtained in the microarray experiments, particularly in addressing the roles of proteins of unknown function. By combining the proposed global approaches with our expertise in genetic, molecular biological and biochemical approaches to metabolism, we will learn more about the role of the SDPADS in prokaryotes, which in turn will provide valuable insights into eukaryotic cell physiology. SPECIFIC AIM #1. PROTEIN MICROARRAY APPROACHES TO IDENTIFYING SUBSTRATES FOR THE SIRTUINDEPENDENT PROTEIN ACETYLATIONIDEACETYLATION SYSTEM (SDPADS). The goal of these studies is to define the extent of the SDPADS regulon. We will use proteome microarray chips developed by Dr. Heng Zhu (TCP-1) to approach this problem from a global perspective. The approach relies on interactions of the acetyI-CoA-dependent protein acetyltransferase (Pat) or the NAD+-dependent Cobb sirtuin deacetylase proteins with their substrates. Two different methods will be used to detect such interactions in addition to performing enzyme-catalyzed transfer of radioabeled cetylgroups from acetyI-CoA to proteins on the chip. We will use technologies developed by Dr. Robert Cotter and Akhilesh Pandey (TCP-4 and TCP-3) for mapping sites of protein acetylation. SPECIFIC AIM #2. MALDI APPROACHES TO STUDYING REGULATION OF EXPRESSION OF GENES ENCODING SDPADS PROTEIN SUBSTRATES. We will use technologies developed by Dr. Akhilesh Pandey (TCP-3) for the quantitative measurement of protein acetylation in complex mixtures. We will use these technologies to reveal changes in the extent of acetylation of proteins of interest as a function of changing physiological conditions. The general strategy will be to clone genes encoding new SDPADS substrates into low-copy number vectors that direct the synthesis of tagged proteins (His, GTS, chitin, etc) that are physiologically functional. Plasmids will be introduced into strains in which the gene of choice is deleted from the chromosome, and strains will be grown under conditions that require the function under study.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 驾驶生物项目c 我们试图回答有关SDPADS的两个核心问题。 首先，哪种蛋白质 - 除乙酰辅酶A之外的合成酶以外 - 构成SDPADS调节剂？其次，这些蛋白质涉及哪些细胞过程？我们将使用中心其他成员开发的强大技术从全球角度来解决这些问题。 MALDI质谱技术将用于鉴定SDPADS Regulon的新蛋白质成员，以确定每种蛋白质中乙酰化赖氨酸残基的精确数量和位置，并监测细胞中乙酰化的SDPADS蛋白在细胞中的丰富度，这是变化的生理条件的功能。 E.线圈的遗传系统的表征很好，它们的使用将大大促进基因产物在各种遗传背景下的生理作用的分析。这种遗传系统的可用性将有助于验证微阵列实验中获得的结果，特别是在解决未知功能的蛋白质的作用方面。通过将所提出的全球方法与我们在代谢的遗传，分子生物学和生化方法方面的专业知识相结合，我们将进一步了解SDPADS在原核生物中的作用，这反过来又将为真核细胞生理学提供宝贵的见解。 特定目标＃1。蛋白微阵列的方法，用于识别SIRTUIDETIDERT蛋白乙酰化乙酰化系统（SDPADS）的底物。这些研究的目的是定义SDPADS调节的程度。我们将使用Heng Zhu博士（TCP-1）开发的Proteome微阵列芯片来解决此问题。该方法依赖于乙酸-COA依赖性蛋白乙酰转移酶（PAT）或NAD+依赖性Cobb Sirtuin脱乙酰基蛋白与底物的相互作用。除了在芯片上从乙酰辅酶A到蛋白质，还将使用两种不同的方法来检测这种相互作用。我们将使用由Robert Cotter博士和Akhilesh Pandey（TCP-4和TCP-3）开发的技术来映射蛋白质乙酰化的位点。 特定目标＃2。 MALDI研究编码SDPADS蛋白底物的基因表达的调节方法。我们将使用Akhilesh Pandey博士（TCP-3）开发的技术来定量测量复杂混合物中的蛋白质乙酰化。我们将使用这些技术来揭示感兴趣的蛋白质乙酰化程度的变化，这是生理条件变化的函数。一般策略将是将编码新SDPADS底物的克隆基因转化为低拷贝数量向量，这些媒介指导了具有生理功能的标记蛋白（HIS，GTS，几丁素等）的合成。质粒将被引入菌株中，其中选择的基因从染色体中删除，并且在需要研究功能的条件下会生长菌株。