DBP-C: ACETYLATION/DEACETYLATION PATHWAYS IN BACTERIA

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 推动生物C项目 我们试图回答有关SDPADS的两个核心问题。首先，除了乙酰-辅酶A合成酶之外，还有哪些蛋白质组成了SDPADS调节子？第二，这些蛋白质参与了哪些细胞过程？我们将使用该中心其他成员开发的强大技术，从全球角度解决这些问题。MALDI质谱学技术将被用于识别SDPADS调节子的新蛋白质成员，确定每种蛋白质中乙酰化赖氨酸残基的准确数量和位置，并监测随着生理条件的变化而在细胞中乙酰化SDPADS蛋白质的丰度。大肠杆菌的遗传系统具有很好的特性，它们的使用将极大地促进分析基因产物在不同遗传背景下的生理作用。这种遗传系统的可用性将有助于验证在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。通过将建议的全球方法与我们在遗传、分子生物学和生化方法方面的新陈代谢专业知识相结合，我们将更多地了解SDPADS在原核生物中的作用，这反过来将为真核细胞生理学提供有价值的见解。 特定目的#1.鉴定SIRTUINDEPENDENT蛋白质ACETYLATIONIDEACETYLATION系统(SDPADS)底物的蛋白质微阵列方法这些研究的目标是确定SDPADS规则的范围。我们将使用由恒珠博士(TCP-1)开发的蛋白质组微阵列芯片来从全球角度来解决这个问题。该方法依赖于依赖于乙酰辅酶A的蛋白乙酰基转移酶(PAT)或依赖于NAD的Cobb sirtuin脱乙酰基酶蛋白与其底物的相互作用。除了在酶催化下将放射性标记的十六烷基基团从乙酰辅酶A转移到芯片上的蛋白质外，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Robert Cotter博士和AkHilesh Pandey博士开发的技术(tcp-4和tcp-3)来绘制蛋白质乙酰化位点的图谱。 特定目的#2.研究SDPADS蛋白底物编码基因表达调控的MALDI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蛋白质的乙酰化程度随着生理条件的变化而发生的变化。一般的策略将是将编码新的SDPADS底物的基因克隆到低拷贝数载体中，以指导具有生理功能的标记蛋白(组氨酸、GTS、甲壳素等)的合成。选择的基因从染色体上删除的菌株将被引入质粒，菌株将在需要研究的功能的条件下生长。

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.