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
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