ADP-Ribosylation and Regulation of Cellular Functions

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Currently there is reasonably convincing support for the hypothesis that reversible modification of proteins by ADP-ribosylation plays a significant role in regulation of cellular functions and signal transduction in eukaryotes. The overall objective of this research is to elucidate at the molecular level the role(s) of ADP- riblosyltransferases (ADPRTases) in signal transduction and regulation of cellular functions. To achieve this goal, it will be necessary to (1) understand how the activity of these enzymes is regulated, (2) identify the proteins which serve as physiologically relevant targets for this post-translational modification, and (3) determine how the modification of these specific proteins controls cellular functions. Progress toward these goals is hampered by the lack of immunological and molecular genetic reagents for studying the modifying enzymes and target proteins involved in ADP- ribosylation reactions. For example, no gene for an endogenous ADPRTase has yet been cloned or sequenced. Likewise, no target protein for in vivo ADP-ribosylation has been identified. One specific goal of this proposal is to generate immunological and molecular genetic reagents to use as probes in addressing some of the important questions surrounding these post-translational modifications of proteins. The parallel and complementary aims of this study are (1) generation of antibodies that specifically recognize ADPRTases and ADP-ribosylated proteins and (2) molecular analysis of these proteins and their genes. %%% Many proteins are structurally modified after synthesis by addition of various small molecules. In some cases these modifications have been shown to play a pivotal role in the mechanism of action of the protein. For example, in many cellular responses to external stimuli the mechanism of signal transduction often involves the activation of an enzyme that adds or removes a phosphate group from some protein. Another type of protein modification is the addition of a molecule, known as adenosine diphosphoribose, derived from a vitamin and a sugar, to specific proteins. This chemical reaction is carried out by an enzyme known as adenosine diphosophoribosyl- transferases (ADPRTases). The overall goal of this research is to elucidate at the molecular level the role(s) of ADP- ribosyltransferases in signal transduction and regulation of cell functions. The near term objectives are to characterize ADP ribosyltransferases and to identify and characterize the physiologically important protein substrates of the ADPRTases. First it is necessary to develop biochemical and immunological tools to proceed with these studies. The results of this research should be an important contribution to understanding of the molecular mechanisms by which cells respond to external stimuli.