Insoine Monophosphate Dehydrogenase (IMPDH) is an essential enzyme in the de novo synthesis of guanine nucleotides and plays an important role in lymphocyt activation and in cellular proliferation and differentiation. Inhibitors of IMPDH are highly effective immunosuppressive agents and have therapeutic potential for the treatment of certain neoplastic diseases. IMPDH catalytic activity is comprised of the activities of 2 highly similar enzymes IMPDH type 1 and 2, which are encoded by distinct genes that are differentlly regulated but have similar structure. The PI hypothesizes that the evolutionary conservation of these two independent genes reflects differential requirements for IMPDH expression during cell growth and development. The overall objective of the proposed studies are to understand 1) the relative roles of these two enzymes in growth, development, and malignant transformation; 2) the regulatio of their respective activities at the levels of transcription and translation; and 3) the consequences of guanine nucleotide depletion for lymphocyte activation and neoplastic transformation. The phenotypes of IMPDH type 1 and heterozygous and homozygous deficient mice resulting from gene knock-outs by recombination will be defined in order to determine whether lymphocyte development and/or activtion is selectively inhibited and whether expression o either isoenzyme can compensate for the loss of the other gene. These animals will be bred with each other and with mice deficient in HPRT activity to determine the relative roles of the de novo and salvage pathways for tissue-specific guanine nucleotide biosynthesis. The effects of loss of the tw isoenzymes on cellular signaling pathways important for oncogenic transformation will also be examined. Critical elements required for the regulation for IMPDH type 2 at the transcriptional level will be indentified, including the protein components that bind to a novel trancription factor binding site, and the possibility of T lymphocyte-specific regulation of IMPDH type 1 expression at the level of translation will be examined. Finally, the effects of guanine nucleotide depletion on cellular signaling pathways in T lymphocytes will be studied with special attention to whether effects on ras-mediated signal transduction explain the inhibition of cell cycle progression induced by inhibition of IMPDH activity.
The aims of the proposed work are to provide a more comprehensive understanding of the regulation and function of IMPDH at the molelcular and cellular levels and to provide a rational foundation for further development and use of inhibitors of this enzyme.
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