The FHIT gene at 3p14.2 is at a translocation that cosegregates with renal cancer and within a region that is homozygously disrupted in many human tumors. HINT and FHIT are the prototypical members of two branches of the newly recognized HIT superfamily of low molecular weight, dimeric nucleotide-binding proteins. HINT proteins have been conserved in evolution since the separation of bacteria, eukarya and archaea. FHIT proteins are a distinct branch found within eukarya that hydrolyze diadenosine polyphosphates such as ApppA Despite their conservation throughout evolution and the implication of FHIT in the etiology or complications of cancer, the function of HIT proteins is not understood in any cell. The P.I. has determined the atomic structure of HINT in its nucleotide-free and nucleotide-bound forms, grown crystals of FHIT, and has begun to generate mutant yeast strains to understand the function of the homologous genes in S cerevisiae. Specifically, it is proposed 1) to determin precise physiological consequences of subtle and gross alterations in the homologous Hnt1 and Hnt2 proteins in yeast and of mammalian HINT and FHIT expressed in yeast, exploring the effects of mutations, inhibitors and interacting proteins; 2) to measure purine mononucleotide binding activity and diadenosine polyphosphate hydrolase activity of purified wild-type and mutant Hnt1 and Hnt2 proteins, comparing their behaviors with mammalian HIT proteins and testing the dependence of cellular functions on these parameters; and 3) to determine the X-ray structure of FHIT crystals that have already been grown, elucidating the moleular basis of FHIT s activity and function. Structural information (aim 3) is already in use in the design of mutants which will provide additional physiological (aim 1) and biochemical (aim 2) information. This integrated strategy is expected to provide a detailed understanding of the functions of HINT and FHIT in eukaryotic cells and a cellular and structural basis for understanding FHIT lesions in tumors.
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