NitFhit--Structure and Molecular Pharmacology

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DESCRIPTION: (Applicant's Description) The Fhit homolog in flies and worms is encoded as a natural fusion protein with a homolog of nitrilase, an enzyme that cleaves indole-3-acetonitrile to produce the plant growth and differentiation factor, indole-3-acetic acid. Animal-type nitrilase homologs (Nit proteins) have been cloned from humans, mice and yeast. Though not fused to the Fhit polypeptide in mammals, mammalian Nit and Fhit have nearly identical expression patterns and Project 1 has shown that Nit appears to link Fhit to other interacting proteins. In view of these linkages between Nit and Fhit, understanding the structure and activity of Nit is seen as part of the problem of understanding the function of Fhit. The Aims of Project 3 are as follows. First, we will determine the 3D structure of NitFhit from diffraction data of native, selenomethionyl and mercurated I222 crystals that have already been obtained. Second, we will obtain substrates and inhibitors of NitFhit with biochemical assays we have developed and characterize the nature of the biochemical interactions. Third, we will generate active-site mutants of Nits and use yeast cells that accumulate Nit substrates to identify and purify the authentic substrates of these enzymes. The 3D structure of Nit-Fhit is expected to reveal the nature of communication between Nit and Fhit, structural and chemical features of nitrilase superfamily enzymes, and the nature of the binding pocket of animal-type nitrilase homologs. Discovery of small molecules that interact with Nit and Fhit is expected to clarify the cellular roles for these enzymes and make possible novel chemical means of disrupting, amplifying or bypassing their functions. Expression of substrate-trapping mutants of Nits may provide an additional way to perturb signaling genetically. Structural and pharmacological characterization of Nit and Fhit are parts of a long-term plan to define molecular targets for tumors with inactivated FHIT genes for genotype-specific chemotherapeutic elimination.