Molecular cloning of deoxyhypusine hydroxylase

Western Illinois University, Macomb IL

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Abstract

DESCRIPTION (applicant's abstract): Hypusine synthesis in the eukaryotic initiation factor 5A (eIF-5A) is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthetase, the deoxyhypusine hydroxylase catalyzes the formation of mature hypusine. The synthesis of hypusine, and therefore of mature eIF-5A, is correlated with proliferation in several eukaryotic cell types. Inhibitors of either of the two hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxhypusine hydroxylase, have been shown to exert antiproliferative effects and are regarded as potential targets for antiproliferative therapy. The objectives of the proposed work are: 1) to purify deoxyhypusine hydroxylase protein from bovine (kidney) using various chromatography and electrophoresis techniques, and to determine its partial amino acid sequence and; 2) to generate antibody and nucleic probes to be used for cloning of a full-length cDNA encoding deoxyhypusine hydroxylase. The long term goals of the research are: (1) to express functional recombinant deoxyhypusine hydroxylase protein in E. coli); (2) to increase our understanding of the structure of deoxyhypusine hydroxylase; (3) to generate probes that will allow the principal investigator to study the distributions and control expression of the protein and the mRNA; and 4) to develop more specific inhibitors of this enzyme, based on structural knowledge gained, to better control hyperproliferative diseases.