Phosphatidylinositol Signaling and Human Disease

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DESCRIPTION (provided by applicant): The major goal of this grant is to define defects in the phosphatidylinositol signaling pathway that result in human diseases. The role of inositol (1,3,4)-P3 5/6-kinase in inhibition of TNF-induced apoptosis will be investigated. Is the effect due to some inositol phosphate product or to phosphorylation of a protein of the apoptotic pathway? Mutant forms of recombinant kinase that lack either inositol or protein kinase activity will be created and tested for their ability to block TNF apoptosis. The enzymes leading to inositol hexaphosphate will be overexpressed in 293 cells or inhibited by RNAi to elucidate functions for the higher inositol phosphates in apoptosis, nuclear export of mRNA, and intracellular vesicle trafficking. The role of the B. pseudomallei protein BobB in virulence of this organism will be studied by determining whether it is an inositol phosphate phosphatase. The substrate specificity of two human homologues of BopB will be determined by cloning cDNA's encoding these proteins and producing recombinant protein in Sf9 cells. Continued studies of the myotubularin gene family (mutations of which cause several different human diseases) will be performed by investigating complexes between myotubularin related proteins 6 and 7 that are enzymatically active with the inactive myotubularin related protein 9. The role of complex formation in the pathogenesis of Lowe syndrome will be studied by determining the function of the inositol polyphosphate 5-phosphatase (OCRL) that is mutated in that disease. The role of OCRL in Golgi vesicle trafficking will be studied and the effect of mutated OCRL on the processing and/or activity of the NBC1 bicarbonate transporter of renal proximal tubules will be investigated. Malfunction of this transporter is suggested by the finding that mutations in NBC1 in humans produce a phenocopy of Lowe syndrome