INOSITOL PHOSPHATES AND HUMAN DISEASE

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This project is a continuation of research supported previously by a SCOR in Thrombosis and is designed to elucidate the phosphatidyl inositol signalling pathway and to determine the role of these intracellular signalling molecules in human disease. The system is ubiquitous and is involved in proliferative responses to growth factors and cytokines. In fact most cellular responses require inositol phosphate messengers. Thus understanding this system is critical to pathways of inflammation, thrombosis and hemostasis, and disorders involving cell proliferation. We have identified, isolated, and cloned cDNA's for several critical enzymes in this pathway from human platelets and human, rat and bovine brain. The first inborn error of inositol metabolism discovered is Lowe's syndrome, also known as oculocerebrorenal syndrome. The protein mutated in this disease is 50% identical to platelet 5-phosphatase type II but its enzymatic function is not yet elucidated. The recombinant protein produced in baculovirus has 5-phosphatase activity and this system will be used to elucidate its properties and substrate specificity. We will also compare tissue distribution and cellular localization of 5-phosphatase and Lowe's protein using immunohistochemistry and in situ RNA hybridization. Inositol polyphosphate 4-phosphatase will also be cloned and expressed in heterologous systems to define its role in cell signalling, cell growth, platelet function, and in the PtdIns 3-kinase pathway. Inositol polyphosphate 1-phosphatase is the prototype of a family of metal dependent Li+ inhibited phosphatases that share a core structure of 155 residues and a metal-binding motif of "DP(i/l)D(g/s)(t/s)." These enzymes are potential targets for the action of Li+ as a therapeutic agent in manic-depressive disease and will be studied by molecular cloning of human homologues, chromosomal localization, determination of substrate specificity and a molecular basis for Li+ inhibition. Inositol polyphosphate 1-phosphatase will be crystallized in the presence of substrates and inhibitory metals to define the mechanism of catalysis and Li + binding site. The structural information will be used to design inhibitors of 1-phosphatase and other family members that may be used to treat psychiatric disorders. X-ray crystal structures will also be determined for 5-phosphatase and 4-phosphatase enzymes. They have no amino acid sequence similarity to the 1-phosphatase family, even though they use similar or identical substrates.