INOSITOL PHOSPHATES AND HUMAN DISEASE

该项目是以前由SCOR支持的研究的延续 旨在阐明磷脂酰肌醇 信号通路，并确定这些细胞内的作用， 人类疾病中的信号分子。该系统无处不在， 参与对生长因子和细胞因子的增殖反应。在 事实上，大多数细胞反应需要肌醇磷酸信使。因此 了解这个系统对炎症的途径至关重要， 血栓形成和止血，以及涉及细胞增殖的病症。我们 已经鉴定、分离和克隆了几种关键酶的cDNA 从人血小板和人、大鼠和牛脑中提取。的 第一个发现的先天性肌醇代谢缺陷是劳氏综合征， 也称为眼脑肾综合征。这种蛋白质发生了突变， 疾病与血小板5-磷酸酶II型有50%相同，但其 酶的功能尚未阐明。产生的重组蛋白 具有5-磷酸酶活性，该系统将用于 阐明其性质和底物特异性。我们还将比较 5-磷酸酶和Lowe's的组织分布和细胞定位 免疫组化和RNA原位杂交检测蛋白表达。肌醇 多磷酸4-磷酸酶也将被克隆并表达在 异源系统，以确定其在细胞信号传导，细胞生长， 血小板功能和PtdIns 3-激酶途径。肌醇 多磷酸1-磷酸酶是一个金属家族的原型， 依赖性Li+抑制的磷酸酶共有155个核心结构 残基和“DP（i/l）D（g/s）（t/s）”的金属结合基序。“这些酶 是Li+作为治疗剂的作用的潜在靶点， 躁狂抑郁症，并将通过人类的分子克隆进行研究 同源物，染色体定位，底物测定 特异性和Li+抑制的分子基础。肌醇 多磷酸1-磷酸酶将在存在下结晶， 底物和抑制性金属，以确定催化机制， Li +结合位点。结构信息将用于设计 1-磷酸酶和其他家族成员的抑制剂， 治疗精神疾病X射线晶体结构也将被 测定5-磷酸酶和4-磷酸酶。它们没有氨基 酸序列相似性的1-磷酸酶家族，即使他们使用 相似或相同的基质。

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.