INOSITOL PHOSPHATES AND HUMAN DISEASE

磷酸肌醇与人类疾病

• 批准号: 2910607
• 负责人: PHILIP W MAJERUS
• 金额: $ 44.64万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910607
• 关键词: X ray crystallography; biological signal transduction; enzyme deficiency; enzyme mechanism; enzyme structure; enzyme substrate; human genetic material tag; human subject; immunocytochemistry; in situ hybridization; inositol phosphates; isozymes; laboratory mouse; laboratory rabbit; molecular cloning; molecular pathology; phosphatase inhibitor; phosphomonoesterases; protein structure function; recombinant proteins

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.

该项目是先前由SCOR支持的研究的继续 在血栓形成中的作用，旨在阐明磷脂酰肌醇 并确定这些细胞内信号转导途径的作用 人类疾病中的信号分子。这个系统无处不在，而且 参与对生长因子和细胞因子的增殖反应。在……里面 事实上，大多数细胞反应都需要肌醇磷酸信使。因此， 了解这一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疾病。我们 已经鉴定、分离和克隆了几种关键酶的cDNAs 在这一途径中，来自人的血小板和人、大鼠和牛脑。这个 第一个被发现的肌醇代谢先天错误是洛氏综合征， 也称为眼脑肾综合征。在这种情况下蛋白质发生了突变 疾病与血小板5-磷酸酶II型有50%的相似之处，但其 酶的功能还没有被阐明。表达的重组蛋白 在杆状病毒中具有5-磷酸酶活性，该系统将用于 阐明其性质和底物专一性。我们还将比较 5-磷酸酶和Lowe‘s的组织分布和细胞定位 用免疫组织化学和原位RNA杂交法检测蛋白质。肌醇 多聚磷酸4-磷酸酶也将被克隆并在 异源系统来定义它在细胞信号传递，细胞生长， 血小板功能，以及在PtdIns 3-激酶途径中。肌醇 多聚磷酸1-磷酸酶是一类金属的原型 依赖的Li+抑制具有155个核心结构的磷酸酶 残基和“DP(i/L)D(g/S)(t/S)”的金属结合基序。这些酶 是Li+作为治疗药物作用的潜在靶点 躁郁症，将通过人类的分子克隆进行研究 同系物、染色体定位、底物测定 Li+抑制的特异性和分子基础。肌醇 多聚磷酸1-磷酸酶将在有 底物和抑制金属，以定义催化和 Li+结合部位。结构信息将用于设计 1-磷酸酶抑制剂和其他可用于 治疗精神疾病。X射线晶体结构也将是 测定5-磷酸酶和4-磷酸酶。他们没有氨基 酸序列与1-磷酸酶家族相似，尽管它们使用 相似的或相同的底物。