MOLECULAR STUDIES OF THE PROTEIN PHOSPHATASE CALCINEURIN

蛋白质磷酸酶钙调磷酸酶的分子研究

基本信息

批准号：
6386280
负责人：
FRANK M RUSNAK
金额：
$ 21.17万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-09-01 至 2004-08-31
项目状态：
已结题

项目摘要

Calcineurin is a Ca - and calmodulin-regulated protein serine/threonine phosphatase and an important component of eukaryotic signal transduction pathways, e.g., the T-cell receptor signal transduction pathway that leads to IL-2 gene transcription. Calcineurin is the target of the immunosuppressant agents, cyclosporin A and FK506, important organ transplantation drugs that inhibit calcineurin phosphatase activity in the presence of the cytoplasmic proteins cyclophililin and FK506- binding protein (FKBP), respectively. Calcineurin is a member of the metallophosphatase superfamily of enzymes that accommodate active site dinuclear metal cofactors. In calcineurin, an Fe-Zn dinuclear metal center has been characterized by x-ray diffraction and spectroscopic techniques. Another member of this family, lambda protein phosphatase, has also been characterized by biochemical and spectroscopic techniques and shown to accommodate a dinuclear metal center. The broad, long-terms goals of this proposal will be to characterize structure/function aspects of calcineurin and lambda protein phosphatase related to the active site dinuclear metal centers and their roles in catalysis and metabolic regulation. In vitro studies have demonstrated that the phosphatase activity of calcineurin is sensitive to the redox state of the bound Fe ion. One of the specific aims will be to extend the link between calcineurin activity and the oxidation state of bound metal ions using different substrates. Recent work suggests that calcineurin phosphatase activity may also be regulated by the cellular redox state. We hypothesize that the phosphatase activity of calcineurin is coupled to the intracellular redox state and that this involves the oxidation state of the active site Fe ion. The specific aims in this regard are to explore redox regulation of calcineurin in vivo. Further specific aims will focus on lambda protein phosphatase as a model for calcineurin and other protein serine/threonine phosphatases to explore the role of the metal ions in this enzyme and their role in the catalytic mechanism. The three dimensional structure of lambda protein phosphatase will be determined by x-ray diffraction methods, and the catalytic mechanism explored using biochemical and kinetic isotope effect studies. Recent studies have implicated oxidative stress in many cellular processes and disease states including apoptosis and cardiovascular disease. Calcineurin has been implicated as a key signaling component in apoptosis and cardiac hypertrophy. The possibility that calcineurin phosphatase activity may be regulated by the cellular redox provides a link between oxidative stress and calcium- dependent signal transduction pathways.

钙调神经磷酸酶是一种钙离子和钙调蛋白调节蛋白丝氨酸/苏氨酸磷酸酶，是真核信号转导途径的重要组成部分，例如导致 IL-2 基因转录的 T 细胞受体信号转导途径。钙调磷酸酶是免疫抑制剂环孢菌素 A 和 FK506 的靶标，这两种药物是重要的器官移植药物，在细胞质蛋白亲环蛋白和 FK506 结合蛋白 (FKBP) 存在的情况下分别抑制钙调磷酸酶活性。钙调神经磷酸酶是金属磷酸酶超家族的成员，可容纳活性位点双核金属辅助因子。在钙调磷酸酶中，Fe-Zn 双核金属中心已通过 X 射线衍射和光谱技术进行了表征。该家族的另一个成员，拉姆达蛋白磷酸酶，也通过生化和光谱技术进行了表征，并显示其可容纳双核金属中心。该提案的广泛、长期目标是表征与活性位点双核金属中心相关的钙调神经磷酸酶和 lambda 蛋白磷酸酶的结构/功能及其在催化和代谢调节中的作用。体外研究表明，钙调神经磷酸酶的磷酸酶活性对结合的铁离子的氧化还原状态敏感。具体目标之一是使用不同的底物扩展钙调神经磷酸酶活性与结合金属离子的氧化态之间的联系。最近的研究表明钙调神经磷酸酶活性也可能受到细胞氧化还原状态的调节。我们假设钙调神经磷酸酶的磷酸酶活性与细胞内氧化还原态耦合，并且这涉及活性位点铁离子的氧化态。这方面的具体目标是探索体内钙调神经磷酸酶的氧化还原调节。进一步的具体目标将集中于 lambda 蛋白磷酸酶作为神经磷酸酶和其他蛋白丝氨酸/苏氨酸磷酸酶的模型，以探索金属离子在该酶中的作用及其在催化机制中的作用。 λ蛋白磷酸酶的三维结构将通过X射线衍射方法确定，并通过生化和动力学同位素效应研究探索催化机制。最近的研究表明氧化应激与许多细胞过程和疾病状态有关，包括细胞凋亡和心血管疾病。钙调神经磷酸酶被认为是细胞凋亡和心脏肥大中的关键信号成分。钙调神经磷酸酶活性可能受细胞氧化还原调节的可能性提供了氧化应激和钙依赖性信号转导途径之间的联系。