MOLECULAR STUDIES OF THE PROTEIN PHOSPHATASE CALCINEURIN

蛋白质磷酸酶钙调磷酸酶的分子研究

• 批准号: 6525644
• 负责人: WHYTE G OWEN
• 金额: $ 21.17万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6525644
• 关键词: X ray crystallography; active sites; apoptosis; biological signal transduction; calcineurin; calcium binding protein; chemical kinetics; electrochemistry; enzyme activity; enzyme mechanism; enzyme substrate; iron; metalloenzyme; oxidation reduction reaction; oxidative stress; phosphatase inhibitor; phosphoprotein phosphatase; protein purification; tissue /cell culture; zinc

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.

钙调蛋白是CA和钙调节蛋白调节的蛋白丝氨酸/苏氨酸磷酸酶，也是真核信号转导途径的重要组成部分，例如，T-Cell受体信号转导途径，导致IL-2基因转录。钙调蛋白是免疫抑制剂Cyclosporin A和FK506，重要器官移植药物的靶标，它们在存在细胞质蛋白质蛋白环磷脂和FK506-结合蛋白（FKBP）的情况下抑制钙调蛋白磷酸酶活性。钙调蛋白是可容纳活性位点双核金属辅助因子的甲基磷酸酶超家族的成员。在钙调蛋白酶中，Fe-Zn的双核金属中心的特征是X射线衍射和光谱技术。该家族的另一个成员Lambda蛋白磷酸酶也以生化和光谱技术为特征，并证明可容纳双核金属中心。该提案的广泛，长期目标是表征与活跃的位点可核金属中心相关的钙调神经酶和lambda蛋白磷酸酶的结构/功能方面及其在催化和代谢调节中的作用。体外研究表明，钙调蛋白的磷酸酶活性对结合FE离子的氧化还原态敏感。具体目的之一是使用不同的底物扩展钙调神经酶活性与结合金属离子的氧化状态之间的联系。最近的工作表明，钙调神经磷酸酶磷酸酶活性也可能受细胞氧化还原态调节。我们假设钙调蛋白的磷酸酶活性与细胞内氧化还原态耦合，这涉及活性位点Fe离子的氧化态。在这方面的具体目的是探索体内钙调蛋白的氧化还原调节。进一步的具体目的将集中于lambda蛋白磷酸酶作为钙调蛋白和其他蛋白质丝氨酸/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。 Lambda蛋白磷酸酶的三维结构将通过X射线衍射方法确定，并使用生化和动力学同位素效应研究探索了催化机制。最近的研究表明，在许多细胞过程和疾病状态（包括凋亡和心血管疾病）中的氧化应激。钙调神经蛋白已被视为凋亡和心脏肥大中的关键信号成分。钙调蛋白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与钙依赖性信号转导途径之间的联系。

项目成果

Differential modulation of cortical synaptic activity by calcineurin (phosphatase 2B) versus phosphatases 1 and 2A.

钙调神经磷酸酶（磷酸酶 2B）与磷酸酶 1 和 2A 对皮质突触活性的差异调节。

• DOI: 10.1016/s0006-8993(96)01305-4
• 发表时间: 1997
• 期刊: Brain research
• 影响因子: 2.9
• 作者: Thomas,GD;O'Rourke,B;Sikkink,R;Rusnak,F;Marban,E;Victor,RG
• 通讯作者: Victor,RG

Calcineurin subunit interactions: mapping the calcineurin B binding domain on calcineurin A.

• DOI: 10.1021/bi00026a016
• 发表时间: 1995-07
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: R. Sikkink;A. Haddy;Sarah H. Mackelvie;P. Mertz;R. Litwiller;F. Rusnak

Conversion of desulforedoxin into a rubredoxin center.

脱硫氧还蛋白转化为红氧还蛋白中心。

• DOI: 10.1006/bbrc.1997.6171
• 发表时间: 1997
• 期刊: Biochemical and biophysical research communications.
• 作者: Yu,L;Kennedy,M;Czaja,C;Tavares,P;Moura,JJ;Moura,I;Rusnak,F
• 通讯作者: Rusnak,F

Expression of Desulfovibrio gigas desulforedoxin in Escherichia coli. Purification and characterization of mixed metal isoforms.

巨大脱硫弧菌脱硫氧还蛋白在大肠杆菌中的表达。

• DOI: 10.1074/jbc.270.35.20273
• 发表时间: 1995
• 期刊: The Journal of biological chemistry
• 作者: Czaja,C;Litwiller,R;Tomlinson,AJ;Naylor,S;Tavares,P;LeGall,J;Moura,JJ;Moura,I;Rusnak,F
• 通讯作者: Rusnak,F

Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.

噬菌体 lambda 蛋白磷酸酶与 Mn(II) 的相互作用：[Mn(II)]2 簇形成的证据。

• DOI: 10.1021/bi982606u
• 发表时间: 1999
• 期刊: Biochemistry.
• 作者: Rusnak,F;Yu,L;Todorovic,S;Mertz,P
• 通讯作者: Mertz,P