Enzymological Aspects Of Neural Functions

神经功能的酶学方面

• 批准号: 7322916
• 负责人: ROBERT W ALBERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7322916
• 关键词: Enzymological; Aspects; Neural; Functions; Enzymological; Aspects; Neural; Functions

The principal goal of this project is to extend our understanding of the mechanisms of enzymes of particular relevance to the nervous system. A study of sodium, potassium pump (Na,K-ATPase) and calcium pump (SERCA ATPase) mechanisms, in collaboration with J. Froehlich (U. Md.), is currently directed at testing a hypothesis that certain of our transient kinetic observations reflect oligomeric interactions of multiple pump molecules during their catalytic cycle. A dependent hypothesis is that such oligomeric interactions contribute to the efficiency of cation transport. Our current evidence suggests that these interactions mediate "out-of-phase" free energy transfers between two or more catalytic subunits, and that these interactions may be a general property of P-type cation pumps examined in their native state. We have recently examined the effects of the small intrinsic membrane protein, phospholamban(PLB), on the transient kinetics and oligomeric state of the SERCA1 and SERCA2a calcium pumps. PLB interacts with both the isoforms SERCA1 and SERCA2a, but is normally co-expressed only with SERCA2a, the cardiac muscle isoform. Activation of SERCA2a by beta-1-agonists increases the efficiency of calcium transport and elevates cardiac sarcoplasmic reticulum calcium stores. This involves PKA-dependent phosphorylation of PLB. PLB-pump interaction decreases the Vmax and the apparent calcium affinity of the pumps, whereas phosphorylation of PLB reverses both effects. Our data suggest that the increased pump efficiency of SERCA2a by beta-1 agonists results from a phosphorylation-induced dissociation of PLB. EPR measurements of spin-labeled SERCA2a indicate that PLB-binding changes the SERCA2a from an oligomeric state to one that is functionally monomeric. Transient kinetic studies indicate that that the skeletal muscle isoform, SERCA1, is an oligomer in the absence of PLB. SERCA1 exhibits a faster turnover and higher Vmax relative to SERCA2a. Oligomeric pump interactions are hypothesized to produce the rapid phase of E2P hydrolysis in SERCA1 that is absent in SERCA2a. We tested whether removal of PLB regulation of SERCA2a converts it to a functionally oligomeric state resembling SERCA1. This was confirmed by measuring the transient kinetic properties of pump phosphorylation using recombinant SERCA2a expressed with or without PLB in insect cells. In parallel experiments the Mahaney lab has found that spin-labeled SERCA2a co-expressed with PLB has a smaller rotational correlation time (63 ms) than either SERCA2a sans PLB (78 ms) or SERCA2a + phosphorylated-PLB (97 ms. The rotational correlation times suggest a more compact structure for SERCA2a-PLB than for SERCA2a sans PLB or SERCA2a + phosphorylated-PLB (Mahaney et al, 2005). We have also collaborated with the Neuronal Cytoskeletal Section of this laboratory to examine the kinetics of cdk5 kinase activation and inhibition. The interaction of this kinase with its different activator proteins and fragments thereof produce complex effects on its biological activity. We have described a fragment (CIP) of the cdk5-activator protein, p35, that produces high-affinity inhibition of cdk5 by selective competition with the p25 activator protein. The p25 form of the cdk5 activator appears in neurons under stress, induces hyperphosphorylation of the microtubule associated protein, tau, and transfection with the inhibitor peptide can prevent this in neuronal cell culture assays (Zheng et al, 2005). We are currently engaged in kinetic studies to further define the mechanism of this selective inhibition. Cdk5 is a member of the cyclin-dependent kinase family. Although cdk5 activator proteins are not cyclins, the activator interfaces with the kinases in this family are largely similar and they induce similar conformational transitions. Thus we are also exploring computational means to obtain information about the interactions of CIP and related inhibitors with the cdk5 kinase.

该项目的主要目标是扩展我们对与神经系统特别相关的酶机制的理解。 目前与J. Froehlich（U. Md。）合作的钠，钾泵（Na，K-ATPase）和钙泵（SERCA ATPase）机制的研究目前旨在测试一个假设，即我们的某些短暂性动力学观察结果反映了他们在抗斗争过程中多泵分子的寡聚体相互作用。一个依赖的假设是，这种寡聚相互作用有助于阳离子运输的效率。我们目前的证据表明，这些相互作用介导了两个或多个催化亚基之间的“相位外”自由能转移，并且这些相互作用可能是在其本地状态检查的P型阳离子泵的一般特性。我们最近研究了小型固有膜蛋白磷脂（PLB）对SERCA1和SERCA2A钙泵的瞬时动力学和低聚状态的影响。 PLB与同工型SERCA1和SERCA2A相互作用，但通常仅与SERCA2A共表达心脏肌肉同工型。 Beta-1-激动剂对SERCA2A的激活提高了钙转运的效率，并提高了心脏肌质网钙储存。这涉及PLB的PKA依赖性磷酸化。 PLB-PUMP相互作用降低了泵的VMAX和明显的钙亲和力，而PLB的磷酸化却逆转了这两种影响。我们的数据表明，β-1激动剂的SERCA2A泵效率提高，是由于PLB的磷酸化诱导的解离。自旋标记的SERCA2A的EPR测量表明，PLB结合将SERCA2A从寡聚状态更改为功能单体的一个。瞬态动力学研究表明，在没有PLB的情况下，骨骼肌同工型SERCA1是低聚物。 SERCA1相对于SERCA2A表现更快，VMAX更高。假设寡聚泵相互作用可产生SERCA2A中不存在的SERCA1中E2P水解的快速相。我们测试了SERCA2A的PLB调节是否将其转换为类似于SERCA1的功能性寡聚状态。通过使用昆虫细胞中有或没有PLB表达的重组SERCA2A测量泵磷酸化的瞬时动力学特性来证实这一点。在并行实验中，Mahaney实验室发现，与PLB共表达的自旋标记的SERCA2A的旋转相关时间（63 ms）比SERCA2A SANS PLB（78 ms）或SERCA2A + phosphorated-PLB（97 ms）（97 ms）（97 ms。磷酸化-PLB（Mahaney等，2005）。 我们还与该实验室的神经元细胞骨架部分进行了合作，以检查CDK5激酶激活和抑制的动力学。该激酶与其不同激活蛋白及其片段的相互作用对其生物学活性产生了复杂的影响。我们已经描述了CDK5激活蛋白P35的片段（CIP），该片段通过与P25激活蛋白的选择性竞争可产生CDK5的高亲和力抑制作用。 CDK5活化剂的p25形式出现在胁迫下的神经元中，诱导微管相关蛋白，tau的高磷酸化，而用抑制剂肽转染可以在神经元细胞培养分析中预防这种磷酸化（Zheng等人，2005年）。 我们目前正在进行动力学研究，以进一步定义这种选择性抑制的机制。 CDK5是Cyclin依赖性激酶家族的成员。尽管CDK5激活蛋白不是细胞周期蛋白，但该家族中与激酶的激活剂界面在很大程度上相似，它们会诱导类似的构象转变。因此，我们还在探索计算方法，以获取有关CIP和相关抑制剂与CDK5激酶相互作用的信息。