Enzymological Aspects Of Neural Functions

神经功能的酶学方面

基本信息

批准号：
7969502
负责人：
ROBERT W ALBERS
金额：
$ 29.88万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
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/7969502
关键词：
Affect Alzheimer&apos s Disease Binding C-terminal Calpain Charge Complex Cyclin-Dependent Kinases Cyclins DNA Sequence Rearrangement Data Enzymes Family Goals Investigation Microtubules Molecular Molecular Conformation Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neurofibrillary Tangles Neurons Neurophysiology - biologic function Pathology Peptide Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Physiological Positioning Attribute Process Property Protein Kinase Proteins Proteolysis Regulation Relative (related person)Relaxation Reporting Stress Structure Surface System Testing Thermodynamics Transfection Work base conformational conversion design enzyme activity enzyme mechanism flexibility hyperphosphorylated tau inhibitor/antagonist insight interest interfacial member molecular dynamics monomer neuropathology prevent tau Proteins

项目摘要

Normally-activated Cdk5 functions in essential neuronal regulatory processes. The occurrence of significant amounts of p25 in neurons appears to result from the abnormally high calpain protease activity in stressed neurons. P25-activated kinase within neurons produces hyperphosphorylated tau protein, which causes tau to dissociate from microtubules and accumulate as a major component of the neurofibrillary tangles that occur in Alzheimer disease. When CIP is expressed in cultured neurons by transfection, it selectively inhibits this pathological activity of cdk5/p25 but not the normal cdk5/p35 activity. Thus when neurons in cullture are stressed to produce p25 and subsequently neurofibrillary tangles, similarly-treated neurons do not produce neurofibrillary tangles if they were first transfected to produce CIP (Zheng et al, 2005). We are engaged in 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. From examination of the crystal structure of the cdk5/p25 complex (Tarricone, et al), when complexed with cdk5, it is evident that,the N- and C- terminals of p25 interact with each other at the molecular surface opposite to that of the p25 interface with cdk5. The truncations that produce CIP delete this interaction but do not eliminate any part of the p25 binding interface with cdk5. These observations suggest that a flexibility hypothesis can explain how truncations can convert p25 activation to selective inhibition. We are employing molecular dynamics simulations of cdk5/p25 and cdk5/CIP complexes to examine the effects of these truncations on the conformations of cdk5/CIP and of the active kinase. Work in progress: Truncations that form CIP from p25 do not remove residues directly involved in the cdk5/p25 interface. Preliminary investigations, some of which were described in the last report, indicate that small changes in this interface can alter the conformation of the kinase substrate-binding groove. This, in turn, may alter the catalytic activity at the kinase phosphorylation site. We are currently acquiring and analysing molecular dynamics data designed to test this and related issues. Large-scale molecular dynamic simulations of the cdk5-p25/CIP complexes, and the corresponding monomers, have been conducted to gain insight into the structure, dynamics, and thermodynamics of the system. Preliminary analyses show that truncation of the N- and C-terminus of p25 leads to structural relaxation and gentle rearrangement of some of the helices in CIP. This relaxation, in turn, elicits larger structural changes in two key loops located at the cdk5-CIP interface, which have been implicated in both cdk5-p25 complex formation, and binding and stabilization of the substrate. In particular, one of these loops contains E240, which is known to interact electrostatically with a conserved charged residue in the substrate (position +3 from the phosphorylation site). The structural changes of this loop repositions E240 relative to the substrate-binding pocket, which may prevent its direct interaction with the substrate. These changes may explain, at least partially, the inhibitory properties of CIP. In addition, crystallographic data show that the active conformation of cdk5 is stabilized by specific interfacial interactions with p25. These stabilizing centers are perturbed in the cdk5-CIP complex, which may destabilize the active conformation of the kinase and further contribute to the inhibitory action of CIP. A thermodynamic study is underway to quantify these processes. Truncation of the N- and C-termini of p25 may thus affect both the active conformation of the kinase and the binding of the substrate, both deleterious to enzyme activity.

正常激活的CDK5在基本神经元调节过程中起作用。神经元中p25大量的发生似乎是由于压力神经元异常高的钙蛋白酶蛋白酶活性引起的。神经元内的P25激活激酶会产生高磷酸化的tau蛋白，这会导致Tau从微管中解离，并积聚为阿尔茨海默氏病中神经纤维缠结的主要组成部分。当CIP通过转染在培养的神经元中表达时，它有选择地抑制CDK5/p25的病理活性，而不是正常的CDK5/p35活性。因此，当将神经元压力以产生p25并随后的神经原纤维缠结时，如果首先转染产生CIP（Zheng等，2005），则类似地处理的神经元不会产生神经原纤维缠结。我们正在研究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。从检查CDK5/p25复合物的晶体结构（Tarricone等）与CDK5复合时，很明显，P25的N和C末端在与P25界面与CDK5相反的分子表面相互相互作用。产生CIP的截断删除了这种相互作用，但不会消除与CDK5的p25结合界面的任何部分。这些观察结果表明，灵活性假设可以解释截断如何将P25激活转化为选择性抑制。我们正在采用CDK5/P25和CDK5/CIP复合物的分子动力学模拟来检查这些截断对CDK5/CIP和活性激酶构象的影响。正在进行的工作：从P25形成CIP的截断不会删除直接参与CDK5/P25接口的残基。初步研究（其中一些在上次报告中进行了描述）表明，该界面的小变化可以改变激酶底物结合凹槽的构象。反过来，这可能会改变激酶磷酸化位点的催化活性。我们目前正在获取和分析旨在测试此问题和相关问题的分子动力学数据。已经进行了CDK5-P25/CIP复合物以及相应的单体的大规模分子动态模拟，以深入了解系统的结构，动力学和热力学。初步分析表明，p25的N端和C端的截断会导致CIP中某些螺旋的结构放松和轻轻的重排。反过来，这种放松在位于CDK5-CIP界面的两个关键环中引起了较大的结构变化，这与CDK5-P25复合物的形成均涉及，以及底物的结合和稳定。特别是，其中一个循环包含E240，已知该循环与底物中的保守带电残基（来自磷酸化位点的位置+3）静电相互作用。该循环重新定位的结构变化相对于底物结合口袋，这可能会阻止其直接与底物相互作用。这些变化可能至少部分解释了CIP的抑制特性。此外，晶体学数据表明，通过特定的界面相互作用与p25稳定CDK5的活动构象。这些稳定中心在CDK5-CIP复合物中受到干扰，这可能会破坏激酶的活跃构象，并进一步有助于CIP的抑制作用。正在进行一项热力学研究以量化这些过程。 p25的N-和C末端的截断可能会影响激酶的活性构象和底物的结合，都对酶活性有害。