Hyper phosphorylation and the plexin CRMP scaffold in Alzheimers Disease

阿尔茨海默病中的过度磷酸化和 plexin CRMP 支架

• 批准号: 10063377
• 负责人: MATTHIAS BUCK
• 金额: $ 44.28万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063377
• 关键词: Actins; Alzheimer' s Disease; Amides; Amyloid beta-Protein; Antibodies; Antigens; Behavior; Binding; Biochemical; Bioinformatics; Biological Markers; Biophysical Process; Brain; Cardiovascular system; Cells; Complex; Computer Models; Development; Diagnostic; Disease; Early Diagnosis; Exploratory/Developmental Grant; Future; Goals; Grain; Grant; Hydrogen; In Vitro; Injury; Knowledge; Label; Laboratories; Learning; Mass Spectrum Analysis; Mediator of activation protein; Memory; Microtubules; Molecular; Monoclonal Antibodies; Mutation; Natural regeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Peptides; Phosphorylation; Phosphotransferases; Physiologic pulse; Process; Proteins; Research; Semaphorin-3A; Spin Labels; Structure; System; Therapeutic; Work; base; biophysical techniques; cell motility; collapsin response mediator protein-2; early detection biomarkers; mimetics; molecular dynamics; molecular modeling; neuron development; plexin; potential biomarker; receptor; reconstitution; response; scaffold; structural biology; tau Proteins

Plexins receptors make guidance decisions for cell migration in cardiovascular and neuronal development, disease, and regeneration. Plexins are also associated with higher brain functions, memory and learning. A protein that has been associated with Alzheimer's, Parkinson's and other neuronal diseases and injuries is the Collapsin Response Mediator Protein (CRMP-2) which interacts with several kinases and becomes hyper- phosphorylated alongside their increased activation. The hyper-phosphorylated CRMP-2 then disrupts the formation of actin and microtubule cytoskeletal structures and it thought to impede Aβ and tau clearance. The intracellular region of plexin is known to interact directly with CRMP and the kinase, Fyn. CRMP can form a bigger complex with Cdk5 and GSK3β also involved in Alzheimer's. Our working hypothesis is that the Fyn- Plexin-CRMP interactions form a scaffold for the association and hyper-activation of several other kinases and that the formation of this complex could be used an early biomarker for the development of neuronal diseases. The proposal has three subaims. Subaim 1) seeks to establish the phosphorylation patters of various kinases on the intracellular domains of plexin-A1,-A2 and –A4 and on CRMP2 (and various complexes) in vitro. 2) The effect that the corresponding phosphomimetic mutations have on the level of activity of the plexins and of the kinases will be studied in vitro. 3) The structure and dynamics of the reconstituted plexin-CRMP-kinase complexes will be examined by a number of biophysical techniques, ranging from 19F NMR, using –CF3 labeled proteins, pulsed EPR, using spin-labeled proteins, to HD-MS (amide hydrogen exchange-mass spectrometry) as well as computational modeling and extensive all-atom/coarse grained molecular dynamics simulations. Finally, 4) the prominent linear phosphorylation motifs that are detected, will be the subject to a bioinformatics search for similar motifs and will be used as an antigen to generate monoclonal antibodies. The knowledge obtained with the plexin-CRMP-kinase complexes will likely provide a new perspective on AD and other neurodegenerative diseases. An antibody (and eventually complex-disrupting-peptides) will likely inform the future development of regeneration/AD-targeted diagnostics and/or therapeutics. As noted in the description of the R21 mechanism, we seek to establish a proof of concept and provide some of the knowledge, if not early leads towards a biomarker. A plexin project has been established in the Buck laboratory for more than a decade (currently in its 3rd R01 grant cycle), but work on CRMP and AD-associated kinases is an entirely new avenue of research in the applicant's laboratory.

丛蛋白受体在心血管和神经元发育中为细胞迁移做出指导决定， 疾病和再生丛蛋白还与更高的大脑功能、记忆和学习有关。一 一种与阿尔茨海默氏症、帕金森氏症和其他神经元疾病和损伤有关的蛋白质是 溃缩蛋白反应介导蛋白（CRMP-2）与几种激酶相互作用， 磷酸化的同时也增加了它们的活化。然后，过度磷酸化的CRMP-2破坏了 肌动蛋白和微管细胞骨架结构的形成，并被认为阻碍Aβ和tau的清除。的 已知丛蛋白的细胞内区域与CRMP和激酶Fyn直接相互作用。CRMP可以形成 与Cdk 5和GSK 3 β形成的更大的复合物也与阿尔茨海默氏症有关。我们的假设是菲恩河- 丛状蛋白-CRMP相互作用形成了一个支架，用于缔合和超活化几种其他激酶， 这种复合物的形成可以用作神经元疾病发展的早期生物标志物。 该提案有三个次级目标。Subaim 1）试图建立各种激酶的磷酸化模式 对丛蛋白-A1、-A2和-A4的细胞内结构域以及对CRMP 2（和各种复合物）的体外作用。2)的 相应的磷酸化模拟物突变对丛蛋白活性水平的影响， 将在体外研究激酶。3)重组丛蛋白CRMP激酶的结构和动力学 复合物将通过许多生物物理技术进行检查，从19 F NMR，使用-CF 3 标记的蛋白质，脉冲EPR，使用自旋标记的蛋白质，到HD-MS（酰胺氢交换-质谱 光谱）以及计算建模和广泛的全原子/粗粒分子动力学 模拟最后，4）检测到的突出的线性磷酸化基序将是一个 生物信息学搜索相似的基序，并将其用作抗原以产生单克隆抗体。的 用丛蛋白-CRMP-激酶复合物获得的知识将可能提供关于AD的新观点， 其他神经退行性疾病。抗体（以及最终的复合物破坏肽）可能会告知 再生/AD靶向诊断和/或治疗的未来发展。中指出 R21机制的描述，我们试图建立一个概念证明，并提供一些 知识，如果不是早期导致生物标志物。在巴克建立了一个丛蛋白项目， 实验室超过十年（目前在其第三个R 01赠款周期），但对CRMP和AD相关的工作 激酶是申请人实验室中的一种全新的研究途径。