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.

plexins受体在心血管和神经元发育中为细胞迁移做出指导决策， 疾病和再生。丛蛋白还与较高的大脑功能，记忆和学习有关。一个 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 折叠蛋白反应介质蛋白（CRMP-2）与几种激酶相互作用，并变为超级 磷酸化并随着它们的激活增加而磷酸化。超磷酸化的CRMP-2然后破坏 肌动蛋白和微管细胞骨架结构的形成，并被认为会阻碍Aβ和TAU清除率。这 已知丛内细胞内区域与CRMP和激酶Fyn直接相互作用。 CRMP可以形成 与阿尔茨海默氏症的CDK5和GSK3β的更大复合物。我们的工作假设是Fyn- Plexin-CRMP相互作用形成了其他几种激酶的关联和过度激活的支架 这种复合物的形成可以用于神经元疾病的发育的早期生物标志物。 该提案有三个子。 Subaim 1）试图建立各种激酶的辐射模式 在Plexin-A1，-a2和–a4的细胞内结构域以及体外CRMP2（以及各种复合物）上。 2） 相应的磷酸化突变对丛蛋白和活性水平的影响 激酶将在体外进行研究。 3）重构的丛蛋白-CRMP-激酶的结构和动力学 复合物将通过许多生物物理技术检查，范围从19f NMR，使用–CF3 使用自旋标记的蛋白与HD-MS（酰胺氢交换 - 质量）标记的蛋白质，脉冲EPR，脉冲EPR 光谱法）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受试者 生物信息学搜索类似的基序，并将用作产生单克隆抗体的抗原。 用plexin-crMP-激酶复合物获得的知识可能会为AD和 其他神经退行性疾病。抗体（有时甚至是复杂的干扰肽）可能会告知 再生/针对性诊断和/或治疗的未来发展。如在 R21机制的描述，我们寻求建立概念证明并提供一些 知识（即使不是早期）会导致生物标志物。笨蛋已经建立了一个丛蛋白项目 实验室已有十多年的 激酶是申请人实验室研究的全新研究途径。