Hyper phosphorylation and the plexin CRMP scaffold in Alzheimers Disease

丛状蛋白受体为心血管和神经元发育中的细胞迁移做出指导决定， 疾病和再生。神经丛蛋白还与更高的大脑功能、记忆力和学习能力有关。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 崩溃蛋白反应介体蛋白(CRMP-2)，它与几种激酶相互作用，成为高... 伴随着它们的激活而被磷酸化。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，认为它阻碍了Aβ和tau的清除。这个 已知丛蛋白的胞内区直接与CRMP和激酶Fyn相互作用。CRMP可以形成一个 更大的与CDK5和Gsk3β的复合体也与阿尔茨海默氏症有关。我们的工作假设是Fyn- 丛状蛋白-CRMP的相互作用形成了一个支架，用于联合和过度激活其他几种激酶和 该复合体的形成可作为神经元疾病发生发展的早期生物标志物。 该提案有三个分目标。Subaim 1)试图建立各种激酶的磷酸化模式 在体外，在丛状蛋白-A1、-A2和-A4的胞内区和CRMP2(以及各种复合体)上。2) 相应的拟磷酸化突变对神经丛蛋白活性水平的影响 激酶将在体外进行研究。3)重组丛状蛋白-CRMP-激酶的结构和动力学 络合物将通过许多生物物理技术进行检查，范围从19F核磁共振，使用-CF3 使用自旋标记蛋白质的脉冲EPR标记蛋白质到HD-MS(酰胺氢交换质量 光谱分析)以及计算模型和广泛的全原子/粗粒分子动力学 模拟。最后，4)检测到的显著线性磷酸化基序将受到 生物信息学寻找相似的基序，并将被用作抗原来产生单克隆抗体。这个 从丛状蛋白-CRMP-激酶复合体中获得的知识将可能为AD和 其他神经退行性疾病。抗体(最终是复杂的扰乱多肽)可能会告诉我们 再生/AD靶向诊断和/或治疗的未来发展。正如在 描述R21机制，我们寻求建立一个概念证明并提供一些 知识即使不是早期的，也会导致生物标记物的出现。在巴克建立了一个Plexin项目 实验室工作了十多年(目前处于第三个R01赠款周期)，但致力于CRMP和AD相关工作 在申请者的实验室中，激酶是一种全新的研究途径。

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.