Roles of Cholesterol and Membrane Nanodomains in the Amyloidogenic Pathway

胆固醇和膜纳米结构域在淀粉样变通路中的作用 胆固醇被认为在淀粉样蛋白-β多肽(Aβ)的形成中起着关键的调节作用。 全长淀粉样前体蛋白被β-分泌酶裂解释放的淀粉样变通路 是跨膜99个残基的C末端结构域(C99)。C99随后被γ-分泌酶切割，释放出Aβ。 膜胆固醇似乎促进了淀粉样蛋白的生成途径，许多证据表明 涉及质膜胆固醇富集区的机制通常被称为脂筏。 根据这个模型，胆固醇促进了APP和C99在脂筏中的分配，这是 富含β分泌酶和γ分泌酶，从而激活淀粉样蛋白裂解。因此，木筏是 建议在空间上将淀粉样蛋白生成途径与体膜相分离。然而，很多 支持这一模型的证据是间接的。检验这一点很重要，这是这项工作的前提 该模型如果正确，则提出了一种通过减少APP/C99的RAFT分配来减少Aβ形成的策略。 另一方面，如果该模型是不正确的，那么支持该模型的现有文献可能需要重新- 评估和追求基于这种模式的治疗策略可能需要被视为 红鲱鱼。这项工作的一个次要前提是，对生物物理和结构进行研究是至关重要的 C99/APP在RAFT和非RAFT相之间分配的生物学基础。这些研究将提供 膜相性质的主要变化对膜蛋白结构的影响 和本地化。此外，对相分配偏好的基础的分子理解 APP和C99可能激发合理的药理学策略来改变这些药物的时相偏好 用于减少Aβ形成的分子。我们注意到，为了保持适当的 (可行！)研究范围将集中在γ-分泌酶和C99。关于全长的研究 APP和β-分泌酶代表了这项工作在完成以下目标后的未来方向。 目的1.验证C99和γ-分泌酶优先定位于膜结构域的假说。 目的2.验证RAFT-缔合改变C99结构的假设。 目的3.确定γ-分泌酶对C99的裂解如何受膜分配的调节， 膜流动性、C99结构和C99-胆固醇复合体的形成。

Project Summary Roles of Cholesterol and Membrane Nanodomains in the Amyloidogenic Pathway Cholesterol is thought to play a key regulatory role in the formation of the amyloid-β polypeptide (Aβ) via the amyloidogenic pathway in which the full length amyloid precursor protein is cleaved by β-secretase to release is transmembrane 99 residue C-terminal domain (C99). C99 is then cleaved by γ-secretase to release Aβ. Membrane cholesterol appears to promote the amyloidogenic pathway, with much evidence pointing to a mechanism involving cholesterol-enriched regions of the plasma membrane often referred to as lipid rafts. According to this model, cholesterol promotes partitioning both of APP and C99 into lipid rafts, which are enriched in β-secretase and γ-secretase, thereby activating amyloidogenic cleavage. Rafts are therefore proposed to spatially segregate the amyloidogenic pathway from the bulk membrane phase. However, much of the evidence in support of this model is indirect. It is the premise of this work that it is important to test this model which, if correct, suggests a strategy for reducing Aβ formation by reducing raft partitioning of APP/C99. On the other hand, if the model is incorrect, the existing literature supporting this model may need to be re- assessed and the pursuit of therapeutic strategies based on this model would possibly need to be viewed as a red herring. A sub-premise of this work is that it is critical to conduct studies of the biophysical and structural biological basis for partitioning of C99/APP between raft and non-raft phases. These studies will provide fundamental insight how major changes in membrane phase properties impact membrane protein structure and localization. Moreover, a molecular understanding of the basis for the phase partitioning preferences of APP and C99 may inspire rational pharmacological strategies for altering the phase preferences of these molecules for the purpose of reducing Aβ formation. We note that in order to maintain an appropriate (feasible!) scope for the studies to be carried out, we will focus on γ-secretase and C99. Studies of full length APP and β-secretase represent a future direction for this work after the completion of the aims below. Aim 1. Test the hypothesis that C99 and γ-secretase preferentially localize to membrane raft domains. Aim 2. Test the hypothesis that raft-association alters the structure of C99. Aim 3. Determine how the cleavage of C99 by γ-secretase is modulated by membrane partitioning, membrane fluidity, C99 structure, and C99-cholesterol complex formation.