Lipid and Drug Effects on the Early Stages of hIAPP Aggregation on Membrane Surfaces Probed by Surface-Selective Chiral SFG Spectroscopy

 描述（由申请人提供）：淀粉样蛋白的聚集与许多疾病相关，包括阿尔茨海默病、帕金森病和II型糖尿病。虽然仍然有一个争论是否聚集是症状或疾病的原因，更好地了解淀粉样蛋白聚集在分子水平上可以提供关键信息的指导策略，在对抗淀粉样蛋白疾病的药物开发。 已知膜在淀粉样蛋白聚集中起关键作用。最近的研究表明，脂质分子是在症状出现之前检测淀粉样疾病的可靠生物标志物。此外，与膜的相互作用可以催化淀粉样蛋白聚集，并且在早期阶段形成的聚集产物可以破坏细胞膜并引起细胞毒性。因此，了解淀粉样蛋白在膜表面的聚集是很有意义的。然而，大多数先前的淀粉样蛋白聚集的分子研究是在溶液相中进行的。因此，关于膜表面在淀粉样蛋白聚集中的作用存在很大的知识缺口。 尽管如此，弥合差距需要表面特异性的物理方法，可以监测淀粉样蛋白与膜表面相互作用后原位和真实的时间的构象变化。挑战是需要表面特异性，其可以消除来自溶液相中的蛋白质和来自水溶剂的信号的干扰，使得可以保留表面信息。在过去的5年中，我们已经开发了表面特异性手性和频产生（cSFG）振动光谱成为一种新的方法，在接口处的蛋白质二级结构的无标记表征。我们成功地使用cSFG来研究与II型糖尿病相关的人胰岛淀粉样多肽（hIAPP）。我们监测了hIAPP与脂质表面相互作用时从无序结构到β-螺旋再到β-折叠的错误折叠动力学。此外，我们进行了从头计算分析cSFG光谱，并获得了膜表面的hIAPP的折叠聚集体的分子取向。 在这里，我们建议使用cSFG方法结合其他表面化学方法来研究hIAPP在膜表面的早期聚集。我们将重点关注（1）脂质组合物，特别是那些被证明可用于检测淀粉样蛋白疾病的生物标志物脂质，（2）候选药物，包括已知抑制原纤维形成的小分子和肽，以及（3）hIAPP的S20 G突变体，唯一与II型糖尿病早发相关的突变体。我们将在不同水平的生物标志物脂质和添加候选药物的情况下测量脂膜上hIAPP和突变体的构象变化率。这些结果将提供对淀粉样蛋白在膜表面早期聚集的机制的理解，为淀粉样蛋白聚集在疾病发病机制中的作用提供见解，并为淀粉样蛋白研究中优先考虑资源提供指导。

 DESCRIPTION (provided by applicant): Aggregation of amyloid proteins is associated with a number of diseases, including Alzheimer's disease, Parkinson's disease, and type II diabetes. Although there is still a debate on whether the aggregation is the symptoms or the cause of the diseases, a better understanding of amyloid aggregation at the molecular level can provide crucial information for steering strategies for drug development in fighting amyloid diseases. Membrane is known to play a crucial role in amyloid aggregation. Recent studies have shown that lipid molecules are reliable biomarkers for detecting amyloid diseases before the onset of symptoms. Moreover, interactions with membrane can catalyze amyloid aggregation and the aggregation product formed at the early stages can disrupt cell membrane and cause cytotoxicity. Thus, it is significant to understand amyloid aggregation on membrane surfaces. However, most previous molecular studies of amyloid aggregation were carried out in solution phase. Hence, there is a large gap of knowledge about the role of membrane surfaces in amyloid aggregation. Nonetheless, bridging the gap requires surface-specific physical methods that can monitor conformational changes in amyloid proteins upon interactions with membrane surfaces in situ and in real time. The challenge is the need of surface-specificity that can eliminate interference of signals from proteins in solution phase and from water solvent such that surface information can be preserved. For the last 5 years, we have developed surface-specific chiral sum frequency generation (cSFG) vibrational spectroscopy into a new approach for label-free characterization of protein secondary structures at interfaces. We successfully used cSFG to study human islet amyloid polypeptide (hIAPP) that is associated with type II diabetes. We monitored the kinetics of the misfolding of hIAPP from disordered structures to -helices and then -sheets upon interactions with lipid surfaces. Also, we preformed ab initio calculations for analyzing cSFG spectra and obtained molecular orientation of the -sheet aggregates of hIAPP on membrane surfaces. Here, we propose to use cSFG method combined with other surface chemistry methods to study the aggregation of hIAPP in early stages on membrane surfaces. We will focus on the effects of (1) lipid compositions, particularly those biomarker lipids proven to be useful for detecting amyloid diseases, (2) drug candidates, including small molecules and peptides, that are known to inhibit fibril formation, and (3) the S20G mutant of hIAPP, the only mutant associated with early onset of type II diabetes. We will measure the rates of conformational changes of hIAPP and the mutant on lipid membranes with various levels of biomarker lipids and addition of the drug candidates. The results will offer mechanistic understanding of amyloid aggregation in the early stages on the membrane surfaces, providing insights into the role of amyloid aggregation in pathogenesis of the diseases and offering guidance in prioritizing resource in amyloid research.