Lopes Dahabada H J, Sinha Sharmistha, Rosensweig Clark, Bitan Gal
Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.
Methods Mol Biol. 2012;849:11-21. doi: 10.1007/978-1-61779-551-0_2.
Assembly of amyloidogenic proteins into toxic oligomers and fibrils is an important pathogenic feature of over 30 amyloid-related diseases. Understanding the structures and mechanisms involved in the assembly process is necessary for rational approaches geared at inhibiting formation of these toxic species. Here, we review the application of photo-induced cross-linking of unmodified proteins (PICUP) to two disease-related amyloidogenic proteins (1) islet amyloid polypeptide (IAPP), whose toxic oligomers are thought to cause the demise of pancreatic β-cells in type-2 diabetes mellitus and (2) α-synuclein, which aggregates into toxic oligomers and precipitates in Lewy bodies in Parkinson's disease. PICUP is an effective method allowing chemical "freezing" of dynamically changing oligomers and subsequent study of the oligomer size distribution that existed before cross-linking. The method has provided insights into the factors controlling early oligomerization, which could not be obtained by other means. We discuss sample preparation, experimental details, optimization of parameters, and troubleshooting.
淀粉样蛋白组装成有毒的寡聚体和纤维是30多种淀粉样相关疾病的一个重要致病特征。了解组装过程中涉及的结构和机制对于旨在抑制这些有毒物质形成的合理方法是必要的。在这里，我们综述了未修饰蛋白质的光诱导交联（PICUP）在两种与疾病相关的淀粉样蛋白上的应用：（1）胰岛淀粉样多肽（IAPP），其有毒寡聚体被认为会导致2型糖尿病中胰腺β细胞的死亡；（2）α-突触核蛋白，它聚集形成有毒寡聚体并在帕金森病的路易小体中沉淀。PICUP是一种有效的方法，可以化学“冻结”动态变化的寡聚体，并随后研究交联前存在的寡聚体大小分布。该方法为控制早期寡聚化的因素提供了见解，而这些因素是通过其他方法无法获得的。我们讨论了样品制备、实验细节、参数优化和故障排除。
