PEPTIDE SYNTHESIS, OLIGOMER PREPARATION AND CONFORMATION-DEPENDENT ANTIBODY CORE

尽管有令人信服的证据表明淀粉样蛋白形成蛋白在疾病中的因果作用， 淀粉样蛋白及其组装状态的存在争议，仍有待建立。原纤维淀粉样蛋白前体 球蛋白是直径约3-10 nm的可溶性球形聚集体， 许多不同类型的淀粉样蛋白的电子和原子力显微镜。其中一个重要的进展是， 我们对前原纤维寡聚体的理解是发现淀粉样蛋白寡聚体有一个共同的肽 骨架结构，这是不同于淀粉样纤维的基础上观察到的构象依赖- ENT抗体特异性识别淀粉样蛋白寡聚体上的共同表位，但不识别原纤维、单体或 天然折叠的蛋白质的许多不同类型的蛋白质。这表明抗体识别一种基因， 一般多肽骨架表位，独立于氨基酸序列，但在氨基酸序列中共享 常见于所有类型的淀粉样蛋白低聚物。抗寡聚体抗体也一般抑制毒性- 在体外检测可溶性低聚物的含量。由于不同的淀粉样蛋白低聚物具有共同的结构， 它们对细胞具有一般毒性，无论它们来自什么蛋白质，这表明它们 在退行性疾病中具有相同的主要毒性机制。越来越多的证据表明- 认为淀粉样蛋白低聚物引起的膜透化可能代表了常见的主要机制， 淀粉样蛋白相关变性疾病的发病机制。 本项目的总体目标是阐明膜透化的机制， 淀粉样蛋白前原纤维低聚物。我支持项目的具体目标，核心C将提供同源- 预纤维Aft低聚物的项目，以及荧光和氘代Aft- gomers。核心C还将提供替代组装状态的制剂，如淀粉样蛋白原纤维和抗- 作为对照的环状原纤维。核心C还提供了特异性地识别蛋白质的构象依赖性抗体。 识别用于阻断膜透化的前原纤维低聚物、原纤维和环形原纤维 以及在实验期间和之后验证低聚物制剂的构象状态。

Even though there is compelling evidence for a causal role of amyloid forming proteins in disease, the type of amyloid and its assembly state is controversial and remains to be established. Prefibrillar amyloid oli- gomers are soluble spherical aggregates of approximately 3-10 nm in diameter that have been observed for many different types of amyloids by electron and atomic force microscopy. One of the significant advances in our understanding of prefibrillar oligomers was the finding that amyloid oligomers have a common peptide backbone structure that is distinct from amyloid fibrils based on the observation that a conformation depend- ent antibody specifically recognizes a common epitope on amyloid oligomers, but not fibrils, monomers or natively folded proteins for many different types of proteins. This indicates that the antibody recognizes a ge- neric polypeptide backbone epitope that is independent of the amino acid sequence, but yet is shared in common among all types of amyloid oligomers. The anti-oligomer antibody also generically inhibits the toxic- ity of soluble oligomers examined in vitro. Since different amyloid oligomers share a common structure and they are generically toxic to cells regardless of what protein they are derived from, this suggests that they have the same primary mechanism of toxicity in degenerative diseases. A growing body of evidence sug- gests that membrane permeabilization by amyloid oligomers may represent the common, primary mecha- nism of pathogenesis of amyloid related degenerative diseases. The overall goal of this program project is to elucidate the mechanism of membrane permeabilization by amyloid prefibrillar oligomers. I support of the specific aims of the projects, Core C will provide homogene- ous preparations of prefibrillar Aft oligomers to the projects as well as fluorescent and deuterated Aftoli- gomers. Core C will also provide preparations of alternative assembly states, such as amyloid fibrils and an- nular protofibrils as controls. Core C also provides conformation dependent antibodies that specifically rec- ognize prefibrillar oligomers, fibrils and annular protofibrils for use in blocking membrane permeabilization and verifying the conformational status of the oligomer preparations during and after the experiments.