Replication mechanism of human prions

描述(由申请人提供)：Pron是由错误折叠的蛋白质组成的，在感染病原体中是前所未有的。哺乳动物的Prion往往在神经系统中大量积聚，导致致命的神经退化。人类Pron病最常见的形式是散发性克雅病(SCJD)。目前尚不清楚sCJD病毒的起源、光谱和结构。该病的特殊表型变异性表明，存在许多不同构象的致病病毒蛋白(PrPSc)，编码不同的CJD病毒株。此外，在大约40%的sCJD大脑中，PrPSc的不同蛋白分解片段共存，这提出了一种有趣的可能性，即构象上不同的sCJD普恩病毒共存于同一宿主中。这反过来又提出了关于sCJD普恩的起源、它们的进化和假设干扰的根本问题。这一应用的重点是破译sCJD患者PrPSc的构象，并阐明人类PrPSc复制的机制。新的构象方法与蛋白质错误折叠循环扩增(PMCA)和构象依赖免疫分析(CDI)相结合，将用于鉴定人类PrPSc中编码不同菌株sCJD蛋白的构象和关键结构域。通过PMCA体外扩增的脑源性PrPSc和PrPSc类构象中氢/氢交换的分析，结合定点自旋标记策略，我们应该能够阐明天然sCJD病毒复制中依赖于应变的构象机制。此外，用串联的CDI和PMCA结合结构数据测量sCJD Prion的复制率应该可以确定大脑PrPSc调节复制率的特定构象特征，从而确定疾病的进展率。这些研究的最终目的是促进我们对控制人类病毒株复制的分子机制的理解，以及PrPSc构象对sCJD表型和疾病传播性的影响。这一见解对于开发针对sCJD PrPSc或其复制的治疗策略至关重要。

DESCRIPTION (provided by applicant): Prions, unprecedented among infectious pathogens, are composed of misfolded proteins. Mammalian prions tend to accumulate at high levels in the nervous system, causing fatal neurodegeneration. The most common form of human prion disorders is sporadic Creutzfeldt-Jakob disease (sCJD). The origin, spectrum, and structure of sCJD prions are unknown. The extraordinary phenotypic variability of the disease suggests the existence of many different conformers of pathogenic prion protein (PrPSc) coding for distinct CJD prion strains. Moreover, the co-existence of different proteolytic fragments of PrPSc in approximately 40% of sCJD brains has raised the intriguing possibility that conformationally distinct sCJD prions coexist in the same host. This, in turn, raises fundamental questions about the origin of sCJD prions, their evolution and hypothetical interference. The focus of this application is to decipher the conformation of PrPSc in sCJD patients and to elucidate the mechanism underlying the replication of human prions. Novel conformational methods in tandem with protein misfolding cyclic amplification (PMCA) and conformation-dependent immunoassay (CDI) will be used to identify the conformations and critical domains in human PrPSc that code for different strains of sCJD prions. The analysis of hydrogen/deuterium exchange in brain-derived PrPSc and PrPSc-like conformers amplified in vitro by PMCA, together with site-directed spin labeling strategy, should allow us to elucidate the strain- dependent conformational mechanism in the replication of natural sCJD prions. Furthermore, measurements of replication rates of sCJD prions with tandem CDI and PMCA combined with structural data should determine the specific conformational features of brain PrPSc that modulate replication rates and thus the progression rate of the disease. The ultimate goal of these studies is to advance our understanding of the molecular mechanisms governing the replication of human prion strains and the impact of PrPSc conformation upon the sCJD phenotype and transmissibility of the disease. This insight is critical for efforts to develop therapeutic strategies targeting sCJD PrPSc or its replication.