STRUCTURAL CHARACTERIZATION OF PRION PROTEINS

朊病毒蛋白的结构表征

• 批准号: 7369016
• 负责人: STANLEY B PRUSINER
• 金额: $ 0.06万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369016
• 关键词: STRUCTURAL; CHARACTERIZATION; PRION; PROTEINS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Prion diseases are unique fatal neurodegenerative diseases caused by novel pathogens termed prions. The only component of prions is an abnormal form of a normal cellular protein termed the prion protein (PrP). The normal form (PrPC) is converted to the pathogenic form (PrPSc) by an as yet unidentified posttranslational process. Mass spectrometry is essential for the detailed analysis of small-scale samples of the prion protein isolated from biological tissues, from recombinant sources and from peptide synthesis. We have developed and refined sensitive methods for the analysis of PrP using ESIMS, MALDI and MS/MS for the characterization of peptides and oligosaccharides from enzyme digests. These analyses are complicated by the hydrophobic, insoluble and strongly aggregating properties, and by problems associated with purification from brain material. One objective is increase the sensitivity for detection of extremely sub-stoichiometric modifications as it is known that one ID50 of infectivity contains tens of thousands of PrP molecules, not all of which are necessarily pathogenic in their own right. It is also necessary to identify possible accessory molecules that might be present in infectious prions. Comparisons are also required between PrPSc from different prion strains to establish whether strain behavior could arise from covalent differences, e.g. in the N-linked oligosaccharides, which might cause cell specific lectins to target a limited range of cell types, resulting in regional distribution of PrPSc. We are using HPLC/MS/MS for much of this characterization. Results obtained so far suggest that the modification of PrPC involved in the formation of PrPSc is likely conformational rather than chemical but this has not been proved unambiguously.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。王室疾病是由称为prions的新病原体引起的独特致命神经退行性疾病。 prions的唯一组成部分是称为prion蛋白（PRP）的正常细胞蛋白的异常形式。正常形式（PRPC）通过尚未识别的翻译后过程转换为致病形式（PRPSC）。质谱对于从生物组织，重组来源和肽合成中分离出的prion蛋白的小规模样品的详细分析至关重要。我们已经开发了使用ESIMS，MALDI和MS/MS来分析PRP的灵敏方法，以从酶消化中表征肽和寡糖。这些分析因疏水性，不溶性和强烈的汇总特性而复杂化，以及与脑物质纯化相关的问题。一个目的是增加检测极端亚化学计量修饰的灵敏度，因为众所周知，一种ID50具有数以万计的PRP分子，并不一定都是病原体。还必须确定可能存在于感染性王室中的辅助分子。从不同的prion菌株中的PRPSC也需要进行比较，以确定应变行为是否可能是由于共价差异引起的，例如在N连接的寡糖中，可能导致细胞特异性凝集素靶向有限的细胞类型，从而导致PRPSC的区域分布。我们正在使用HPLC/MS/MS进行大部分特征。到目前为止获得的结果表明，与PRPSC形成有关的PRPC的修饰可能是构象的，而不是化学的，但尚未明确证明。