INTERACTIONS OF PRIONS WITH OTHER PROTEINS AND GLYCOSAMINOGLYCANS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 （合作者：波士顿大学生物化学系Harris教授）。 朊病毒疾病包括罕见的神经系统疾病，如克雅氏病和Gerstmann-Str <$ussler综合征。朊病毒疾病是由正常的细胞朊病毒蛋白（PrPC）（一种功能未知的膜糖蛋白）构象转化为PrPSc（一种错误折叠的PrPC亚型）引起的。尽管有令人信服的证据证明PrPC的构象转换成PrPSc，我们目前的了解朊病毒疾病的神经变性的主要机制仍然是有限的。 虽然人们通常认为PrPSc是所有朊病毒疾病的标志，但其神经毒性很可能依赖于细胞表面功能性PrPC分子的存在。了解PrPC的生理功能的逻辑方法是识别其相互作用伙伴。多个实验室都采用了这一策略，确定了大量的候选人。然而，到目前为止，没有一个拟议的候选人显示出与PrPC的明确联系，或提供了解其功能的线索。此外，对PrPC生理活性的重要见解也来自不同突变的表征（CR PrP，PrPC缺失残基105-125）。为了阐明导致 CR PrP毒性，以及野生型PrPC的拯救活性，我们正在研究野生型PrPC的相互作用伴侣（蛋白质和GAG）， CR PrP和PrP23-31.为此目的，相互作用伙伴正在确定质谱法。从含有野生型或突变型PrPC的样品（使用磁珠免疫沉淀）进行自下而上的方法。通过MALDI-TOF MS和nanoLC-MS/MS在LTQ-Orbitrap（Thermo-Fisher）上分析消化的肽。由于低丰度的相互作用伙伴，我们已经优化了样品制备，以避免污染。最佳条件为：1）超滤（10 kD）磁珠蛋白，2）磁珠直接消化，3）纯化，4）质谱分析。MALDI-TOF质谱显示野生型和突变型PrPC之间略有差异。在未来的工作中，将在LTQ-Orbitrap（Thermo-Fisher）上通过nanoLC-MS/MS分析这些样品，以鉴定蛋白质。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. (Collaborator Pr. Harris group, Department of Biochemistry, Boston University). Prion diseases include rare neurological disorders such as Creutzfeldt-Jakob disease and Gerstmann-Str¿ussler syndrome. Prion diseases are caused by the conformational conversion of the normal, cellular prion protein (PrPC), a membrane glycoprotein of unknown function, into PrPSc, a misfolded isoform of PrPC. Despite compelling evidence about the conformational conversion of PrPC into PrPSc, our current understanding of the primary mechanisms of neurodegeneration in prion disease is still limited. Although it is commonly assumed that PrPSc is a hallmark of all prion diseases, its neurotoxicity most probably relies on the presence of functional PrPC molecules at the cell surface. A logic approach for gaining insights into the physiological function of PrPC is to identify its interaction partners. This strategy has been pursued by multiple laboratories identified a plethora of candidates. However, so far none of the proposed candidates has shown a definitive link with PrPC, or provided a clue for understanding its function. Moreover, important insights into the physiological activity of PrPC came also from the characterization of different mutations (CR PrP, PrPC deleted for residues 105-125). In order to elucidate the mechanism responsible for CR PrP toxicity, and for wild type PrPC rescuing activity, we are investigating the interaction partners (proteins and GAGs) of wild type PrPC, CR PrP, and PrP23-31. Towards this purpose, interaction partners are being identified by mass spectrometry. A bottom-up approach is performed from samples (immunoprecipitated using magnetic beads) containing the wild type or mutant PrPC. Digested peptides have been analyzed by MALDI-TOF MS and by nanoLC-MS/MS on the LTQ-Orbitrap (Thermo-Fisher). Because of the low abundance of the interaction partners, we have optimized the sample preparation to avoid contamination. The best conditions werre found to be: 1) ultrafiltration (10 kD) of the proteins attached to the magnetic beads, 2) digestion directly on the magnet beads, 3) purification of the peptides and 4) analysis of the samples by mass spectrometry. MALDI-TOF mass spectra showed slight differences between the wild type and the mutant PrPC. In future work, these samples will be analyzed by nanoLC-MS/MS on LTQ-Orbitrap (Thermo-Fisher) to identify the proteins.