Phosphorylation of Prion Protein as a novel mechanism for conversion

朊病毒蛋白的磷酸化作为一种​​新的转化机制

• 批准号: 7908715
• 负责人: ANDREA C LEBLANC
• 金额: $ 13.09万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908715
• 关键词: Affect; Animals; Antibodies; Biology; Brain; Cattle; Cell Fractionation; Cell Line; Cell model; Cells; Congo Red; Cyclin-Dependent Kinase 5; Disease; Electron Microscopy; Endopeptidase K; Epitopes; Exploratory/Developmental Grant; Goals; Hamsters; Human; Human Cell Line; Immune Sera; Immunohistochemistry; Immunoprecipitation; In Vitro; Induced Mutation; Infection; Knowledge; Label; Methods; Molecular; Mus; Nature; Neuroblastoma; Neurons; Orthophosphate; Peptide Hydrolases; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Phosphotransferases; Predisposition; Prevention; Prion Diseases; Prions; Proline; Proteins; Recombinants; Relative (related person); Research; Resistance; Scrapie; Site; Staining method; Stains; Structure; System; Techniques; Testing; Therapeutic; Western Blotting; in vivo; mutant; non-prion; novel; overexpression; public health relevance; research study

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine if phosphorylation is involved in normal or pathological prion protein (PrP) biology. Endogenously expressed normal cellular prion protein converts into a proteinase K resistant (PKRES) and fibrillar form when neurons in brain are exposed to prion transmissible material or express a familial mutant form of prion protein. The reason for this conversion is unclear. We show here new preliminary evidence that purified recombinant human, mouse and hamster prion protein can be phosphorylated by the neuronal specific and proline-directed cyclin dependent kinase-5 (cdk5) in vitro. A fragment of the phosphorylated prion protein becomes resistant to proteinase K as it often does in prion diseases. Furthermore, electron microscopy shows the formation of fibrillar and globular aggregate structures in phosphorylated prion protein. Therefore, phosphorylation could be a logical mechanism for the conversion of prion protein observed in disease states and could even be involved in the transmissible nature of the prion protein. While in vitro phosphorylation is easily tested, it is much more difficult to determine if phosphorylation of a specific protein occurs in vivo. Therefore, the goal of this exploratory application is to determine if phosphorylation exists in vivo. We will assess phosphorylation with a three-prong approach: using phospho-columns and western blot analyses, generating phospho-PrP specific antibodies to assess phospho-PrP by western blotting or immunohistochemistry, and immunoprecipitating prion protein from gamma-32P- orthophosphate-labeled cells. We will perform these techniques in systems known to be sensitive to transmissible prions and capable of generating protease-resistant prion protein: normal or mutant prion protein- transfected mouse neuroblastoma N2A cell lines, and human and mouse brains affected by prion diseases. Our preliminary evidence indicates cdk5-independent phosphorylation of PrP in N2A cells and in scrapie- infected mouse brains. Therefore, another goal of this application is to determine which other kinases are involved in prion protein phosphorylation, whether phosphorylation of prion protein at different sites generates identical or different protease resistant fragments, and whether phosphorylated prion protein is transmissible or induced by scrapie exposure. If positive, the result of the present application would not only add novel and potentially important mechanisms to the current state of knowledge of prion protein conversion in vivo, but could have a very high impact on how we view and treat prion diseases. PUBLIC HEALTH RELEVANCE: Prion diseases are transmitted through the conversion of the normally inoffensive prion protein of a host (human, bovine, Elk) into a disease-causing protein. The mechanism for the conversion of this protein is unknown. The present application examines one possible mechanism that would explain the conversion of prion protein and offer new avenues for the prevention or treatment of prion diseases.

描述（由申请人提供）：该提案的长期目标是确定磷酸化是否参与正常或病理prion蛋白（PRP）生物学。当大脑中的神经元暴露于可传播材料或表达家族性突变蛋白的家族性突变形式时，内源表达的正常细胞蛋白会转化为蛋白酶K耐药（PKRE）和原纤维形式。这种转换的原因尚不清楚。我们在这里显示了新的初步证据表明，纯化的重组人，小鼠和仓鼠prion蛋白可以在体外被神经元特异性和指导的细胞周期蛋白依赖性激酶5（CDK5）磷酸化。磷酸化的prion蛋白的碎片对蛋白酶K具有抗性，就像在prion疾病中一样。此外，电子显微镜显示了磷酸化的prion蛋白中原纤维和球状骨料结构的形成。因此，磷酸化可能是在疾病状态下观察到的prion蛋白转化的逻辑机制，甚至可能参与prion蛋白的可传染性。尽管体外磷酸化很容易测试，但要确定特定蛋白的磷酸化是否在体内发生要困难得多。因此，该探索性应用的目的是确定体内是否存在磷酸化。我们将通过三键方法评估磷酸化：使用磷酸化 - 殖民地和蛋白质印迹分析，生成磷酸化特异性抗体，通过蛋白质印迹或免疫组织化学评估磷酸化抗体，以及免疫沉淀的prion蛋白来自γ-32P-磷酸磷酸根磷酸根磷酸根磷酸盐蛋白。我们将在已知对可传染性prions敏感的系统中执行这些技术，并能够产生抗蛋白酶的prion蛋白：正常或突变的prion蛋白转染的小鼠神经母细胞瘤N2A细胞系，以及受prion疾病影响的人和小鼠大脑。我们的初步证据表明，N2A细胞和被抓感染的小鼠大脑中PRP的CDK5无关磷酸化。因此，该应用的另一个目标是确定哪些其他激酶参与prion蛋白磷酸化，不同位点的prion蛋白的磷酸化是否会产生相同或不同的蛋白酶耐药片段，以及磷酸化的prion蛋白是否可以通过刮屑暴露来传播或诱导。如果肯定，本应用的结果不仅会为体内的prion蛋白转化率的当前知识状态增加新颖而潜在的重要机制，而且对我们如何看待和治疗prion疾病会产生很大的影响。公共卫生相关性：通过将宿主（人类，牛，麋鹿）的正常非正式pr蛋白蛋白转化为引起疾病的蛋白质，使病毒疾病传播。该蛋白转化的机制尚不清楚。本应用研究了一种可能解释prion蛋白转化的可能机制，并为预防或治疗prion疾病提供了新的途径。