Molecular Regulation of Alpha-Synuclein Misfolding and Toxicity in Two Yeast Mode

两种酵母模式下α-突触核蛋白错误折叠和毒性的分子调控

• 批准号: 7848666
• 负责人: SHUBHIK KUMAR DEBBURMAN
• 金额: $ 1.45万
• 依托单位: LAKE FOREST COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7848666
• 关键词: Antioxidants; Apoptosis; Binding; Biochemistry; Biological Assay; Biology; Cell Death; Cell membrane; Cells; Cellular biology; Centrifugation; Communities; Complementary DNA; Cookbook; Cytoplasm; Degradation Pathway; Dimethyl Sulfoxide; Disease; Educational Curriculum; Enzymes; Evaluation; Face; Fatty Acids; Fission Yeast; Functional disorder; Future; Genes; Genetic; Glutathione; Goals; Grant; Growth; Head; Human; Knock-out; Laboratories; Life; Link; Lipid Binding; Lysosomes; Measures; Mediating; Membrane; Methods; Microscopy; Midbrain structure; Mitochondria; Modeling; Molecular; Molecular Genetics; Multivesicular Body; Mutation; Necrosis; Neurodegenerative Disorders; Optics; Oxidants; Oxidative Stress; Parkinson Disease; Pathology; Pathway interactions; Phospholipid Interaction; Phospholipids; Plasmids; Principal Investigator; Production; Property; Proteins; Public Health; Publishing; Regulation; Research; Saccharomyces cerevisiae; Saccharomycetales; Science; Scientist; Staining method; Stains; Stress; Supporting Cell; Symptoms; Testing; Time; Toxic effect; Training; United States; Vacuole; Western Blotting; Yeast Model System; Yeasts; alpha synuclein; base; career; catalase; combinatorial; comparative; density; design; dopaminergic neuron; experience; expression vector; genetic manipulation; in vivo; multicatalytic endopeptidase complex; mutant; neurotoxicity; polymerization; programs; protein aggregation; protein misfolding; synuclein

Parkinson's disease (PD) is a common and fatal neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein a-synuclein, its impaired degradation, and oxidant damage are all hypotheses for the molecular cause of this selective neurotoxicity. In published findings with budding yeast (Sharma et al. 2006) and fission yeast (Brandis et al. 2006) models for a-synuclein misfolding and toxicity, we provided genetic and live cell support for these hypotheses, while uncovering unexpected yeast-specific a-synuclein property differences. In budding yeast, expression of a-synuclein alone does not cause toxicity, but the addition of either proteasomal dysfunction or mitochondrial oxidative stress is synthetic lethal. This lethality does not correlate well with a-synuclein aggregation. Instead, a-synuclein localizes primarily to the yeast plasma membrane even when toxic. In fission yeast, a-synuclein misfolds and aggregates within the cytoplasm in an exquisitely time and concentration-dependent manner, providing crucial live cell evidence for a mechanism that follows the nucleation-polymerization model. Despite the extensive aggregation, a-synuclein is not toxic. Even at low concentrations, it does not localize to the plasma membrane. By continuing such comparative analysis using both yeast models and by employing genetic manipulation in living cells, the following specific questions that all centrally focus on the molecular determinants within a-synuclein and cellular pathways that regulate its misfolding, lipid binding, degradation, and toxicity can be examined. What is the significance of the newly discovered familial PD mutation E46K in vivo? Does a-synuclein membrane localization in vivo involve specific phospholipids and is membrane interaction required for in vivo toxicity? Does a- synuclein contain domains that confer plasma membrane localization and aggregation in vivo? Can cytoplasmic oxidative stress also cause a-synuclein-mediated lethality or is lethality limited to mitochondrial stress? Lastly, does the lysosome also degrade a- synuclein and by what mechanism? The aims are: 1) To test the hypothesis that a-synuclein mutant E46K is significantly toxic to cells and binds phospholipids in vivo, E46K will compared to wild-type, A30P, A53T, and combination familial mutants (E46K/A53T, A30P/E46K, and A30P/E46K/A53T), in localization, aggregation, and toxicity in both yeasts. 2) To test the hypothesis that specific phospholipid composition and total phospholipid content is critical to a-synuclein membrane association and toxicity, a- synuclein localization (via GFP microscopy), aggregation (via GFP microcopy, Western blotting, and differential centrifugation), and toxicity (via growth curves, serial plating, and yeast cell death stains) will be evaluated in budding yeasts genetically compromised for the production of the major membrane phospholipids, and in both yeasts, supplemented with fatty acids or DMSO to increase overall phospholipid content. 3) To test the hypothesis that specific aggregation domains and lipid-binding domains mediate a-synuclein properties, N- and C-terminus fragments, along with specific point mutants will be tested for their localization, aggregation, and toxicity in both yeast models. 4) To test the hypothesis that cytoplasmic oxidative stress also contributes to a- synuclein-mediated toxicity, a-synuclein's localization, aggregation, and toxicity will be evaluated in budding yeasts compromised for major cytoplasmic antioxidant enzymes, including catalases, glutathione regulating enzymes, and DJ1, using single knockout strains, and multiple knockouts, where available. 5) To test the hypothesis that the lysosome pathway also degrades a-synuclein, the localization, aggregation, and toxicity of a-synuclein will be evaluated in budding yeast strains knocked out for genes encoding proteins that make up the well-studied multivesicular body (MVB) pathway to the yeast vacuole, which serves as its lysosome. Pilot undergraduate projects underway have already provided initial evidence to support some of these hypotheses. Once completed, these related studies, intentionally diverse in scope and designed to attract a diverse and large set of undergraduates to my lab, will together clarify the molecular bases for the regulation of the normal biology and pathobiology of a-synuclein. They will also expand the usefulness of multiple yeast models to study diverse protein misfolding diseases. Project Description Page 7 Principal Investigator/Program Director (Last, first, middle): DebBurman, Shubhik, Kumar PROJECT RELEVANCE The growing list of neurodegenerative diseases, including Parkinson's disease, represents a burgeoning public health concern in the United States. None of them are curable and most are fatal. Despite symptom diversity, many of these diseases share a common mechanism of pathology. Therefore, discoveries made with this Parkinson's disease grant may also impact progress for the other diseases. Undergraduate educators in the United States face significant challenges in preparing diverse graduates for a technologically sophisticated and scientifically interdisciplinary 21st-century community. Curricula that integrate more research and research-like experiences for non-majors and science majors in lieu of cookbook laboratory experiences, graduate a more diverse and larger group of well-prepared undergraduates that enter the scientific workforce. A major focus of this proposal is to provide rigorous scientific training to many undergraduates headed for future biomedical careers. Therefore, this proposal's major relevance is that it seeks to engage talented and diverse undergraduates in substantial and original research experiences, where they will contribute as scientists to biomedical discoveries linked to a major public health concern.

帕金森病 (PD) 是一种常见且致命的神经退行性疾病，由中脑多巴胺能神经元选择性丧失引起。 α-突触核蛋白的错误折叠和聚集、其降解受损和氧化损伤都是这种选择性神经毒性的分子原因的假设。在已发表的芽殖酵母（Sharma et al. 2006）和裂殖酵母（Brandis et al. 2006）模型的α-突触核蛋白错误折叠和毒性研究中，我们为这些假设提供了遗传和活细胞支持，同时发现了意想不到的酵母特异性α -突触核蛋白性质差异。在芽殖酵母中，单独表达α-突触核蛋白不会引起毒性，但蛋白酶体功能障碍或线粒体氧化应激的增加是合成致死的。这种致死率与α-突触核蛋白聚集没有很好的相关性。相反，即使有毒，α-突触核蛋白也主要定位于酵母质膜。在裂殖酵母中，α-突触核蛋白以精确的时间和浓度依赖性方式在细胞质内错误折叠和聚集，为遵循成核聚合模型的机制提供了重要的活细胞证据。尽管广泛聚集，α-突触核蛋白并无毒性。即使在低浓度下，它也不会定位于质膜。通过使用酵母模型继续进行这种比较分析并在活细胞中采用遗传操作，以下具体问题都集中在α-突触核蛋白内的分子决定因素以及调节其错误折叠、脂质结合、降解和毒性的细胞途径中被检查。新发现的家族性PD突变E46K在体内有何意义？体内α-突触核蛋白膜定位是否涉及特定的磷脂？体内毒性是否需要膜相互作用？ α-突触核蛋白是否含有在体内赋予质膜定位和聚集的结构域？细胞质氧化应激是否也会导致α-突触核蛋白介导的致死作用，或者致死作用仅限于线粒体应激？最后，溶酶体是否也降解α-突触核蛋白？通过什么机制？目的是： 1) 为了检验α-突触核蛋白突变体 E46K 对细胞具有显着毒性并在体内结合磷脂的假设，将 E46K 与野生型、A30P、A53T 和组合家族突变体（E46K/A53T、A30P/ E46K 和 A30P/E46K/A53T），在两种酵母中的定位、聚集和毒性。 2) 检验特定磷脂组成和总磷脂含量对α-突触核蛋白膜结合和毒性、α-突触核蛋白定位（通过 GFP 显微镜）、聚集（通过 GFP 显微镜、蛋白质印迹和差速离心）至关重要的假设，以及将在因生产主要膜磷脂而基因受损的芽殖酵母中评估毒性（通过生长曲线、连续平板接种和酵母细胞死亡染色），并且在两种酵母中，补充脂肪酸或DMSO以增加总磷脂含量。 3) 为了测试特定聚合结构域和脂质结合结构域介导α-突触核蛋白特性的假设，将在两种酵母模型中测试N-和C-末端片段以及特定点突变体的定位、聚集和毒性。 4) 为了检验细胞质氧化应激也导致α-突触核蛋白介导的毒性的假设，将在主要细胞质抗氧化酶（包括过氧化氢酶、谷胱甘肽调节酶和DJ1，使用单敲除菌株和多重敲除（如果有）。 5）为了检验溶酶体途径也降解α-突触核蛋白的假设，将在芽殖酵母菌株中评估α-突触核蛋白的定位、聚集和毒性，该酵母菌株敲除编码构成经过充分研究的多泡体的蛋白质的基因（ MVB）通往酵母液泡的途径，作为其溶酶体。正在进行的试点本科项目已经提供了支持其中一些假设的初步证据。一旦完成，这些相关研究有意在范围上多样化，旨在吸引多样化的大量本科生到我的实验室，将共同阐明α-突触核蛋白正常生物学和病理生物学调节的分子基础。他们还将扩展多种酵母模型的用途，以研究多种蛋白质错误折叠疾病。项目描述第 7 页 首席研究员/项目主任（姓、名、中）：DebBurman、Shubhik、Kumar 项目相关性 包括帕金森氏病在内的神经退行性疾病日益增多，代表了美国日益增长的公共卫生问题。它们都无法治愈，而且大多数都是致命的。尽管症状多种多样，但许多疾病具有共同的病理机制。因此，帕金森病资助的发现也可能影响其他疾病的进展。美国的本科教育工作者在为技术先进、科学跨学科的 21 世纪社区培养多元化毕业生方面面临着重大挑战。课程为非专业和科学专业整合了更多研究和类似研究的经验，而不是食谱实验室经验，毕业后将培养出更加多样化、规模更大的准备充分的本科生，他们将进入科学劳动力队伍。该提案的一个主要重点是为许多走向未来生物医学职业的本科生提供严格的科学培训。因此，该提案的主要意义在于，它寻求让才华横溢、多元化的本科生参与实质性和原创性的研究经验，他们将作为科学家为与重大公共卫生问题相关的生物医学发现做出贡献。