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.

帕金森氏病（PD）是一种常见且致命的神经退行性疾病，是由于中脑多巴胺能神经元的选择性丧失而导致的。蛋白A-突触核蛋白的错误折叠和聚集，其降解受损和氧化剂损伤都是这种选择性神经毒性的分子原因的假设。在发表的酵母（Sharma等人，2006年）和裂变酵母（Brandis等，2006）模型中，我们为A-核蛋白错误折叠和毒性模型提供了遗传和实时细胞支持，同时发现了意外的酵母A-核素特异性A-核素的性质差异。在萌芽的酵母中，单独的A-突触核蛋白的表达不会引起毒性，而是添加蛋白酶体功能障碍或线粒体氧化应激是合成的致命致死性的。这种杀伤力与A-核蛋白的聚集不太相关。取而代之的是，即使有毒时，A-核蛋白也主要定位于酵母质膜。在裂变酵母中，A-突触核蛋白的折叠和聚集体以精确的时间和浓度依赖性方式在细胞质中，为遵循成核聚合模型的机制提供了至关重要的活细胞证据。尽管有广泛的聚集，但A-核蛋白没有毒性。即使在低浓度下，也不会定位于质膜。通过使用酵母模型和在活细胞中采用遗传操纵的遗传操纵，通过以下特定问题，所有这些问题都集中在A-突触核蛋白和细胞途径内的分子决定因素和细胞途径中，可以检查其错误折叠，脂质结合，降解和毒性。新发现的家族PD突变E46K在体内有什么意义？ A-突触核蛋白膜在体内是否涉及特定的磷脂，并且体内毒性需要膜相互作用吗？ A-突触核蛋白是否包含在体内赋予质膜定位和聚集的域？细胞质氧化应激还可以引起A-突触核蛋白介导的致死性还是杀伤力仅限于线粒体应激？最后，溶酶体是否还会降解a-突触核蛋白，并通过什么机制降解？目的是：1）检验以下假设：a-核蛋白突变体E46K对细胞有显着毒性并结合体内磷脂，E46K将与野生型，A30P，A53T和组合家族突变体进行比较两种酵母中的聚集和毒性。 2）检验以下假说：特定的磷脂组成和总磷脂含量对于A-核蛋白膜的关联和毒性和毒性至关重要萌芽的酵母在基因生产主要膜磷脂的基因上，并在两种酵母中都补充了脂肪酸或DMSO，以增加总体磷脂含量。 3）为了测试以下假设：特定的聚集结构域和脂质结合结构域介导a-核蛋白特性，N-和C末端片段以及特定点突变体的定位，聚集和毒性将在两种酵母模型中进行测试。 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多个淘汰赛，如果有可用的地方。 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.正在进行的试点本科项目已经提供了初步证据来支持其中一些假设。完成后，这些相关的研究有意地在范围上进行了多样化，并旨在吸引我实验室的多样化和大量的本科生，将共同阐明调节A-核蛋白正常生物学和病理学的分子碱基。他们还将扩大多种酵母模型的实用性，以研究多种蛋白质错误折叠疾病。项目说明第7页首席研究员/计划主管（最后，第一，中间）：库马尔项目的DEBBURMAN，库马尔项目相关性的神经退行性疾病的越来越多，包括帕金森氏病，代表了美国蓬勃发展的公共卫生问题。它们都无法治愈，大多数是致命的。尽管症状多样性，但其中许多疾病具有常见的病理机制。因此，用这种帕金森氏病赠款做出的发现也可能影响其他疾病的进展。美国的本科教育者在为一个技术复杂且科学跨学科的21世纪社区准备不同的毕业生方面面临重大挑战。将更多的研究和研究经验整合到非硕士和科学专业的课程中，以代替食谱实验室的经验，毕业了进入科学劳动力的更多多样化和更大的准备好的本科生。该提案的主要重点是为许多前往未来生物医学职业的本科生提供严格的科学培训。因此，该提议的主要意义是，它试图参与有才华和多样化的本科生，以实质性和原始的研究经验，在那里他们将作为科学家作为与主要公共卫生有关的生物医学发现做出贡献。