The impact of polyQ-expansion and suppression of huntingtin-deficiency phenotypes in the model organism Dictyostelium

PolyQ 扩增和抑制亨廷顿蛋白缺陷表型对模式生物网基网柄菌的影响

基本信息

批准号：
9813233
负责人：
Frederic Chain
金额：
$ 45.62万
依托单位：
UNIVERSITY OF MASSACHUSETTS LOWELL
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9813233
关键词：
Affect Amino Acids Ammonia Animal Model Apoptosis Apoptotic Autophagocytosis Autophagosome Autopsy Biochemical Bioinformatics Biological Assay Biological Models Brain Catabolic Process Catabolism Cell physiology Cells Chemicals Chimeric Proteins Chloroquine Complement Conflict (Psychology)Confocal Microscopy Coupled Cytopathology Cytoplasmic Organelle Data Defect Development Dictyostelium Dictyostelium discoideum Disease model Drug Metabolic Detoxication Etiology Eukaryota Eukaryotic Cell Evaluation Genes Genetic Genetic Screening Genomic DNA Homeostasis Human Huntington Disease Huntington gene Huntington protein Hypersensitivity Impairment Insertional Mutagenesis Inverse Polymerase Chain Reaction Length Mammalian Cell Measures Mediating Metabolic Methodology Methods Methylnitronitrosoguanidine Microscopic Modeling Molecular Monitor Mutagenesis Mutate Mutation Nitrogen Nitrosoguanidines Nutrient Onset of illness Orthologous Gene Pathway interactions Phenotype Population Heterogeneity Process Proteins Proteolysis Recycling Regulation Role Site Starvation Stress Structure Students Suppressor Genes Symptoms System Testing Therapeutic Intervention Time Ubiquitin Work cell type deprivation genome sequencing in vivo innovation insight loss of function multicatalytic endopeptidase complex mutant novel nutrient deprivation polyglutamine pressure response restriction enzyme therapeutic development therapeutic target undergraduate student whole genome

项目摘要

PROJECT SUMMARY/ABSTRACT Poly-glutamine (Q) expansion mutation in the protein huntingtin (HTT) causes Huntington’s disease (HD). How the mutation imparts an unknown effect on full-length HTT function has yet to be determined. Importantly, postmortem brain ammonia levels are elevated in HD cases, indicating aberrant protein catabolism and seems to occur prior to symptoms of HD. Although HTT expression occurs in all cell types, the metabolic mechanisms of protein catabolism and amino acid-homeostasis in response to nutrient-deprivation controlled by HTT are poorly understood and under-studied. This proposal focuses on defining the conserved cellular pathways regulated by HTT and the impact of polyQ-expansion on these cellular processes. Strategies that use novel model systems represents an innovative approach to understanding both normal and mutant HTT function. Dictyostelium genetics may also identify conserved genetic modifiers as therapeutic targets for the treatment of HD. Data suggests that HTT has an ancestral role in the regulation of protein recycling and clearance in eukaryotic cells, yet it is unclear how HTT controls these cellular processes. HTT deficiency in Dictyostelium results in strong organismal defects indicative of altered protein catabolism that confers hypersensitivity to chemicals that alter autophagic flux. Importantly, both expression of human HTT in Dictyostelium htt- cells or ammonia-detoxification treatments independently rescue htt- phenotypes that suggests the presence of genetic modifiers of conserved HTT-dependent catabolic processes. Undergraduate students will use an array of well- characterized phenotypic assays, the power of Dictyostelium genetics to test protein-clearance mechanisms and perform complementary, non-biased suppressor screens coupled with whole-genome sequencing to define key upstream and downstream effectors that regulate HTT-dependent catabolic pathways in the cell. In Aim 1, the lab will perform chemical mutagenesis screens to saturation using N-methyl-N′ -nitro-N-nitrosoguanidine (NTG) plus restriction enzyme-mediated random insertional mutagenesis (REMI) to mutate genes in htt- cells, and screen for suppressors of autophagic defects and developmental sensitivity to NH3+ and chloroquine. In Aim 2, students will test the hypothesis that HTT regulates autophagy and/or the ubiquitin proteasome system. This aim will quantify autophagic and proteasomal machinery in both htt- cells, suppressor mutants and cells expressing normal or mutant human HTT using established molecular, biochemical and microscopic methods. Targeting specific human homologs identified in the screen could be a viable way to suppress aberrant catabolic phenotypes in mutant HTT cells. The sustained impact from this approach, implemented by ethnically and economically diverse populations of undergraduate students will help circumvent a significant barrier to understanding HTT normal function. Moreover, orthologs of identified suppressor genes can be studied in relevant mammalian models and importantly, students at UMass Lowell will contribute to a deeper understanding of the normal role of HTT and the impact of polyQ expansion on regulating protein catabolism within the cell.

项目概要/摘要亨廷顿蛋白 (HTT) 中的聚谷氨酰胺 (Q) 扩展突变如何导致亨廷顿病 (HD)。该突变对全长 HTT 功能的影响尚待确定。 HD 病例死后脑氨水平升高，表明蛋白质分解代谢异常，并且似乎尽管 HTT 表达出现在所有细胞类型中，但其代谢机制 HTT 控制的营养剥夺对蛋白质分解代谢和氨基酸稳态的影响该提案的重点是定义保守的细胞途径。 HTT 的调节以及 PolyQ 扩增对这些细胞过程的影响使用新颖的策略。模型系统代表了理解正常和突变 HTT 功能的创新方法。盘基网柄菌遗传学还可以将保守的基因修饰剂识别为治疗靶点 HD. 数据表明，HTT 在调节蛋白质循环和清除方面发挥着祖传作用。真核细胞，但目前尚不清楚 HTT 如何控制盘基网柄菌中的这些细胞过程。导致强烈的生物缺陷，表明蛋白质分解代谢改变，从而导致过敏重要的是，盘基网柄菌 htt- 细胞或细胞中人类 HTT 的表达。氨解毒治疗独立地挽救了 htt 表型，表明存在遗传性保守的 HTT 依赖性分解代谢过程的修饰剂本科生将使用一系列良好的方法。特征表型测定，盘基网柄菌遗传学测试蛋白质清除机制的能力并进行互补的、无偏见的抑制子筛选以及全基因组测序来定义在目标 1 中，调节细胞中 HTT 依赖性分解代谢途径的关键上游和下游效应器。该实验室将使用 N-甲基-N'-硝基-N-亚硝基胍进行化学诱变筛选至饱和 (NTG) 加上限制性内切酶介导的随机插入诱变 (REMI) 来突变 htt- 细胞中的基因，并筛选自噬缺陷抑制剂以及对 NH3+ 和氯喹的发育敏感性。 2、学生将检验 HTT 调节自噬和/或泛素蛋白酶体系统的假设。目标将量化 htt 细胞、抑制突变体和细胞中的自噬和蛋白酶体机制使用已建立的分子、生物化学和显微镜方法表达正常或突变的人类 HTT。针对筛选中确定的特定人类同源物可能是抑制异常分解代谢的可行方法突变 HTT 细胞的表型。这种方法的持续影响，由种族和群体实施。本科生群体的经济多样化将有助于规避一个重大障碍此外，可以研究已识别的抑制基因的直向同源物。相关的哺乳动物模型，重要的是，麻省大学洛厄尔分校的学生将有助于更深入的理解 HTT 的正常作用以及 PolyQ 扩增对调节细胞内蛋白质分解代谢的影响。