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

• 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
• 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

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.

项目总结/摘要 亨廷顿蛋白（HTT）中的多聚谷氨酰胺（Q）扩增突变导致亨廷顿病（HD）。如何 该突变对全长HTT功能的影响尚不清楚。重要的是， HD患者死后脑氨水平升高，表明异常的蛋白质代谢， 发生在HD症状之前。虽然HTT表达发生在所有类型的细胞中，但代谢机制 HTT控制的蛋白质代谢和氨基酸稳态对营养剥夺的反应， 不被理解和研究。这项建议的重点是确定保守的细胞途径 由HTT调节和polyQ扩增对这些细胞过程的影响。使用小说的策略 模型系统代表了理解正常和突变HTT功能的创新方法。 网骨藻遗传学还可以鉴定保守的遗传修饰剂作为治疗靶点，用于治疗 HD.数据表明，HTT在调节蛋白质回收和清除中具有祖先作用， 然而，目前还不清楚HTT如何控制这些细胞过程。网骨藻HTT缺乏症 导致强烈的生物缺陷，表明改变的蛋白质催化剂，赋予对 改变自噬流的化学物质重要的是，人HTT在网骨藻htt-细胞中的表达或 氨解毒处理独立地拯救提示存在遗传缺陷的HTT表型。 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- 表征的表型分析，Dictyosteoblasts遗传学的力量，以测试蛋白质清除机制， 并进行互补的，无偏见的抑制筛选，再加上全基因组测序，以确定 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物。在目标1中， 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，直至达到饱和 (NTG)加上限制性酶介导的随机插入诱变（REMI）以突变htt-细胞中的基因， 并筛选自噬缺陷和对NH 3+和氯喹的发育敏感性的抑制因子。在Aim中 学生将测试HTT调节自噬和/或泛素蛋白酶体系统的假设。这 aim将对htt细胞，抑制突变体和细胞中的自噬和蛋白酶体机制进行定量。 使用已建立的分子、生物化学和显微方法表达正常或突变的人HTT。 靶向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细胞中的表型。这一方法的持续影响， 经济上多样化的本科生群体将有助于绕过一个重大障碍， 了解HTT的正常功能。此外，鉴定的抑制基因的直向同源物可以在 相关的哺乳动物模型，重要的是，学生在马萨诸塞州洛厄尔将有助于更深入的了解 HTT的正常作用和polyQ扩增对调节细胞内蛋白质催化剂的影响。