Using suppressor analysis to elucidate peroxisome biogenesis and peroxin function

使用抑制分析阐明过氧化物酶体生物合成和过氧化物酶功能

• 批准号: 9755456
• 负责人: Roxanna Llinas
• 金额: $ 4.5万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9755456
• 关键词: Alleles; Arabidopsis; Back; Biogenesis; Bioinformatics; Biological; Bypass; Carrier Proteins; Cells; Complex; Critical Pathways; Data; Defect; Degenerative Disorder; Development; Disease; Docking; Enzymes; Equipment and supply inventories; Ethyl Methanesulfonate; Event; Fatty Acids; Functional disorder; Gene Targeting; Genes; Genetic; Genetic Recombination; Genome; Goals; Growth; Growth and Development function; Human; Impairment; Interphase Cell; Lead; Mammals; Membrane; Mentors; Metabolic; Metabolic Pathway; Metabolism; Mitochondria; Modeling; Molecular; Mutagens; Mutation; Nuclear Protein; Organelles; Organism; PHEX protein; Parents; Patients; Physiologic Monitoring; Physiological; Plants; Process; Protein Import; Proteins; Reaction; Reactive Oxygen Species; Recovery; Research Personnel; Role; Suppressor Genes; System; Testing; Therapeutic; Translating; Ubiquitin; Ubiquitination; Yeasts; causal variant; design; enzyme substrate; functional restoration; genome sequencing; improved; insight; mutant; novel; oxidation; oxidative damage; peroxisome; peroxisome membrane; protein transport; receptor; receptor recycling; recruit; repaired; response; tool; ubiquitin-protein ligase; undergraduate student; whole genome

Project summary/Abstract Using suppressor analysis to elucidate peroxisome biogenesis and peroxin function The import of matrix enzymes that catalyze metabolic reactions supporting growth and development is central to peroxisome biogenesis and function. The current model for peroxisomal matrix protein import provides a framework of peroxisomal proteins (peroxins) involved and the associations these peroxins make to import proteins into the peroxisome. However, this model lacks mechanistic details of the associations among these peroxins, contributions of non-transport peroxins to import, and possibly unidentified peroxins. This project proposes using mutant suppression screens to identify new peroxisomal components, reveal novel genetic interactions between known peroxins, and provide more detailed understanding of known associations. Mutations in distinct steps in matrix protein import were chosen for suppression screens: pex12-1, a mutant of a peroxin of the ubiquitin ligating complex that aids in peroxisomal receptor recycling, and pex14-1 and pex14-6, mutants of a docking complex peroxin that recruits peroxisomal receptors and aids in matrix protein insertion. In each screen, the initial mutant (pex12-1, pex14-1, or pex14-6) was mutagenized and then screened for lines with beneficial secondary mutations that alleviated one or more peroxisomal defects. Numerous mutant suppressors already have been isolated through these screens. For each prioritized suppressor, this project aims to identify the mutation causing suppression through whole- genome sequencing, bioinformatics, and recombination mapping. Suppression mechanisms will be elucidated by combining different mutations and comparing impacts on peroxisome function by monitoring physiological, cell biological, and molecular responses. Characterizing the novel mutations identified in these screens and determining the mechanisms through which they restore or bypass peroxisome dysfunction will elucidate the roles of the suppressor gene and the targeted peroxin in peroxisome function and refine our understanding of matrix protein import. Furthermore, because this project is designed to uncover means of repairing or circumventing peroxisomal dysfunction in mutants of PEX12 and PEX14 and because peroxins often have conserved function among diverse species, the results of this proposal may inform therapies for peroxisome biogenesis disorder patients with mutations that impair docking (PEX14) or ubiquitination (PEX12) of receptor peroxins. !

项目概要/摘要 利用抑制子分析阐明过氧化物酶体的生物发生和过氧化物酶的功能 催化支持生长和发育的代谢反应的基质酶的进口是 对过氧化物酶体的生物发生和功能至关重要。过氧化物酶体基质蛋白输入的现有模型 提供了一个框架的过氧化物酶体蛋白（过氧化物）参与和协会，这些过氧化物 将蛋白质输入过氧化物酶体。然而，该模型缺乏关联的机械细节 在这些过氧化物中，非运输性过氧化物和可能未经鉴定的过氧化物的进口量。 该项目建议使用突变抑制筛选来识别新的过氧化物酶体组分，揭示 已知过氧化物之间的新的遗传相互作用，并提供更详细的了解已知的 协会.选择基质蛋白输入中不同步骤中的突变进行抑制筛选： pex12 - 1，一种泛素连接复合物的过氧化物酶的突变体，其有助于过氧化物酶体受体的再循环， 以及pex 14 - 1和pex 14 - 6，它们是一种停靠复合物过氧化物酶的突变体，该过氧化物酶募集过氧化物酶体受体， 有助于基质蛋白插入。在每次筛选中，将初始突变体（pex12 - 1、pex14 - 1或pex14 - 6）进行突变。 诱变，然后筛选具有有益的次级突变的品系，所述次级突变减轻了一种或多种 过氧化物酶体缺陷通过这些筛选已经分离出许多突变的抑制基因。为 每一个优先的抑制，这个项目的目的是确定突变引起抑制通过整个， 基因组测序、生物信息学和重组作图。抑制机制将是 通过结合不同的突变并通过监测比较对过氧化物酶体功能的影响来阐明 生理学、细胞生物学和分子反应。 表征在这些筛选中鉴定的新突变，并通过以下方式确定机制： 他们恢复或旁路过氧化物酶体功能障碍将阐明抑制基因的作用， 有针对性的过氧化物酶体功能的过氧化物酶和完善我们的理解，基质蛋白的进口。此外，委员会认为， 因为这个项目旨在揭示修复或规避过氧化物酶体功能障碍的方法， PEX12和PEX14的突变体，并且由于过氧化物酶在不同物种中通常具有保守的功能， 该建议的结果可以为具有突变的过氧化物酶体生物合成障碍患者的治疗提供信息， 削弱受体过氧化物的对接（PEX14）或泛素化（PEX12）。 !