The Roles of Polymerase Gamma Accessory Subunit Gene Mutations in Human Disease.

聚合酶γ辅助亚基基因突变在人类疾病中的作用。

• 批准号: 9265468
• 负责人: Matthew J Young
• 金额: $ 24.9万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9265468
• 关键词: Acetaminophen; Address; Adult; Affect; Age of Onset; Aging; Alleles; Alzheimer' s Disease; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Award; Biochemical; Biochemistry; Bioenergetics; Biological; Biomedical Research; Breast Cancer Treatment; Catalytic Domain; Cell Line; Cell model; Cells; Code; Complex; DNA Maintenance; DNA Repair; DNA biosynthesis; DNA copy number; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Defect; Development; Disease; Dominant-Negative Mutation; Employment; Engineering; Environment; Environmental Risk Factor; Exposure to; Functional disorder; Gene Mutation; Genes; Genetic; Genomic Instability; Genotype; Genus Hippocampus; Green Fluorescent Proteins; Health; Histidine; Human; Human Cell Line; In Vitro; Knowledge; Laboratory Research; Leadership; Libraries; Link; Malignant Neoplasms; Measurement; Measures; Mentors; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Molecular; Monitor; Mutation; Neurodegenerative Disorders; Nuclear; Nucleosides; Onset of illness; Oxygen Consumption; Pathogenesis; Pathogenicity; Pharmaceutical Preparations; Phase; Phenotype; Play; Polymerase; Preclinical Drug Evaluation; Preventive Intervention; Prospective Studies; Proteins; Public Health; Recombinants; Reporting; Research; Research Personnel; Respiration; Reverse Transcriptase Inhibitors; Role; Scientist; Structure; Syndrome; Tacrine; Tamoxifen; Techniques; Technology; Testing; Therapeutic; Therapeutic Effect; Time; Toxin; Training; Translations; Variant; Yeasts; base; career development; combat; diabetic; drug discovery; early childhood; expression vector; extracellular; fundamental research; gene product; high throughput screening; human disease; improved; in vivo; mitochondrial dysfunction; mitochondrial genome; negative affect; novel; novel strategies; public health relevance; stable cell line; tool; trafficking; troglitazone

DESCRIPTION (provided by applicant): The human mitochondrial DNA (mtDNA) polymerase, (Polg) is composed of two subunits encoded by two nuclear genes: 1. the POLG gene encoding the catalytic subunit, p140 and 2. The POLG2 gene encoding the homodimeric accessory subunit, p55. Polg is the sole cellular mtDNA polymerase for mtDNA replication and repair. Mitochondrial diseases, including those linked to POLG2 mutations, are devastating disorders that comprise a continuum of overlapping phenotypes and the age of onset of these diseases range from early childhood to late adulthood. Currently there are no cures for these diseases. To date, al reported POLG2 mutations associated with human mitochondrial disease occur as heterozygous mutations. In vivo dysfunction associated with POLG2 mutations likely results from dominant negative p55 variants that negatively affect or block the function of wild-type gene products, namely p140 and p55. Major barriers to this field of research include the lack of characterization of heterodimeric p55 variants and the limitations of biochemical approaches that exclude analysis of pathogenic alleles. Another barrier in the field is the absence of cell models harboring POLG2 mutations to determine and characterize cellular mechanisms of respiratory dysfunctions. During the K99/Mentored phase Dr. Young's aim is to determine dominant negative mechanisms of POLG2 mutations. Dr. Young has developed and will exploit p55 heterodimeric variants to determine dominant negative mechanisms in vitro. In alliance with heterodimeric studies Dr. Young has developed stable human cell lines with POLG2 mutations to determine dominant negative mechanisms in vivo. Dr. Young will receive structured training in qualitative analysis of mtDNA deletions and quantitative measurements of mtDNA copy number to investigate cellular dysfunctions of mtDNA maintenance linked to POLG2 mutations. In the R00/Independent phase of this award Dr. Young will study prospectively human stable cell line models to address the second aim of determining mechanisms of respiratory defects associated with mitochondrial disease mutations. He will exploit already developed human cell lines harboring POLG2 mutations and develop new cell models to determine mechanisms of respiration deficiency. These studies are essential to fundamental research and determining the biological mechanisms of the mitochondrion that respond to disease mutations. The applicant's long-term objective is targeted at exposure research and determining how drugs and toxins affect mtDNA maintenance and respiration in wild-type and disease states, so called mitochondrion-environment interactions. The principle investigator, Dr. Young, is well suited to carry out the proposed research plans based on his prior training in the mitochondrial research field. His prior training includes yeast mitochondrial genetics, development of human stable cell lines harboring POLG2 mutations, and biochemical characterization of Polg variants. The proposed training and career development will enable Dr. Young to become an independent biomedical research scientist and prepare him for a leadership role managing a mitochondrial research laboratory.

描述（由申请人提供）：人类线粒体 DNA (mtDNA) 聚合酶 (Polg) 由两个核基因编码的两个亚基组成：1. 编码催化亚基 p140 的 POLG 基因和 2. 编码同源二聚体的 POLG2 基因附属亚基，p55。 Polg 是唯一用于 mtDNA 复制和修复的细胞 mtDNA 聚合酶。线粒体疾病，包括与 POLG2 突变相关的疾病，是一种毁灭性的疾病，包含一系列重叠的表型，这些疾病的发病年龄范围从儿童早期到成年晚期。目前这些疾病还没有治愈方法。迄今为止，所有报道的与人类线粒体疾病相关的 POLG2 突变均以杂合突变形式出现。与 POLG2 突变相关的体内功能障碍可能是由显性失活 p55 变异引起的，这些变异对野生型基因产物（即 p140 和 p55）的功能产生负面影响或阻断。该研究领域的主要障碍包括缺乏异二聚体 p55 变体的表征以及排除致病等位基因分析的生化方法的局限性。该领域的另一个障碍是缺乏含有 POLG2 突变的细胞模型来确定和表征呼吸功能障碍的细胞机制。在 K99/Mentored 阶段，Young 博士的目标是确定 POLG2 突变的显性失活机制。 Young 博士已经开发并将利用 p55 异二聚体变体来确定体外显性负作用机制。 Young 博士与异二聚体研究相结合，开发了具有 POLG2 突变的稳定人类细胞系，以确定体内的显性负作用机制。 Young 博士将接受 mtDNA 缺失定性分析和 mtDNA 拷贝数定量测量方面的结构化培训，以研究与 POLG2 突变相关的 mtDNA 维持的细胞功能障碍。在该奖项的 R00/独立阶段，Young 博士将前瞻性地研究人类稳定细胞系模型，以解决确定与线粒体疾病突变相关的呼吸缺陷机制的第二个目标。他将利用已经开发的含有 POLG2 突变的人类细胞系，并开发新的细胞模型来确定呼吸缺陷的机制。这些研究对于基础研究和确定线粒体响应疾病突变的生物学机制至关重要。申请人的长期目标是进行暴露研究，并确定药物和毒素如何影响野生型和疾病状态下线粒体DNA的维持和呼吸，即所谓的线粒体-环境相互作用。首席研究员 Young 博士基于他之前在线粒体研究领域的培训，非常适合执行拟议的研究计划。他之前接受的培训包括酵母线粒体遗传学、携带 POLG2 突变的人类稳定细胞系的开发以及 Polg 变体的生化表征。拟议的培训和职业发展将使杨博士成为一名独立的生物医学研究科学家，并为他担任管理线粒体研究实验室的领导角色做好准备。