Investigating the Role of Seryl-tRNA Synthetase in Mitochondrial Biology and Human Recessive Disease

研究 Seryl-tRNA 合成酶在线粒体生物学和人类隐性疾病中的作用

• 批准号: 10750183
• 负责人: Christina Del Greco
• 金额: $ 4.08万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-26 至 2025-09-25
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750183
• 关键词: Acceleration; Address; Affect; Alkalosis; Alleles; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Awareness; Binding; Biological Assay; Biological Process; Biology; Catalytic Domain; Cell Line; Cell Survival; Cell model; Cell physiology; Cells; Central Nervous System; Classification; Complement; Cytoplasm; Data; Diagnosis; Dimerization; Disease; Doxycycline; Enzymes; Excision; Foundations; Frequencies; Genetic; Genetic Predisposition to Disease; Genotype; Genus Hippocampus; Goals; Haploid Cells; Hereditary Disease; Heterogeneity; Human; Human Genome; Hyperuricemia; Impairment; Individual; Inherited; Kidney Failure; Knowledge; Lead; Libraries; Life; Ligation; Maps; Metabolic; Mitochondria; Molecular; Mutation; Neurologic; Nuclear; Open Reading Frames; Oxygen Consumption; Paresis; Pathogenicity; Patients; Phenotype; Prognosis; Proteins; Pulmonary Hypertension; Reporting; Research; Research Training; Role; Science Policy; Serine; Serine-tRNA Ligase; Severities; Syndrome; System; Tertiary Protein Structure; Testing; Therapeutic; Tissues; Transfer RNA; Transfer RNA Aminoacylation; Translations; Variant; Yeasts; cell growth; cellular transduction; clinical heterogeneity; clinical phenotype; design; disease phenotype; early onset; genetic variant; human disease; improved; infancy; insight; loss of function; mutation screening; next generation sequencing; protein function; rapid diagnosis; spasticity

ABSTRACT My long-term professional goal is to study rare human inherited diseases with an emphasis on: (1) defining disease mechanisms and developing therapeutics; and (2) increasing research and awareness through participating in science policy. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that charge tRNA with cognate amino acids in the cytoplasm and mitochondria; 17 of the 37 nuclear-encoded ARSs act exclusively in the mitochondria. All 17 mitochondrial ARSs have been implicated in human recessive diseases with a broad range of clinical phenotypes, often affecting tissues with high energy demands. Interestingly, certain mitochondrial ARSs cause variable tissue-specific effects, which can depend on the specific genetic variants that a patient carries. One important example of this observation is mitochondrial seryl-tRNA synthetase (SARS2), which has been implicated in disease phenotypes ranging from progressive spastic paresis to HUPRA syndrome (Hyper Uricemia, Pulmonary hypertension, Renal failure in infancy, and Alkalosis). Currently, there are no genetic or molecular explanations for this clinical heterogeneity. Furthermore, the number of known pathogenic variants is limited, resulting in a large gap in our knowledge of the allelic heterogeneity in SARS2-related disease. I will pursue two specific aims to address these issues. Under Aim 1, I will perform massively parallel cell growth assays on all possible variants in the SARS2 open reading frame to assess the functional consequences of each variant. These studies will be performed in haploid (Hap1) cells that harbor: (a) a randomly integrated, 1xFLAG-tagged, doxycycline-inducible wild-type copy of SARS2; and (b) an endogenous SARS2 null allele. I will transduce these cells with lentiviral libraries containing all possible SARS2 variants and will quantitate the frequencies of each allele (via next-generation sequencing) in the presence of doxycycline (i.e., in the presence of wild-type SARS2-1xFLAG) and in the absence of doxycycline (i.e., in the absence of SARS2-1xFLAG). Variants that are reduced in frequency after doxycycline removal will be classified as loss-of-function alleles. Under Aim 2, I will generate clonal Hap1 cell lines that carry individual, known pathogenic SARS2 variants that are associated with distinct clinical phenotypes (i.e., progressive spastic paresis versus HUPRA syndrome). These cell lines will contain an integrated, inducible SARS2-1xFLAG that can be used to maintain cell viability. I will employ these cell lines to assess the effect of each variant on protein translation, mitochondrial function, and interactions with potential SARS2 binding partners. Overall, these studies will: (a) reveal essential regions of the SARS2 protein toward a better understanding of enzyme biology; (b) provide a complete panel of loss-of-function SARS2 variants toward rapid patient diagnosis; and (c) provide insight into the molecular mechanisms of SARS2-related disease toward defining genotype:phenotype correlations and developing therapeutics.

抽象的 我的长期专业目标是研究罕见的人类遗传疾病，重点是：（1）定义 疾病机制和发展治疗学； （2）通过 参加科学政策。氨基酰基-TRNA合成酶（ARSS）是为tRNA电荷的必需酶 与细胞质和线粒体中的同源氨基酸； 37个核编码ARS法案中的17 仅在线粒体中。所有17个线粒体ARS都与人类隐性有关 具有广泛临床表型的疾病通常会影响能量高需求的组织。 有趣的是，某些线粒体ARS会引起可变的组织特异性效应，这可能取决于 患者携带的特定遗传变异。该观察结果的一个重要例子是线粒体 Seryl-tRNA合成酶（SARS2），与疾病表型有关 对Hupra综合征的痉挛性麻痹（高尿素，肺动脉高压，婴儿期肾衰竭和 碱性病）。当前，对于这种临床异质性，没有遗传或分子解释。 此外，已知的致病变异的数量受到限制，导致我们对 SARS2相关疾病中的等位基因异质性。我将追求两个具体的目的来解决这些问题。 在AIM 1下，我将对SARS2打开的所有可能变体进行大规模平行的细胞生长测定 阅读框架以评估每个变体的功能后果。这些研究将在 藏有的单倍体（HAP1）细胞：（a）随机整合的，1XFLAG标签，强力霉素诱导的野生型 SARS2的副本； （b）内源性SARS2无效等位基因。我将用慢病毒库将这些细胞转导 包含所有可能的SARS2变体，并将量化每个等位基因的频率（通过下一代 测序）在有多西环素的情况下（即，在存在野生型SARS2-1XFLAG的情况下）和 缺乏强力霉素（即在没有SARS2-1XFLAG的情况下）。频率降低后的变体 强力霉素的去除将被归类为功能丧失等位基因。在AIM 2下，我将产生克隆HAP1 带有个体的，已知的致病SARS2变体的细胞系，与不同的临床相关 表型（即进行性痉挛性麻痹与Hupra综合征）。这些细胞系将包含 可用于维持细胞活力的集成，可诱导的SARS2-1XFLAG。我将使用这些细胞线 评估每个变体对蛋白质翻译，线粒体功能以及与与之相互作用的影响 潜在的SARS2约束伙伴。总体而言，这些研究将：（a）揭示SARS2的基本区域 蛋白质对酶生物学有更好的了解； （b）提供一个完整的功能丧失 SARS2变体用于快速患者诊断； （c）提供有关分子机制的见解 SARS2相关的疾病针对定义基因型：表型相关和发展治疗。