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.

摘要 我的长期职业目标是研究罕见的人类遗传性疾病，重点是：（1）定义 疾病机制和开发治疗方法;（2）通过以下方式增加研究和认识 参与科学政策。氨酰-tRNA合成酶（ARSs）是转运tRNA的必需酶 与细胞质和线粒体中的同源氨基酸; 37个核编码的ARS中的17个 只存在于线粒体中。所有17个线粒体ARS都与人类隐性遗传有关。 具有广泛临床表型的疾病，通常影响具有高能量需求的组织。 有趣的是，某些线粒体ARS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。这种观察的一个重要例子是线粒体 丝氨酰-tRNA合成酶（SARS2），它与疾病表型有关， 痉挛性轻瘫至HUPRA综合征（高尿酸血症、肺动脉高压、婴儿期肾衰竭和 碱中毒）。目前，这种临床异质性还没有遗传学或分子学解释。 此外，已知的致病性变体的数量是有限的，导致我们在以下方面的知识存在很大差距： SARS 2相关疾病等位基因异质性我将努力实现两个具体目标来解决这些问题。 在目标1下，我将对SARS2开放的所有可能的变体进行大规模平行细胞生长测定。 阅读框架来评估每个变体的功能后果。这些研究将在 单倍体（Hap1）细胞，其具有：（a）随机整合的、1xFLAG标记的、多西环素诱导的野生型 SARS 2拷贝;和（B）内源性SARS 2无效等位基因。我将用慢病毒库来克隆这些细胞 包含所有可能的SARS2变体，并将定量每个等位基因的频率（通过下一代 测序）在多西环素存在下（即，在存在野生型SARS 2 - 1xFLAG的情况下）和在 不存在强力霉素（即，在没有SARS2 - 1xFLAG的情况下）。变异体的出现频率降低， 多西环素去除将被分类为功能丧失等位基因。在目标2下，我将产生克隆Hap1 携带个体的、已知的致病性SARS2变异体的细胞系，这些变异体与不同的临床表现相关， 表型（即，进行性痉挛性轻瘫与HUPRA综合征）。这些细胞系将含有 整合的，可诱导的SARS2 - 1xFLAG，可用于维持细胞活力。我将利用这些细胞系 评估每种变体对蛋白质翻译、线粒体功能以及与蛋白质相互作用的影响 潜在的SARS2结合伴侣。总体而言，这些研究将：（a）揭示SARS的重要区域2 （B）提供一个完整的功能丧失的面板 SARS2变体用于快速患者诊断;以及（c）提供对SARS2变体的分子机制的深入了解。 SARS2相关疾病对基因型的定义：表型相关性和开发治疗。