Administrative Supplement Equipment Request for GM135577

GM135577 行政补充设备请求

基本信息

批准号：
10798490
负责人：
SAMUEL T LAMITINA
金额：
$ 7.76万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-09 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10798490
关键词：
Administrative Supplement Affect Alleles Anabolism Animals Architecture Caenorhabditis elegans Cell physiology Cells Cellular Stress Complex Cultured Cells Defect Dehydration Disease Environment Enzymes Equipment Exhibits Extracellular Matrix Gene Expression Genes Genetic Screening Glucose Glycerol Health Homologous Gene Human Mammalian Cell Mammals Mediating Messenger RNA Molecular Mutation Nematoda Nuclear Proteins O-GlcNAc transferase Osmosis Pathway interactions Phenocopy Physiological Processes Play Poly A Polyadenylation Post-Translational Protein Processing Protein Biosynthesis Proteins RNA Processing Role Signal Transduction Stress Testing Tissues Up-Regulation biological adaptation to stress human tissue in vivo mRNA Translation mechanical properties mutant novel posttranscriptional protein complex response sensor

项目摘要

Project Summary Cellular stress responses play essential roles in cell and organismal survival and contribute to a wide range of physiological processes and diseases in humans. The molecular architecture of most stress response pathways are well defined. A striking exception to this is osmotic stress response, where the relevant stress sensors and signaling mechanisms in animals are poorly understood. Most studies of the osmotic stress response use cultured cells, where in vivo complexities, i.e. the extracellular matrix, tissue mechanical properties, etc., are not replicated. To better mimic these conditions, we study the osmotic stress response in a live animal, the nematode C. elegans. Like humans, C. elegans responds to osmotic stress by metabolizing glucose to produce organic osmolytes, such as glycerol. We performed an unbiased forward genetic screen to identify mutants that exhibit no induction of osmolyte biosynthesis genes (Nio genes) and discovered multiple alleles of nio-2, which encodes the sole C. elegans homolog of the O-GlcNAc transferase (ogt-1; OGT in humans). OGT post-translationally O-GlcNAcylates Ser/Thr residues of cytosolic and nuclear proteins but also exhibits important GlcNAcylation independent functions. Mammalian cells lacking OGT do not survive, but C. elegans lacking ogt- 1 are viable and fertile, providing a unique opportunity to study the role of ogt-1 in cellular physiology. ogt-1 mutants are unable to adapt and grow in hypertonic environments and exhibit reduced organic osmolyte levels and no induction of the osmolyte biosynthesis protein GPDH-1. However, osmotic induction of osmolyte biosynthesis gene mRNAs is normal, suggesting that ogt-1 functions post-transcriptionally. These defects can be rescued by expression of wild type or catalytically inactive human OGT, showing that non-canonical functions of OGT in the osmotic stress response are conserved from C. elegans to humans. We also discovered mutations in interacting components of a conserved 3’ mRNA processing complex that phenocopy ogt-1. We hypothesize that non-canonical functions of ogt-1 facilitate upregulation of stress-induced mRNA translation via interactions with 3’ RNA processing complex proteins during osmotic stress. To test this hypothesis, we will determine the temporal, functional, and regulatory requirements for ogt-1 in the osmotic stress response (Aim 1), identify the specific gene expression mechanism(s) that are affected by ogt-1 (Aim 2), and determine if ogt-1 regulates the osmotic stress response via interactions with 3’ mRNA cleavage and polyadenylation components also identified in our Nio screen. (Aim 3). Our studies will delineate a novel paradigm in stress signaling and reveal new mechanisms by which OGT impacts cell physiology.

项目摘要细胞应激反应在细胞和有机存活中起着重要作用，并有助于人类的各种物理过程和疾病。分子结构大多数应力响应途径都定义得很好。罢工例外是渗透压力反应，其中相关的应力传感器和动物的信号传导机制很差理解齿。渗透压反应的大多数研究都使用培养的细胞，其中体内复杂性，即细胞外基质，组织机械性能等。为了更好地模仿这些条件，我们研究活动物中的渗透应激反应，线虫C.秀丽隐杆线虫。像人类一样，秀丽隐杆线虫通过代谢来应对渗透压葡萄糖产生有机渗透剂，例如甘油。我们表现了一个公正的前锋遗传筛查以鉴定不存在渗透质生物合成基因的突变体（nio 基因）并发现了Nio-2的多个等位基因，该等位基因编码唯一的秀丽隐杆线虫同源物 O-GLCNAC转移酶（人类中的OGT-1； OGT）。 OGT翻译后O-Glcnacylates 胞质和核蛋白的Ser/THR残基，但也表现出重要的Glcnacylation 独立功能。缺乏OGT的哺乳动物细胞无法生存，但是缺乏OGT-的秀丽隐杆线虫 1是可行且肥沃的，为研究OGT-1在细胞中的作用提供了独特的机会生理。 OGT-1突变体无法在高渗环境中适应和生长并展示有机渗透量降低，没有诱导渗透液生物合成蛋白GPDH-1。然而，渗透诱导渗透质生物合成基因mRNA是正常的，表明 OGT-1在转录后功能。这些缺陷可以通过表达野生类型来检索或催化性不活跃的人OGT，表明OGT在渗透中的非传统功能秀丽隐杆线虫对人类的压力反应是保守的。我们还发现了突变保守的3'mRNA加工复合物的相互作用成分，该复合物是OGT-1。我们假设OGT-1的非规范功能最喜欢的压力诱导的mRNA上调通过在渗透应激期间与3’RNA加工复杂蛋白的相互作用进行翻译。到检验该假设，我们将确定 OGT-1在渗透应力反应中（AIM 1），确定特定的基因表达机制受OGT-1影响（AIM 2）的影响，并确定OGT-1是否调节渗透应力反应通过与3'mRNA裂解和聚腺苷酸化成分的相互作用也可以在我们的NiO中鉴定屏幕。（目标3）。我们的研究将描述应力信号的新型范式，并揭示了新的范式 OGT影响细胞生理的机制。