DEFINING RAPTOR-MEDIATED MECHANISMS OF HYPOXIC INJURY

定义猛禽介导的缺氧损伤机制

基本信息

批准号：
10732078
负责人：
C. Michael Crowder
金额：
$ 67.85万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10732078
关键词：
Acute Affect Aging Alleles Autophagocytosis Biochemical Biological Biological Assay Biological Process Biology CRISPR/Cas technology Caenorhabditis elegans Cancer Biology Cell Death Cell Hypoxia Cell physiology Cells Chromosome Mapping Chronic Clinical Complex Data Defect Development FRAP1 gene Foundations Future Generations Genes Genetic Growth Hypoxia Injury Label Lesion Malignant Neoplasms Mammals Mediating Metabolism Methods Modeling Mutagenesis Mutate Mutation Names Nature Nematoda Nutrient Orthologous Gene Output Pathway interactions Pharmaceutical Preparations Phenotype Phosphorylation Protein-Serine-Threonine Kinases Proteins Proteome Proteomics Publishing Purine-Nucleoside Phosphorylase RNA Interference Reagent Reporter Reporting Resistance Role Serine Signal Pathway Signal Transduction Sirolimus Site Suppressor Mutations Temperature Testing Time Translations Work Yeasts candidate identification causal variant conditional mutant experimental study forward genetics gain of function genetic approach genetic manipulation genome sequencing improved inhibitor knock-down loss of function loss of function mutation metabolomics mutant mutation screening novel response tool whole genome

项目摘要

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that is activated by nutrients and energy, phosphorylating substrates to promote and coordinate anabolic metabolism. mTOR was identified about thirty years ago in a yeast screen for mutants resistant to growth inhibition by the drug rapamycin; shortly thereafter the highly conserved mammalian ortholog was discovered independently by biochemical methods. Over the past thirty years, biochemical and genetic approaches have identified numerous evolutionarily conserved components of the mTOR signaling pathway as well as the proteins forming the two distinct mTOR complexes, mTORC1 and mTORC2. mTORC1 is composed of three core components, mTOR, Raptor, and mLST8 (mammalian lethal with SEC13 protein 8); Rictor replaces Raptor in mTORC2. mTORC1 is inhibited by rapamycin whereas mTORC2 is relatively insensitive. mTORC1 has been intensely studied because it functions as a central regulator of the cell’s response to nutrients and energy and thereby impacts clinically important conditions such as cancer, aging, and hypoxic injury. Through an unbiased mutant screen in C. elegans for hypoxia resistant mutants, we have identified a missense partial-loss-of-function mutation in daf- 15, which encodes the sole C. elegans ortholog of Raptor (Ce-Raptor). A CRISPR/CAS9-generated mutant with the identical lesion confirmed the mutation as conferring hypoxia resistance. Our discovery that reduction of function of Ce-Raptor imparts resistance to hypoxic injury is consistent with published data using mTORC1 inhibitors in mammalian models. Our Ce-Raptor mutant called daf-15(gc67) is unique among published metazoan Raptor mutants in that it is conditional; Raptor pathway functions can be turned off and on again by simply varying temperature. daf-15(gc67) is essentially wild type at the standard culture temperature of 20°C, hypoxia resistant at 22°C, and developmentally arrested at 25°C, the phenotype of daf-15 null mutants. The graded temperature-conditional nature of the daf-15(gc67) phenotypes overcomes a critical barrier in the field by providing a genetic reagent that allows temporal and tunable genetic control of Raptor and mTORC1 function for the first time in metazoans. This project will use this unique reagent to answer key questions about how and when Raptor regulates hypoxic injury. In Aim 1, we will combine genetic and proteomic methods to test specific hypotheses and discover the Raptor-regulated proteins that determine how and when Raptor controls hypoxic injury. Using our conditional mutant, we have completed a screen for suppressors of Raptor loss of function. In Aim 2, we will identify mutated genes suppressing Raptor loss-of-function and thereby discover regulators of Raptor-mediated hypoxic sensitivity. Through these two aims, we will define how Raptor regulates hypoxic sensitivity. Given the unbiased nature of the suppressor screen and proteomic studies, our project has potential to identify novel components of the mTORC1 pathway and thereby more broadly advance our fundamental understanding of mTORC1 signaling pathways, including those regulating cancer and aging.

雷帕霉素（MTOR）的机械靶标是一种丝氨酸/苏氨酸激酶，被养分和能量，磷酸化底物以促进和协调合成代谢代谢。确定了mTOR 大约30年前，在酵母筛查中，突变体对药物雷帕霉素的抑制作用具有抗性；不久此后，高度组成的哺乳动物直系同源物是通过生化方法独立发现的。在过去的三十年中，生化和遗传方法在进化上已经确定了许多进化 MTOR信号通路的配置组件以及形成两个不同mTOR的蛋白质复合物，mtorc1和mtorc2。 MTORC1由三个核心组成部分组成，MTOR，RAPTOR和 MLST8（Sec13蛋白8的哺乳动物致命）； Rictor取代了MTORC2中的猛禽。 mtorc1被雷帕霉素，而mTORC2相对不敏感。 MTORC1已被积极研究，因为它充当细胞对营养和能量反应的中心调节剂，从而对临床产生影响癌症，衰老和低氧损伤等重要条件。通过C中的公正突变屏幕。秀丽隐杆线虫耐多氧突变体，我们已经确定了DAF-的错义部分功能突变 15，编码Raptor（CE-Raptor）的鞋类秀丽隐杆线虫直系同源物。 CRISPR/CAS9生成的突变体由于相同的病变证实了该突变是会议缺氧抗性。我们的发现减少 CE反铁的功能赋予对低氧损伤的抵抗力与使用MTORC1发表的数据一致哺乳动物模型中的抑制剂。我们称为DAF-15（GC67）的CE Raptor突变体在已发表的多宗猛禽突变体是有条件的；猛禽路径功能可以通过只是改变温度。在20°C的标准培养温度下，DAF-15（GC67）本质上是野生型在22°C下耐药性低，并在25°C（DAF-15无效突变体的表型）下捕集。 DAF-15（GC67）表型的渐变温度条件性质克服了现场的关键障碍通过提供一种遗传试剂，该试剂允许对Raptor和MTORC1进行暂时且可调的遗传控制在后生动物中首次功能。该项目将使用该独特的试剂来回答有关的关键问题猛禽如何和何时调节低氧损伤。在AIM 1中，我们将结合遗传学和蛋白质组学方法测试特定的假设，并发现猛禽调节的蛋白质，这些蛋白质决定了如何以及何时猛禽控制低氧损伤。使用条件突变体，我们已经完成了猛禽补充的屏幕功能丧失。在AIM 2中，我们将确定抑制猛禽功能丧失的突变基因，从而发现猛禽介导的低氧灵敏度的调节剂。通过这两个目标，我们将定义如何猛禽调节低氧灵敏度。鉴于抑制器筛查和蛋白质组学研究的无偏性，我们项目有可能识别MTORC1途径的新成分，从而更广泛地进步我们对MTORC1信号通路的基本理解，包括那些癌症和衰老的人。