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) 的机制靶标是一种丝氨酸/苏氨酸激酶，可被营养物质和确定了促进和协调合成代谢的能量、磷酸化底物。大约三十年前，在酵母中筛选对雷帕霉素药物生长抑制具有抗性的突变体；此后，通过生化方法独立发现了高度保守的哺乳动物直向同源物。在过去的三十年里，生物化学和遗传学方法已经鉴定出许多进化论 mTOR 信号通路的保守成分以及形成两个不同 mTOR 的蛋白质 mTORC1 和 mTORC2 复合体由三个核心成分组成：mTOR、Raptor 和 mLST8（哺乳动物致死性 SEC13 蛋白 8）；Rictor 取代 mTORC1 中的 Raptor，被抑制。雷帕霉素而 mTORC2 相对不敏感，因此已得到深入研究。作为细胞对营养物质和能量的反应的中央调节器，从而影响临床通过对 C. 的无偏突变筛选，发现了癌症、衰老和缺氧损伤等重要条件。线虫抗缺氧突变体，我们在 daf- 中发现了错义部分功能丧失突变 15，编码 Raptor 的唯一秀丽隐杆线虫直系同源物 (Ce-Raptor) CRISPR/CAS9 生成的突变体。具有相同病变的突变证实了我们的发现，即降低了耐缺氧性。 Ce-Raptor 赋予对缺氧损伤的抵抗力的功能与使用 mTORC1 发表的数据一致我们的 Ce-Raptor 突变体 daf-15(gc67) 在已发表的研究中是独一无二的。后生动物 Raptor 突变体是有条件的，可以通过以下方式关闭和再次打开：在标准培养温度 20°C 下，daf-15(gc67) 本质上是野生型，在 22°C 下具有耐缺氧性，在 25°C 下发育停滞，这是 daf-15 无效突变体的表型。 daf-15(gc67) 表型的分级温度条件性质克服了该领域的关键障碍通过提供一种遗传试剂，可以对 Raptor 和 mTORC1 进行临时和可调的遗传控制该项目将使用这种独特的试剂来回答有关的关键问题。 Raptor 如何以及何时调节缺氧损伤在目标 1 中，我们将结合遗传学和蛋白质组学方法来研究。测试特定的假设并发现 Raptor 调节的蛋白质，这些蛋白质决定 Raptor 如何以及何时使用我们的条件突变体，我们已经完成了 Raptor 抑制子的筛选。在目标 2 中，我们将识别抑制 Raptor 功能丧失的突变基因。通过这两个目标，我们将发现 Raptor 介导的缺氧敏感性的调节因子。考虑到抑制子筛选和蛋白质组学研究的公正性，我们的研究该项目有潜力识别 mTORC1 通路的新成分，从而更广泛地取得进展我们对 mTORC1 信号通路的基本了解，包括调节癌症和衰老的信号通路。