DEFINING RAPTOR-MEDIATED MECHANISMS OF HYPOXIC INJURY

定义猛禽介导的缺氧损伤机制

• 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
• 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

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）的机制靶点是丝氨酸/苏氨酸激酶，其被营养物激活， 能量，磷酸化底物，以促进和协调合成代谢。鉴定了mTOR 大约30年前，在一个酵母筛选突变体抵抗药物雷帕霉素的生长抑制;不久， 此后，通过生物化学方法独立地发现了高度保守的哺乳动物直系同源物。 在过去的30年里，生物化学和遗传学方法已经从进化上鉴定出许多 mTOR信号通路的保守组分以及形成两种不同mTOR的蛋白质 复合物，mTORC1和mTORC2。mTORC1由三个核心组件组成，mTOR，Raptor和 mLST8（具有SEC13蛋白8的哺乳动物致死性）; Rictor取代mTORC 2中的Raptor。mTORC1被以下物质抑制 雷帕霉素，而mTORC 2相对不敏感。mTORC1已经被深入研究，因为它 作为细胞对营养素和能量反应的中央调节器， 重要的条件，如癌症，衰老和缺氧损伤。通过无偏突变体筛选，在C. elegans的耐缺氧突变体，我们已经确定了一个错义部分丧失功能的突变daf- 15，编码唯一的C. Raptor的elegans直系同源物（Ce-Raptor）。CRISPR/CAS9产生的突变体 与相同的病变证实了突变赋予耐缺氧性。我们发现， Ce-Raptor的功能赋予对缺氧损伤的抵抗力与使用mTORC 1的已发表数据一致 哺乳动物模型中的抑制剂。我们的Ce-Raptor突变体称为daf-15（gc67）是唯一的， 后生动物猛禽突变体，因为它是有条件的;猛禽途径的功能可以关闭和再次打开， 简单地改变温度。daf-15（gc67）在20 ℃的标准培养温度下基本上是野生型， 在22 ° C下耐缺氧，并且在25 ° C下发育停滞，daf-15无效突变体的表型。的 DAF-15（GC 67）表型的分级温度条件性质克服了该领域的关键障碍 通过提供允许Raptor和mTORC1的时间和可调遗传控制的遗传试剂， 首次在后生动物中发挥作用。该项目将使用这种独特的试剂来回答以下关键问题 Raptor如何以及何时调节缺氧损伤。在目标1中，我们将联合收割机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，决定如何以及何时猛禽 控制缺氧损伤。使用我们的条件突变体，我们已经完成了猛禽抑制剂的筛选 功能丧失。在目标2中，我们将鉴定抑制Raptor功能丧失的突变基因， 发现Raptor介导的缺氧敏感性调节因子。通过这两个目标，我们将定义猛禽如何 调节低氧敏感性。考虑到抑制基因筛选和蛋白质组学研究的无偏性， 该项目有可能识别mTORC 1通路的新组分，从而更广泛地推进 我们对mTORC1信号通路的基本理解，包括那些调节癌症和衰老的信号通路。