Defining RAPTOR-mediated mechanisms of hypoxic injury - the mechanistic target of rapamycin (MTOR) is a serine/threonine kinase that is activated by nutrients and energy, phosphorylating substrates to…

Defining RAPTOR-mediated mechanisms of hypoxic injury - 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.