RESUMO
Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.
Assuntos
Microscopia Crioeletrônica , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/ultraestrutura , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/ultraestrutura , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Aminoácidos/deficiência , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/ultraestruturaRESUMO
The popularization of microwave raised concerns about its influence on health including cognitive function which is associated greatly with dendritic spines plasticity. SNK-SPAR is a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity. In this study, Wistar rats were exposed to microwaves (30â¯mW/cm2 for 6â¯min, 3 times/week for 6â¯weeks). Spatial learning and memory function, distribution of dendritic spines, ultrastructure of the neurons and their dendritic spines in hippocampus as well as the related critical molecules of SNK-SPAR pathway were examined at different time points after microwave exposure. There was deficiency in spatial learning and memory in rats, loss of spines in granule cells and shrinkage of mature spines in pyramidal cells, accompanied with alteration of ultrastructure of hippocampus neurons. After exposure to 30â¯mW/cm2 microwave radiation, the up-regulated SNK induced decrease of SPAR and PSD-95, which was thought to cause the changes mentioned above. In conclusion, the microwave radiation led to shrinkage and even loss of dendritic spines in hippocampus by SNK-SPAR pathway, resulting in the cognitive impairments.