Structural characterization of the ragulator complex, a mediator of the amino acid signaling to mTORC1

Abstract

The signaling pathways commonly deregulated in cancer frequently involve the PI3k-Akt-mTOR pathway. The mTOR kinase, identified as the target of rapamycin, plays an essential role in the regulation of cell growth, integrating signals from both local and systemic nutrient availability, growth factors and several types of stress. In the context of the nutrient responsive branch, the regulation of mTOR by amino acid availabilty stands as a conserved yet poorly understood pathway. Recent studies have demonstrated that amino acids signaling is mediated by the Rag GTPases and the Ragulator complex, which anchors these GTPases to the lysosomal surface and promotes their activation by exchanging GDP for GTP. In this project, our main purpose is to study the molecular mechanism of mTOR activation by amino acids through the structural characterization of the Ragulator complex. We intend to reconstruct the Ragulator complex in vitro from recombinant proteins and to elucidate its structure using a combination of experimental techniques (X-ray scattering at low angle, crystallography and mass spectrometry) with computational modeling and docking. The structural model obtained will be validated by mutagenesis experiments combined with subcellular localization and interaction assays of the subunits. Our aims rely on our previous studies and a solid international collaboration with Dr. David Sabatini (MIT/USA), a distinguished researcher in the field of mTOR and responsible for the identification of the Ragulator complex. We expect the results to bring very relevant contributions to the field of study and to shed light on fundamental regulatory mechanisms of these proteins, further allowing a possible application in developing new modulators of the mTOR pathway activity. (AU)