DC 1: Novel regulators of RAG GTPase mediated Non-Canonical (NC) mTORC1 signaling

Project part of Work Package 1

Objectives : Non-Canonical mTORC1 (NC-mTORC1) signaling controls the function of MiT-TFE transcription factors through substrate recruitment, mediated by RagC/D and Folliculin (FLCN), independently of the TSC-Rheb axis. Our discovery represents a paradigm shift in the field. However, we still lack knowledge of components, regulators and mechanisms by which this pathway contributes to mTORopathies such as Birt-Hogg-Dubé (BHD) syndrome and TSC. The doctoral candidate will perform interactome analysis of FLCN, FNIP1 and RagC by IPs or bimolecular complementation affinity purification (BiCAP) and LC-MS/MS at FTELE MS Facility. Candidate interactors will be validated by co-IP and a validated multiparametric high-content assay at TIGEM High Content Screening Facility (HCSF), to test TFEB nuclear localization and canonical mTORC1 activity (pS6). Hits inducing TFEB nuclear translocation without affecting pS6 (i.e. NC-mTORC1) will be characterized based on the biochemical structure and function, and regarding their tumorigenic potential (proliferation, clonogenic assays, tumor sphere formation) in cell and animal models of mTORopathies.

Expected Results: Doctoral candidate will provide new insights into the functioning of NC-mTORC1 machinery and reveal targets for therapeutic intervention.

Enrolment in Doctoral degree(s): University of Naples “Federico II”.

Planned secondments: 3 months at UPCité (INEM) – Paris, France for TFEB involvement in TSC animal models. 3 months at Mimetas (Leiden, the Netherlands) to establish FLCN mutant kidney on chip