Biology subjects

Napolitano, G.

Publications and source records attributed to Napolitano, G..

4 recordsLinked to original sources

bioRxiv2025

Phosphoproteomics reveals novel BCR::ABL1-independent mechanisms of resistance in chronic myeloid leukemia

BCR::ABL1 drives chronic myeloid leukemia (CML) disease and treatment, as revealed by the success of tyrosine kinase inhibitor (TKI) therapy. However, additional poorly characterized molecular pathways, acting as BCR::ABL1 independent mechanisms, play crucial roles in CML, contributing to leukemic stem cells (LSCs) persistence, TKI resistance and disease progression. Here, by combining high sensitive mass spectrometry (MS)-based phosphoproteomics with the SignalingProfiler pipeline, we obtained two signaling maps offering a comprehensive description of the BCR::ABL1 dependent and independent pro-survival signalling mechanisms. We leveraged these maps to unbiasedly and systematically discover therapeutic vulnerabilities, by implementing the Druggability Score computational algorithm. By this strategy, and in combination with in vitro and in ex vivo functional assays, we show a crucial role of acquired FLT3-dependency in resistant CML models. In conclusion, we reposition FLT3, one of the most frequently mutated drivers of acute leukemia, as a potential therapeutic target for TKI resistant CML patients.

systems biology↗

bioRxiv2024

Structural basis for growth factor and nutrient signal integration on the lysosomal membrane by mTORC1

Mechanistic target of rapamycin complex 1 (mTORC1), which consists of mTOR, Raptor, and mLST8, receives signaling inputs from growth factor signals and nutrients. These signals are mediated by the Rheb and Rag small GTPases, respectively, which activate mTORC1 on the cytosolic face of the lysosome membrane. We biochemically reconstituted the activation of mTORC1 on membranes by physiological submicromolar concentrations of Rheb, Rags, and Ragulator. We determined the cryo-EM structure and found that Raptor and mTOR directly interact with the membrane at anchor points separated by up to 230 [A] across the membrane surface. Full engagement of the membrane anchors is required for maximal activation, which is brought about by alignment of the catalytic residues in the mTOR kinase active site. The observations show at the molecular and atomic scale how converging signals from growth factors and nutrients drive mTORC1 recruitment to and activation on the lysosomal membrane in a three-step process, consisting of (1) Rag-Ragulator-driven recruitment to within [~]100 [A] of the lysosomal membrane, (2) Rheb-driven recruitment to within [~]40 [A], and finally (3) direct engagement of mTOR and Raptor with the membrane. The combination of Rheb and membrane engagement leads to full catalytic activation, providing a structural explanation for growth factor and nutrient signal integration at the lysosome.

biochemistry↗

bioRxiv2022

Structural basis for mTORC1-dependent regulation of the lysosomal and autophagic transcription factor TFEB

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino-acid-mediated activation of the RagC GAP FLCN. TFEB lacks the TOR signaling (TOS) motif responsible for the recruitment of other mTORC1 substrates. We used cryo-electron microscopy (cryo-EM) to determine the structure of TFEB as presented to mTORC1 for phosphorylation. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first via a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB in a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. Mutation of the clamp drives TFEB constitutively into the nucleus whilst having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. This structure presents the phosphorylatable Ser residues of TFEB to the mTORC1 active site in a suitable geometry for their phosphorylation. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

cell biology↗

bioRxiv2018

mTOR-dependent phosphorylation controls TFEB nuclear export

The transcriptional activation of catabolic processes during starvation is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient re-feeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data reveal that modulation of TFEB nuclear export via phosphorylation plays a major role in the modulation of TFEB localization and activity.

cell biology↗