Biology subjects

Monfregola, J.

Publications and source records attributed to Monfregola, J..

4 recordsLinked to original sources

bioRxiv2026

HKDC1 contributes to aberrant lysosome-mitochondria contact in Niemann-Pick disease type C

Niemann-Pick disease type C (NPC) is a neurovisceral lysosomal storage disorder comprising two clinically indistinguishable but genetically distinct subtypes caused by mutations in NPC1, or NPC2. The specific impact of each deficiency on cellular homeostasis remains poorly defined due to the phenotypic heterogeneity of patient-derived models and a lack of isogenic platforms for comparative study. Here we established isogenic ARPE19 models of NPC1 and NPC2 deficiency that faithfully recapitulate hallmark pathologies, including homogeneous lysosomal expansion and lipid sequestration. Direct comparison of these isogenic lines revealed a fundamental divergence in organelle crosstalk: while both genotypes exhibit comparable lipid accumulation, expanded mitochondria-lysosome contact sites (MLCs) are observed exclusively in NPC1-/- cells. Using StARD3-targeted proximity labelling and quantitative proteomics, we identified the mitochondrial protein HKDC1 as an MLC regulator. We demonstrate that HKDC1 is markedly upregulated in NPC1-/- cells and that its overexpression drives MLC expansion in wild-type cells. Thus our study uncovers a homeostatic role for HKDC1-mediated organelle remodelling and demonstrates the power of isogenic modelling for identifying novel regulators of organelle architecture and potential therapeutic targets.

cell biology↗

bioRxiv2024

Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

Endosomes have emerged as major signaling hubs where different internalized ligand-receptor complexes are integrated and the outcome of signaling pathways are organized to regulate the strength and specificity of signal transduction events. Ezrin, a major membrane-actin linker that assembles and coordinates macromolecular signaling complexes at membranes, has emerged recently as an important regulator of lysosomal function. Here, we report that endosomal-localized EGFR/Ezrin complex interacts with and triggers the inhibition of the Tuberous Sclerosis Complex (TSC complex) in response to EGF stimuli. This is regulated through activation of the AKT signaling pathway. Loss of Ezrin was not sufficient to repress TSC complex by EGF and culminated in translocation of TSC complex to lysosomes triggering suppression of mTORC1 signaling. Overexpression of constitutively active EZRINT567D is sufficient to relocalize TSC complex to the endosomes and reactivate mTORC1. Our findings identify EZRIN as a critical regulator of autophagy via TSC complex in response to EGF stimuli and establish the central role of early endosomal signaling in the regulation of mTORC1. Consistently, Medaka fish deficient for Ezrin exhibit defective endo-lysosomal pathway, attributable to the compromised EGFR/AKT signaling, ultimately leading to retinal degeneration. Our data identify a pivotal mechanism of endo-lysosomal signaling involving Ezrin and its associated EGFR/TSC complex, which are essential for retinal function.

cell biology↗

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↗