Biology subjects

Demetriades, C.

Publications and source records attributed to Demetriades, C..

10 recordsLinked to original sources

bioRxiv2026

ATM-dependent RHEB phosphorylation couples DNA damage to lysosomal mTORC1 signaling to orchestrate the cellular response to genotoxic stress

Cells dynamically adapt to environmental stressors by rewiring signaling networks that coordinate growth, metabolism and genome maintenance. The DNA damage response (DDR) and mTORC1 signaling pathways govern DNA repair and cell growth, respectively, but how these pathways intersect remains incompletely understood. Here, we identify RHEB, the most direct mTORC1 activator, as a substrate of the DDR kinase ATM. Strikingly, we find that, although genotoxic stress differentially regulates mTORC1 activity--reducing the phosphorylation of its lysosomal target TFEB, while enhancing phosphorylation of its cytoplasmic target S6K--the DDR-induced phosphorylation of RHEB specifically controls the lysosomal mTORC1 signaling branch. Preventing RHEB phosphorylation impairs TFEB nuclear translocation and lysosome biogenesis upon DNA damage. Functionally, the RHEB phosphorylation-dependent TFEB response is required for proliferative recovery following genotoxic stress. These findings uncover an ATM-RHEB-mTORC1-TFEB signaling axis that links DNA damage to selective mTORC1 outputs, revealing a mechanism which enables cells to adapt to genotoxic cues.

cell biology↗

bioRxiv2026

TSC1 phosphorylation by lysosomal mTORC1 establishes a minimal autoregulatory feedback loop

mTORC1 lies at the center of an intricate signaling network that allows cells to homeostatically respond to a multitude of intra- and extracellular cues. Although this network is dynamically rewired to prevent excessive mTORC1 activation under permissive conditions, the mechanisms that fine-tune its activity remain incompletely understood. Here, we identify TSC1 (tuberous sclerosis complex 1), a core subunit of the mTORC1-inhibitory TSC complex, as a direct mTORC1 substrate, thus establishing an autoregulatory circuit in mTOR signaling. Notably, TSC1 combines features of both canonical and non-canonical/lysosomal mTORC1 substrates: while its phosphorylation depends on Rheb activation and growth factor signaling, it also requires an intact lysosomal LAMTOR-Rag GTPase supercomplex. In turn, TSC1 phosphorylation selectively influences lysosomal mTORC1 signaling, as expression of a phospho-dead TSC1 mutant is associated with lower levels of TFEB phosphorylation, increased nuclear TFE3 translocation, and enhanced lysosome biogenesis, while phosphorylation of S6K1, a non-lysosomal canonical substrate, remains largely unaffected. Mechanistically, phosphorylation of TSC1 by mTORC1 promotes its stability, with a non-phosphorylatable mutant undergoing proteasomal degradation. In sum, these findings reveal a minimal feedback loop within the mTOR network that orchestrates compartmentalized signaling to selectively control the activation of processes downstream of lysosomal mTORC1 signaling.

cell biology↗

bioRxiv2025

The tumor suppressor CYLD acts as a deubiquitinase for mTOR to constrain its activity

Proper control of mTOR (mechanistic/mammalian target of rapamycin) signaling is relevant for health, disease and ageing. Information from intra- and extra-cellular signaling cues is transmitted to mTOR through an intricate signaling network that impinges on the Rag and Rheb GTPases to regulate its localization and activity. Interestingly, although mTOR is a heavily ubiquitinated protein, the role of this post- translational modification (PTM) in regulating its activation status remains poorly understood. Here, through an unbiased RNAi screen, we identified the tumor suppressor CYLD deubiquitinase (DUB) as a direct negative regulator of both mTORC1 and mTORC2 activities. Mechanistically, CYLD interacts with mTOR and removes non-degradative, K63-linked ubiquitin (Ub) chains from multiple of its residues. Consequently, CYLD loss-of-function cells are characterized by mTORC1/2 hyperactivation, elevated rates of protein synthesis, increased cell size, and resistance to serum-starvation-induced activation of cell death pathways. Moreover, silencing of cyld-1, the C. elegans CYLD ortholog, fully reverses the extended lifespan of low- TORC1-activity mutant worms. Finally, we find that inactivation of CYLD is associated with hyperactivation of mTORC1 also in skin biopsies from CYLD cutaneous syndrome (CCS) patients. In sum, our findings highlight CYLD as a sentinel of mTOR hyperactivation via direct control of its ubiquitination, and suggest that dysregulated mTOR activity may contribute to the development and progression of CCS tumors.

cell biology↗

bioRxiv2025

Resilience and restoration from fasting-refeeding mediated by a nutrient-regulated linker histone

Intermittent fasting and fasting-refeeding regimens can slow biological aging across taxa1. Shifts between fed and fasted states activate ancient nutrient-sensing pathways which alter cellular and epigenetic states to promote longevity2-4. Yet how biological age trajectories progress during fasting-refeeding, and how nutrient-sensing pathways reprogram epigenetic state remain largely unknown. Here we observe increases in predicted biological age of Caenorhabditis elegans during prolonged fasting in adult reproductive diapause, followed by extraordinary reduction of biological age during refeeding. We identify hil-1/H1-0 as an evolutionarily conserved nutrient-regulated linker histone which mediates adaptations to fasting and refeeding downstream of FOXO and TFEB transcription factors. In C. elegans and human cell culture, hil-1/H1-0 upregulation during low-nutrient states promotes long-term survival and subsequent refeeding-induced recovery. Restoration of C. elegans after prolonged fasting is improved by enhancing the natural downregulation of hil-1 specifically during refeeding. Our study identifies HIL-1/H1.0 as part of an ancestral epigenetic switch during fasting-refeeding that reprograms metabolic and cellular states underlying resilience and restoration.

genetics↗

bioRxiv2024

GRASP55 Safeguards Proper Lysosome Function by Controlling Sorting of Lysosomal Enzymes at the Golgi

Lysosomes are multifunctional organelles that play important roles in cellular recycling, signaling, and homeostasis, relying on precise trafficking and activation of lysosomal enzymes. While the Golgi apparatus plays a central role in lysosomal enzyme sorting, the mechanisms linking Golgi function to lysosomal activity remain incompletely understood. Here, we identify the Golgi-resident protein GRASP55, but not its paralog GRASP65, as a key regulator of lysosome function. More specifically, we demonstrate that loss of GRASP55 expression leads to missorting and secretion of lysosomal enzymes, lysosomal dysfunction and bloating. GRASP55 deficiency also disrupts lysosomal mTORC1 signaling, reducing the phosphorylation of its lysosomal substrates, TFEB and TFE3, while sparing its non-lysosomal targets. Mechanistically, GRASP55 interacts with GNPTAB, a critical enzyme required for mannose 6-phosphate (M6P) tagging of lysosomal enzymes, and is necessary for its correct trafficking and stability. These findings reveal an essential role for GRASP55 in Golgi-lysosome communication and lysosomal enzyme trafficking, and suggest that GRASP55/GORASP2 may act as a susceptibility gene for lysosomal storage disorder (LSD)-like conditions. Overall, this work underscores the importance of Golgi-mediated protein sorting in lysosome function and lysosomal mTORC1 signaling, and provides insights into the molecular basis of LSD-related pathologies.

cell biology↗

bioRxiv2023

Spatially and Functionally Distinct mTORC1 Entities Orchestrate the Cellular Response to Amino Acid Availability

Amino acid (AA) availability is a robust determinant of cell growth, through controlling mTORC1 activity 1. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes (reviewed in 2,3). Importantly, however, 15 years after its initial discovery, the teleonomy of the proposed lysosomal regulation of mTORC1, and where mTORC1 acts on its effector proteins remain enigmatic 4. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes due to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs, and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease 2, our findings will likely open new directions toward the identification of function-specific mTORC1 regulators, and suggest new targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.

cell biology↗

bioRxiv2022

Comprehensive Evaluation of the Specificity of Rapamycin as an mTOR Inhibitor

Rapamycin is a macrolide antibiotic that functions as an immunosuppressive and anti-cancer agent, and displays robust anti-ageing effects in multiple organisms including humans. Importantly, rapamycin analogs (rapalogs) are of clinical importance against certain cancer types and neurodevelopmental diseases. Although rapamycin is widely perceived as an allosteric inhibitor of mTOR (mechanistic target of rapamycin), the master regulator of cellular and organismal physiology, its specificity has not been thoroughly evaluated so far. In fact, previous studies in cells and in mice suggested that rapamycin may be also acting independently from mTOR to influence various cellular functions. Here, we generated a gene-edited cell line, that expresses a rapamycin-resistant mTOR mutant (mTORRR), and assessed the effects of rapamycin treatment on the transcriptome and proteome of control or mTORRR-expressing cells. Our data reveal a striking specificity of rapamycin towards mTOR, demonstrated by virtually no changes in mRNA or protein levels in rapamycin-treated mTORRR cells, even following prolonged drug treatment. Overall, this study provides the first comprehensive and conclusive assessment of rapamycins specificity, with important potential implications for ageing research and human therapeutics.

cell biology↗

bioRxiv2022

Malonyl-CoA is an ancient physiological ATP-competitive mTORC1 inhibitor

Cell growth is regulated primarily by the mammalian/mechanistic Target of Rapamycin Complex 1 (mTORC1) that functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways 1. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid (FA) biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here, we show that mTORC1 senses the capacity of a cell to synthesize FAs by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is very direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When ACC1 (acetyl-CoA carboxylase 1) is hyperactive or FASN (fatty acid synthase) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with ACC1 and FASN. Our findings represent a conserved, unique, homeostatic mechanism whereby impaired FA biogenesis leads to reduced mTORC1 activity to coordinatively link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the first-described example of a physiological metabolite that directly inhibits the activity of a signalling kinase by competing with ATP for binding.

cell biology↗

bioRxiv2022

Decreased spliceosome fidelity inhibits mTOR signalling and promotes longevity via an intron retention event

Changes in splicing fidelity are associated with loss of homeostasis and ageing1-3, yet only a handful of splicing factors have been shown to be causally required to promote longevity 1-3, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within RNP-6/PUF60, a spliceosome component promoting weak 3 splice site recognition, which causes aberrant splicing, elevated stress responses, and enhances longevity in Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6 interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover a single intron retention event in egl-8/phospholipase C B4 as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulate mTORC1 signaling to control organismal life span. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates mTOR signaling and suggest a potential therapeutic strategy to delay ageing.

genetics↗

bioRxiv2020

G3BP1 tethers the TSC complex to lysosomes and suppresses mTORC1 in the absence of stress granules

G3BP1 (Ras GTPase-activating protein-binding protein 1) is widely recognized as a core component of stress granules (SG), non-membranous RNA-protein-assemblies required for cellular survival under stress. We report that in the absence of SG, G3BP1 acts as lysosomal anchor of the Tuberous Sclerosis Complex (TSC) protein complex. By tethering the TSC complex to lysosomes, G3BP1 suppresses signaling through the metabolic master regulator mTORC1 (mechanistic target of rapamycin complex 1). Like the known TSC complex subunits, G3BP1 suppresses phenotypes related to mTORC1 hyperactivity in the context of tumors and neuronal dysfunction. Thus, G3BP1 is not only a core component of SG but also a key element of lysosomal TSC-mTORC1 signaling. HighlightsThe bona fide stress granule component G3BP1 O_LIis a key element of the TSC-mTORC1 signaling axis. C_LIO_LItethers the TSC complex to lysosomes. C_LIO_LIprevents mTORC1 hyperactivation by metabolic stimuli. C_LIO_LIsuppresses mTORC1-driven cancer cell motility and epileptiform activity. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/044081v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@77cb4forg.highwire.dtl.DTLVardef@c4d1a0org.highwire.dtl.DTLVardef@19e83acorg.highwire.dtl.DTLVardef@1d6a378_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗