Biology subjects

Khaled, M.

Publications and source records attributed to Khaled, M..

8 recordsLinked to original sources

bioRxiv2026

Endosomal membrane state governs trafficking and cell migration under TFEB control

Transcription Factor EB (TFEB) regulates the biogenesis of lysosomes, which are acidic organelles of the endosomal network. While its role in cellular clearance is well established, the pleiotropic functions of TFEB across diverse cellular processes, as well as its activation in several cancer types, remain incompletely understood. Investigating TFEB in a bladder cancer model, we found that its depletion selectively impaired cell migration and adhesion. This phenotype was associated with the retention of the adhesion molecule integrin {beta}5 (ITG{beta}5), in central intracellular compartments identified as multivesicular bodies (MVBs) upon TFEB knockdown (KD). Mechanistically, TFEB regulated cellular lipid composition and membrane fluidity of acidic endosomes that controlled ITG{beta}5 trafficking. Remarkably, exogenous supplementation with a monounsaturated fatty acid (MUFA) that increased MVB membrane fluidity was sufficient to phenocopy the intracellular trapping of ITG{beta}5 in 2D culture and 3D tumoroids. We further showed that TFEB-dependent maintenance of MVB membrane fluidity relies on ESCRT-0 subcomplex, HGS/Hrs, which retains cholesterol at MVBs. Together, these findings reveal how transcriptional programs shape endosomal membrane states to control cargo trafficking and cell behavior.

cell biology↗

bioRxiv2026

Solvation Shapes the Conformational Landscape of a Therapeutically Relevant SMN2 Splice-Site Defect

The SMN2 exon 7 5 splice-site/U1 snRNA duplex contains an A-1 bulge that weakens splice-site recognition and represents a therapeutically relevant RNA connectivity defect, yet its conformational landscape and coupling to solvation remain poorly understood. Here, we performed enhanced-sampling Hamiltonian replica-exchange molecular dynamics simulations of the SMN2 splice-site duplex using four explicit-solvent models (OPC, TIP4P-Ew, TIP3P, and SPC/E) and characterized the sampled ensemble using linear and machine-learned latent representations. Across representations, the A-1 defect consistently populated three metastable conformational states distinguished by local duplex geometry, base stacking, hydrogen-bonding patterns, and solvent exposure. The relative populations of these states, together with first-shell hydration and Na+ distributions around the defect, varied substantially across water models, demonstrating that hydration and ion organization actively shape the equilibrium between locally accommodated and solvent-exposed conformations of the SMN2 splice-site bulge. Our results shed light on the conformational components of this therapeutic RNA target and highlight the impact of solvation model as an important consideration for molecular simulations of RNA splice-site recognition and small-molecule repair.

biophysics↗

bioRxiv2025

A hydrophobic cluster controls long-range allostery in the TRMT2A RNA recognition motif

TRMT2A has emerged as a disease-modifying target in polyglutamine (PolyQ) models, yet the conformational preferences and allostery of its RNA recognition motif (RRM) remain poorly resolved. Here we combine extensive atomistic molecular dynamics with Markov state modeling (BHMSM), transition path theory, and structure-based pocket analysis to map the conformational landscape of the human TRMT2A RRM. We resolve six metastable states and show that a hydrophobic cluster centered on F92-W134-L133 governs their interconversion. We further identify residues contributing to RNA strand recognition and reveal state-specific cryptic pockets consistent with reported TRMT2A RRM small molecule inhibitors. Together, these results support a hinge-gate model in which a soft, defectenabled 2 segment and a loop 5 hydrophobic cluster coordinate long-range communication between the RNP face and opposite side, yielding testable mutational predictions and state-specific opportunities for allosteric control of TRMT2A in polyQ disease contexts.

biophysics↗

bioRxiv2024

USP9X is a mechanosensitive deubiquitinase that controls tumor cell invasiveness and drug response through YAP stabilization

Post-translational modification by ubiquitin is crucial for protein turnover. Deubiquitinases (DUBs) remove ubiquitin chains from target proteins to prevent their degradation by the proteasome, thus acting as gatekeepers of protein homeostasis alongside the ubiquitin-proteasome system (UPS). Tumor cells exhibit remarkable plasticity, enabling them to adapt to anticancer treatments and the conditions of the tumor microenvironment, including mechanical cues from the extracellular matrix (ECM). However, the role of DUBs in mechanotransduction remains unexplored. To identify DUBs involved in cancer cell mechanosignaling, we used melanoma cells grown on collagen matrices with varying stiffnesses and an activity-based ubiquitin probe to profile DUB activities. Our approach, combined with quantitative proteomics, revealed that ubiquitin-specific protease 9X (USP9X) is sensitive to ECM stiffness through discoidin domain receptors (DDR)/actomyosin signaling pathway. In silico analysis further indicated that the mechanosensor YAP is part of the USP9X interactome, and USP9X expression correlates with the YAP transcriptional signature in melanoma. We hypothesized that mechanical signals regulate YAP levels through USP9X DUB activity. Consistently, low collagen stiffness reduced YAP expression, and siRNA-mediated depletion or pharmacological inhibition of USP9X decreased YAP protein expression in tumor cells. Conversely, knockdown of the ubiquitin E3 ligase {beta}TrCP increased YAP protein levels. Affinity purification of polyubiquitinated proteins using Tandem Ubiquitin Binding Entities (TUBEs) showed that combined USP9X and proteasome inhibition increased YAP poly-ubiquitination, revealing that USP9X deubiquitinates YAP to prevent its proteasomal degradation. Targeting USP9X impaired stiffness-mediated responses, including YAP nuclear translocation and transcriptional activity, cell migration and invasion, and drug resistance. An experimental metastasis assay showed that stable knockdown of USP9X impaired melanoma cell lung colonization. Finally, targeting USP9X in a syngeneic BRAF-mutant melanoma model counteracted targeted therapy-induced ECM remodeling, enhanced treatment efficacy, and delayed tumor relapse. Our findings reveal a novel role of USP9X in cancer cell mechanobiology and drug resistance through stiffness-dependent stabilization of the oncoprotein YAP, proposing USP9X as a targetable "mechano-DUB" in cancer.

cancer biology↗

bioRxiv2024

Amyloid Morphology is Encoded in H-bonds and Electrostatics Interactions Ruling Protein Phase Separation

Ion-protein interactions regulate biological processes and are the basis of key strategies of modulating protein phase diagrams and stability in drug development. Here, we report the mechanisms by which H-bonds and electrostatic interactions in ion-protein systems determine phase separation and amyloid formation. Using microscopy, small-angle X-ray scattering, circular dichroism and atomistic molecular dynamics (MD) simulations, we found that anions specifically interacting with insulin induced phase separation by neutralising the protein charge and forming H-bond bridges between insulin molecules. The same interaction was responsible for an enhanced insulin conformational stability and resistance to oligomerisation. Under aggregation conditions, the anion-protein interaction translated into the activation of a coalescence process, leading to amyloid-like microparticles. This reaction is alternative to conformationally-driven pathways, giving rise to elongated amyloid-like fibrils and occurs in the absence of preferential ion-protein binding. Our findings depict a unifying scenario in which common interactions dictated both phase separation at low temperatures and the occurrence of pronounced heterogeneity in the amyloid morphology at high temperatures, similar to what has previously been reported for protein crystal growth.

biophysics↗

bioRxiv2021

Amyloid-β peptide dimers undergo a random coil to β-sheet transition in the aqueous phase but not at the neuronal membrane

AO_SCPLOWBSTRACTC_SCPLOWThe aggregation of amyloid {beta}-peptides into neurotoxic oligomers is a key feature in the development of Alzheimers disease. Mounting evidence suggests that the neuronal cell membrane is the main site of oligomer-mediated neuronal toxicity. To gain a detailed understanding of the mutual effects of amyloid-{beta} oligomers and the neuronal membrane, we carried out a total of 12 {micro}s all-atom molecular dynamics (MD) simulations of the dimerization of the full-length A{beta}42 peptide in the presence of a lipid bilayer mimicking the in vivo composition of neuronal membranes. The conformational changes of A{beta}42 resulting from its dimerization and interactions with the neuronal membrane are compared to those occurring upon its dimerization in the aqueous phase, which is also tested by 12 {micro}s of MD simulations. We find that the interactions with the neuronal membrane decrease the order of the A{beta}42 dimer by attenuating its propensity to form a {beta}-sheet structure. The main lipid interaction partners of A{beta}42 are the surface-exposed sugar groups of the gangliosides GM1. A{beta}42 dimerization in solution, on the other hand, is characterized by a random coil to {beta}-sheet transition that seems to be on-pathway to amyloid aggregation. As the neurotoxic activity of amyloid oligomers increases with oligomer order, the results suggest that GM1 is neuroprotective against A{beta}-mediated toxicity by inhibiting the formation of ordered amyloid oligomers.

biophysics↗

bioRxiv2019

Tumors escape immunosurveillance by overexpressing the proteasome activator REGγ

The success of CD8+ T cell based cancer immunotherapy emphasizes the importance of understanding the mechanisms of generation of MHC-I peptide ligands and possible pathways of tumor cell escape from immunosurveillance. Recently, we showed that peptides generated in the nucleus during the pioneer round of mRNA translation (pioneer translation products, or PTPs) can be a potentially important source of tumor specific peptides, given the presence of aberrant splicing and transcription associated with oncogenesis. Here we show that cancer cells up-regulation of the REG{gamma} proteasome regulator results in increased destruction of PTP-derived peptides in the nucleus thus subverting immunosurveillance. These findings add to understanding of the role of REG{gamma} in antigen processing and identify it as a druggable target for improving the efficacy of cancer immunotherapy.\n\nSignificanceWith the clear success of CD8+ T cell based immunotherapy, it is critical to understand i) how tumor cells generate MHC-I peptide antigens? and ii) the various mechanisms used by cancer cells to evade immunosurveillance. One of them is to up-regulate the REG{gamma} proteasome regulator which results in an increase destruction of MHC-I peptides in the nucleus thus subverting immunosurveillance.

immunology↗

bioRxiv2017

OCT4 Impedes Cell Fate Redirection By The Melanocyte Lineage Master Regulator MITF

Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has never been systemically examined. Here we discovered that amongst different developmental stages, embryonic stem cells (ESCs) were resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. We generated a transgenic system and found that in ESCs, the pluripotency master regulator, OCT4, counteracts pro-differentiation induced by MITF by physical interference with MITF transcriptional activity. We further found that ESCs must be released from OCT4-maintained pluripotency prior to ectopically induced differentiation. Moreover, OCT4 induction in various differentiated cells repressed their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggested that OCT4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.

developmental biology↗