Biology subjects

Mesquita, F. S.

Publications and source records attributed to Mesquita, F. S..

7 recordsLinked to original sources

bioRxiv2026

The luminal and cytosolic domains of CLIMP-63 cooperate to set ER sheet width

The endoplasmic reticulum (ER) synthesized and folds secreted and transmembrane protein in regions composed of flattened sheets with a defined luminal width of [~]50 nm. CLIMP-63 has been proposed to be the principal determinant of this spacing, but the molecular mechanism by which it controls luminal width remains unclear. The luminal domain of CLIMP-63 has been proposed to act as a fixed-length spacer through antiparallel coiled-coil dimerization between molecules on opposing membranes. Here we revise this model. Using systematic cysteine trapping across the luminal domain, we demonstrate that CLIMP-63 assembles into parallel trimers. An AlphaFold-guided screen of CLIMP-63 orthologs combined with cryo-electron microscopy reveals that the luminal domain forms an elongated trimeric rod with intrinsic conformational flexibility at hinge regions. This flexibility is functionally required: replacing the human luminal domain with a more rigid ortholog causes complete ER vacuolation. Higher-order assembly of CLIMP-63 trimers beyond the trimer depends on the cytosolic tail rather than on luminal trans interactions, and the tails role in higher order assembly is separable from its other architectural functions. Together, these findings show that luminal trimeric extension and cytosolic tail-mediated clustering cooperate to determine ER sheet width.

cell biology↗

bioRxiv2026

Src-dependent tyrosine-phosphorylation of NM2A has a protective role against bacterial pore-forming toxins

Pore-forming toxins (PFTs) are key bacterial virulence factors that disrupt host plasma membrane (PM) integrity, triggering cellular stress and initiating repair mechanisms. The cytolysin Listeriolysin O (LLO), secreted by Listeria monocytogenes, has well established roles in infection, yet the host signaling responses to LLO-induced damage remain poorly understood. Here, we identify a previously unrecognized protective pathway in which LLO triggers rapid activation of the tyrosine kinase Src, leading to phosphorylation of the non-muscle myosin II heavy chain NMHC2A at tyrosine 158. While Src activation and NMHC2A tyrosine phosphorylation have been observed during Listeria infection, we demonstrate here that both responses are directly driven by LLO. This phosphorylation event does not alter NMHC2A motor activity in vitro but is required for cytoskeletal reorganization and efficient responses to PM damage. Using Caenorhabditis elegans, we further show that phosphorylation of the NMHC2A homolog NMY-2 at the conserved tyrosine 163 is required for survival under PFT-induced stress and heat shock, revealing an evolutionarily conserved defense mechanism. Together, our findings establish Src-mediated NMHC2A phosphorylation as a critical link between PFT-induced PM damage sensing and actomyosin remodeling, advancing our understanding of host defense against bacterial toxins. Author SummaryPathogenic bacteria often produce toxins that create pores in the membranes of host cells, compromising cell survival and helping the infection spread. To counter this, host cells must quickly detect and repair the damage. We studied how cells respond to Listeriolysin O (LLO), produced by the human pathogenic bacterium Listeria monocytogenes. We found that LLO activates the host Src kinase, which in turn modifies a key structural protein, non-muscle myosin II (NMII). Although this modification does not affect NMIIs normal motor function, it is crucial for organizing the cellular skeleton and protecting the cells membrane after toxin damage. Importantly, we found that this protective mechanism is conserved in the worm Caenorhabditis elegans, where it also defends against a similar bacterial toxin and even heat stress. Our findings reveal a new way that cells use existing structural proteins to rapidly respond to external threats, providing insight into how organisms protect themselves from harmful microbes and environmental stress.

microbiology↗

bioRxiv2025

Molecular Regulation and Physiological Role of 1 GOLPH3-mediated Golgi retention

The Golgi complex serves as the central hub of the biosynthetic pathway, where anterograde and retrograde trafficking converge. How cargo and Golgi-resident proteins traverse this organelle has long been debated. Recent studies have identified a molecular machinery that sorts resident proteins into retrograde-directed COPI vesicles during cisternal maturation. Golgi phosphoprotein 3 (GOLPH3) is a key component of this system; however, its physiological relevance and regulatory mechanisms remain poorly defined. Here, we show that GOLPH3 depletion in mice disrupts both protein and lipid glycosylation, causes partially penetrant embryonic lethality, and severely impairs growth and bone mineralization. At the molecular level, we find that GOLPH3 is regulated by functionally antagonistic S-acylation events that control the topology of its membrane association. To mediate retrograde trafficking of Golgi-resident glycosyltransferases, GOLPH3 must bind their cytosolic tails. This occurs via a negatively charged surface region, which is correctly oriented only in one of the S-acylated GOLPH3 conformations. Together, these findings reveal a lipid-mediated regulatory mechanism for intra-Golgi trafficking and establish the critical role of GOLPH3 in vertebrate development.

cell biology↗

bioRxiv2025

Identification of the Lipid Oxygen Radical Defense pathway and its epigenetic control

Membrane phospholipids are vulnerable to oxidative radicals, and uncontrolled lipid peroxidation affects cell viability. Cells have evolved quality control and defense mechanisms, of which the genetic regulation is not fully understood. Here, we identify what we have coined the Lipid Oxygen Radical Defense (LORD) pathway. It is epigenetically repressed by a complex comprising the KRAB-zinc finger protein ZNF354A, the scaffold protein KAP1/TRIM28, the histone methyltransferase SETDB1, and the transcriptional activator ATF2. Upon lipid peroxide accumulation, p38- and JNK-dependent phosphorylation of ATF2, KAP1/TRIM28, and ZNF354A leads to disassembly of the repressive complex, releasing ZNF354A from specific DNA loci and activating a network of protective genes, including NRF2 targets. The pathway affects the cellular sensitivity to oxidative stress and ferroptosis, revealing a novel layer of epigenetic control in lipid quality control and damage repair. This positions the LORD pathway as a promising therapeutic target for diseases linked to chronic inflammation, neurodegeneration and cancer.

cell biology↗

bioRxiv2023

SARS-CoV-2 shifts transcription of host gene to increase Spike acylation and boost infectivity

SARS-CoV-2 infection requires Spike protein mediating fusion between the viral and cellular membranes. The fusogenic activity of Spike requires its post-translational lipid modification by host S-acyltransferases, predominantly ZDHHC20. Previous observations indicate that SARS-CoV-2 infection augments the S-acylation of Spike when compared to transfection. Here, we find that SARS-CoV-2 infection triggers a change in the transcriptional start site of the zddhc20 gene, both in cells and in an in vivo infection model, resulting in a 67-amino-acid-long N-terminally extended protein with 37-times higher Spike acylating activity, leading to enhanced viral infectivity. Furthermore, we observed the same induced transcriptional change in response to other challenges, such as chemically induced colitis, indicating that SARS-CoV-2 hijacks an existing cell damage response pathway to generate more infectious viruses.

cell biology↗

bioRxiv2021

S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

SARS-CoV-2 virions are surrounded by a lipid bilayer which contains membrane proteins such as Spike, responsible for target-cell binding and virus fusion, the envelope protein E and the accessory protein Orf3a. Here, we show that during SARS-CoV-2 infection, all three proteins become lipid modified, through action of the S-acyltransferase ZDHHC20. Particularly striking is the rapid acylation of Spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics and biochemical approaches, we show that this massive lipidation controls Spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid rich lipid nanodomains, in the early Golgi where viral budding occurs. ZDHHC20-mediated acylation allows the formation of viruses with enhanced fusion capacity and overall infectivity. Our study points towards S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

cell biology↗

bioRxiv2020

Src-dependent NM2A tyrosine-phosphorylation regulates actomyosin dynamics

Non-muscle myosin 2A (NM2A) is a key cytoskeletal enzyme that along with actin assembles into actomyosin filaments inside cells. NM2A is fundamental in cellular processes requiring force generation such as cell adhesion, motility and cell division, and plays important functions in different stages of development and during the progression of viral and bacterial infections. We previously identified a novel tyrosine phosphorylation on residue 158 (pTyr158) in the motor domain of NM2A. This phosphorylation is dependent on Src kinase and is promoted by Listeria monocytogenes infection of epithelial cells, however its role is unknown. Here we show that Listeriolysin O (LLO), the pore-forming toxin (PFT) secreted by L. monocytogenes, is sufficient to trigger NM2A pTyr158 by activating Src, an upstream regulator of actomyosin remodeling. We further address the role of NM2A pTyr158 on the organization and dynamics of the actomyosin cytoskeleton and find that, by controlling the activation of the NM2A, the status of the pTyr158 alters cytoskeletal organization, dynamics of focal adhesions and cell motility. In vitro, we observe that non-phosphorylatable and phospho-mimetic versions of NM2A at Tyr158 display motor and ATPase activities similar to the wild-type NM2A, which indicates that the phenotype of these mutants in cells is independent of their ability to translocate actin filaments. Importantly, we find the regulation of this phosphorylation site to be of physiological relevance in Caenorhabditis elegans, in particular in response to intoxication by a PFT and to heat shock. We conclude that the control of the phosphorylation status at NM2A Tyr158 is a conserved trait that contributes to the regulation of actomyosin dynamics and the ability of cells to respond to bacterial infection. We propose Src-dependent NM2A pTyr158 as a novel layer of regulation of the actomyosin cytoskeleton.

cell biology↗