Biology subjects

Messens, J.

Publications and source records attributed to Messens, J..

14 recordsLinked to original sources

bioRxiv2026

PTPRF is a stress responsive cytoskeletal checkpoint that coordinates metabolic adaptation in hepatocytes and β cells

Cytoskeletal remodeling is essential for adaptation to nutrient availability, yet how cells coordinate actin dynamics with glucose homeostasis in metabolic organs remains unclear. Here, we identify a pathway linking metabolic stress to actin reorganization in hepatocytes and pancreatic {beta} cells. This mechanism involves transcriptional repression of the receptor protein tyrosine phosphatase PTPRF by spliced XBP1, a key unfolded protein response factor. In hepatocytes, PTPRF loss under dietary stress enhances insulin signaling, increases mitochondrial respiration and reduces steatosis. Proteomic analyses show that PTPRF interacts with regulators of actin polymerization and cell junctions, and its deletion promotes actin filament organization, shifting metabolism toward oxidative pathways. In {beta} cells, PTPRF deficiency similarly enhances actin polymerization and augments glucose-stimulated insulin secretion in obesity. Collectively, these findings place PTPRF as a nutrient-responsive regulator of cytoskeletal remodeling that coordinates hepatic metabolism and {beta}-cell function, highlighting its potential as a therapeutic target for improving systemic glucose control.

physiology↗

bioRxiv2026

Disease-linked mutations in hnRNPA2B1 accelerate condensate maturation and promote amyloid aggregation

Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is a multifunctional RNA-binding protein that undergoes liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles across different cellular contexts. However, dysregulated phase separation can drive the transition from functional condensates to pathological protein aggregates. Two mutations in the low-complexity domain (LCD) of hnRNPA2B1 (D302V and P310L) have been linked to neurodegenerative diseases. How these mutations alter the interplay between LLPS and aggregation remains poorly understood. Here, we show that these disease-linked mutations accelerate condensate maturation and promote amyloid-like aggregation of hnRNPA2B1, with fibrils emerging from condensate-like cores. To dissect the mechanistic link between phase separation, aggregation, and disease, we focused on a conserved 25-amino-acid region within the LCD harboring both mutation sites. Deletion of this region markedly impairs LLPS and abolishes aggregation, revealing its contribution to both processes. Isolated peptides derived from this region have an intrinsic propensity for amyloid-like fibril formation, which is enhanced by the mutations. Although unable to phase separate, these peptides remodel condensate morphology and nucleate aggregation of the LCD. Together, our results identify a sequence-encoded mechanism linking disease-associated mutations to altered condensate behavior and amyloid aggregation, providing molecular insights into how aberrant phase transitions may contribute to hnRNPA2B1-associated disease.

biochemistry↗

bioRxiv2026

Challenges in reconstituting the peroxiredoxin 2:STAT3 transient redox-relay complex in vitro

Peroxiredoxin 2 (Prdx2) mediates redox signaling by transferring oxidative equivalents to target proteins such as STAT3, a redox-sensitive transcription factor implicated in inflammation and cancer. Although this interaction has been demonstrated in cells, reconstituting the Prdx2:STAT3 complex in vitro remains challenging due to its transient and redox-dependent nature. Here we test various conditions to stabilize the complex between taggless Prdx2 and the core fragment of STAT3 (CF-STAT3), including oxidants, detergents, the facilitator Annexin A2, anaerobic environments, and CovalX crosslinking. Complex formation was assessed via mass photometry, analytical size-exclusion chromatography (SEC), SEC-MALS, and electron microscopy (EM). No stable complex was observed under standard conditions. Anaerobic environments briefly stabilized the interaction, but cryo-EM could not resolve the structure. CovalX crosslinking yielded short-lived but homogeneous complexes. We found that Prdx2 is highly susceptible to hyperoxidation at its peroxidatic cysteine, particularly in the presence of DTT or excess H2O2, resulting in loss of function. Maintaining non-reducing conditions during purification preserved Prdx2 in an oxidation-competent state, promoting formation of the disulfide bond between the peroxidatic and resolving cysteines and thereby enabling reproducible detection of a weak complex with CF-STAT3. Our findings establish a framework for studying redox-relay protein complexes in vitro and highlight the importance of oxidation state management during protein handling.

molecular biology↗

bioRxiv2025

Pervanadate-induced oxidation relieves autoinhibition of SRC protein tyrosine kinase

Dynamic regulation of protein tyrosine phosphorylation (pTyr) by phosphatases (PTPs) and kinases enables cells to sense and respond to environmental changes. The widely used chemical probe Pervanadate (PV) induces accumulation of high levels of pTyr in cells, an effect primarily attributed to its properties as a PTP inhibitor. This led to the assertion that PTPs are the master gatekeeper of intracellular pTyr homeostasis. Here, we use diverse approaches to reveal that PV disrupts cellular redox homeostasis and directly activates SRC family tyrosine kinases via oxidation of specific cysteine residues. Using mass spectrometry and biophysical approaches, we show that oxidation activates SRC by disrupting autoinhibition and altering phosphopeptide binding by its SH2 domain. We further establish that redox-sensitive cysteine residues are essential for SRC to promote cellular overgrowth. Our findings call for a re-evaluation of PV-based experiments and provide compelling evidence that oxidation is a crucial mechanism in controlling the oncogenic properties of SRC.

molecular biology↗

bioRxiv2025

Heterooligomerization drives structural plasticity of eukaryotic peroxiredoxins

Peroxiredoxins are highly conserved thiol peroxidases essential for peroxide detoxification, redox signaling, and chaperone activity. Prx1/AhpC-type peroxiredoxins are found throughout the eukaryotic kingdom, where multiple isoforms frequently coexist within the same cell and even in the same subcellular compartment. Long thought to form exclusively homooligomeric structures, we reveal that heterooligomerization is a conserved and important feature of eukaryotic Prx1/AhpC-type peroxiredoxins. We demonstrate that heterooligomer formation modulates peroxoredoxin oligomeric state and enhances structural stability. In yeast, Tsa1-Tsa2 peroxiredoxin heterodecamers form in response to oxidative stress and incorporated Tsa2 stabilizes the decameric state. Beyond yeast, we show that human PRDX1 and PRDX2, as well as plant and parasitic peroxiredoxins, engage in functional heterooligomerization. These findings challenge the long-held paradigm of peroxiredoxin homooligomerization and reveal a novel mechanism for regulating redox homeostasis. Our study provides new insights into peroxiredoxin structural plasticity with broad implications for redox biology, stress responses, and cellular adaptation.

biochemistry↗

bioRxiv2024

Thermal Proteome Profiling reveals rapid proteomic responses to redox changes in specific cellular compartments

Hydrogen peroxide (H2O2) functions as a secondary messenger in cellular redox signaling, acting mainly via oxidation of protein thiols. Its spatially and temporally regulated activity within cells is essential for maintaining proper redox balance, and disruptions in these patterns can lead to oxidative stress and various related pathologies. Redox proteomics, which examines the impact of H2O2 at the proteome level, typically focuses only on thiol oxidation, overlooking broader proteomic alterations and the significance of subcellular localization in these redox processes. In this study, we address these open questions by combining chemogenetics with Thermal Proteome Profiling (TPP) to map global proteome response to compartmentalized H2O2 production. We identified hundreds of proteins with altered thermostability and/or abundance upon localized H2O2 generation in the cytosol, nucleus, and the ER lumen, highlighting their roles in cellular responses to localized H2O2. We identified proteins such as MAP2K1, PARK7, TRAP1, and UBA2 to be highly sensitive to localized H2O2 production. Furthermore, we validated their altered thermostability and found that these changes are controlled via dysregulated protein-protein interactions. This study provides a valuable resource for researchers exploring redox-mediated signal transduction and offers novel insights that could be harnessed in treating oxidative stress-induced pathologies.

systems biology↗

bioRxiv2024

The nuclear sulfenome of Arabidopsis: spotlight on histone acetyltransferase GCN5 regulation through functional thiols

Partial reduction of oxygen during energy generating metabolic processes in aerobic life forms results in the production of reactive oxygen species (ROS). In plants, ROS production is heightened during periods of both abiotic and biotic stress, which imposes a significant overload on the antioxidant systems. Hydrogen peroxide (H2O2) holds a central position in cellular redox homeostasis and signalling, playing an important role by oxidising crucial cysteines to sulfenic acid (-SOH), considered as a biologically relevant post-translational modification (PTM). Until now, the role of the nucleus in the cellular redox homeostasis has been relatively underexplored. The regulation of histone-modifying enzymes by oxidative PTMs on redox-active cysteines or tyrosine residues is particularly intriguing as it allows the integration of redox signalling mechanisms with chromatin control of transcriptional activity. One of the most extensively studied histone acetyltransferases is the conserved GENERAL CONTROL NONDEPRESSIBLE 5 (GCN5) complex. This study investigated the nuclear sulfenome in Arabidopsis thaliana by expressing a nuclear variant of the Yeast Activation Protein-1 (YAP) probe, identifying 225 potential redox-active nuclear proteins subject to sulfenylation. Mass spectrometry analysis further confirmed the sulfenylation of GCN5 at specific cysteine residues, with their functional significance and impact on the protein-protein interaction network assessed through cysteine-to-serine mutagenesis. HighlightProtein cysteine thiols are post-translationally modified under oxidative stress. Through the in vivo capturing of nuclear proteins undergoing sulfenylation in Arabidopsis, we highlight the functionality of particular cysteines in the histone acetyltransferase GCN5.

plant biology↗

bioRxiv2024

The NAPstar family of NADP redox state sensors highlights glutathione as the primary mediator of anti-oxidative electron flux

The NADPH/NADP+ redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools. Here, we introduce NAPstars, a family of genetically encoded, fluorescent protein-based NADP redox state biosensors. NAPstars offer real-time, specific, pH-resistant measurements, across a broad-range of NADP redox states, with subcellular resolution. We establish NAPstar measurements in yeast, plants and mammalian cell models, revealing a conserved robustness of cytosolic NADP redox homeostasis. NAPstars uncovered NADP redox oscillations linked to the cell cycle in yeast and illumination- and hypoxia-dependent NADP redox changes in plant leaves. By selectively impairing the glutathione and thioredoxin anti-oxidative pathways under acute oxidative challenge, NAPstars demonstrated an unexpected role for the glutathione system as the primary mediator of anti-oxidative electron flux that is conserved across eukaryotic kingdoms.

biochemistry↗

bioRxiv2023

Protein tyrosine phosphatase receptor kappa regulates glycolysis and de novo lipogenesis to promote hepatocyte metabolic reprogramming in obesity

Fat accumulation, de novo lipogenesis, and glycolysis are key drivers of hepatocyte reprogramming and the consequent metabolic dysfunction-associated steatotic liver disease (MASLD). Here we report that obesity leads to dysregulated expression of hepatic protein-tyrosine phosphatases (PTPs). PTPRK was found to be increased in steatotic hepatocytes in both humans and mice, and positively correlated with PPAR{gamma}-induced lipogenic signalling. High-fat-fed PTPRK knockout mice displayed reduced weight gain and hepatic fat accumulation. Phosphoproteomic analysis in primary hepatocytes and hepatic metabolomics identified fructose-1,6-bisphosphatase 1 and glycolysis as PTPRK targets in metabolic reprogramming. Silencing PTPRK in hepatoma cell lines resulted in reduced colony-forming ability and PTPRK knockout mice developed smaller tumours after diethylnitrosamine-induced hepatocarcinogenesis. Our study defines a novel role for PTPRK in regulating hepatic glycolysis, lipid metabolism, and tumour development. PTPRK inhibition may provide therapeutic possibilities in obesity-associated liver diseases. HighlightsO_LIHepatic receptor-type PTPs are increased in MASLD C_LIO_LIPTPRK is expressed in hepatocytes and upregulated in obesity C_LIO_LIPTPRK deficiency reduces body fat mass and liver steatosis in diet-induced obesity C_LIO_LIPTPRK regulates hepatic glycolysis and lipogenesis, promoting tumorigenesis C_LI

cancer biology↗

bioRxiv2022

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (~96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

systems biology↗

bioRxiv2021

Monitoring oxidative inflammatory processes in live cells and tissue with Hypocrates, a genetically encoded biosensor for hypochlorite

Hypochlorous acid, an aggressive oxidant, is important in immune defense against pathogens. The current lack of tools to monitor the dynamics of hypochlorous acid in live cells and tissue hinders a better understanding of inflammatory processes. We engineered a genetically encoded biosensor, Hypocrates, for the visualization of hypochlorous acid. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from E. coli. We determined sensitivity, selectivity, reaction rates, and the X-ray structure of this ratiometric redox biosensor, and tested the response of Hypocrates in HeLa Kyoto cells at varying hypochlorite concentrations. By combining Hypocrates with the biosensor HyperRed, we visualized the dynamics of hypochlorous acid and hydrogen peroxide in a zebrafish tail fin injury model.

immunology↗

bioRxiv2020

Charge Interactions in a Highly Charge-depleted Protein

Interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations, and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions. SignificanceElectrostatic forces are important for protein folding and are favored targets of protein engineering. However, despite the many advances in the field of protein electrostatics, the prediction of changes in protein structure and function upon introduction or removal of titratable residues is still complicated. In order to provide a basic understanding of protein electrostatics we here characterize a highly charge-depleted protein and its titratable variants by a combination of NMR spectroscopy and constant pH molecular dynamics simulations. Our investigations reveal how strongly interacting residues engaged in salt bridging, can be characterized. Furthermore, our study may also enrich and facilitate the understanding of dehydration of salt-bridges and its potential effect on protein stability.

biochemistry↗

bioRxiv2020

Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

plant biology↗

bioRxiv2020

Redox response of iron-sulfur glutaredoxin GRXS17 activates its holdase activity to protect plants from heat stress

Living organisms use a large panel of mechanisms to protect themselves from environmental stress. Particularly, heat stress induces misfolding and aggregation of proteins which are guarded by chaperone systems. Here, we examine the function the glutaredoxin GRXS17, a member of thiol reductases families in the model plant Arabidopsis thaliana. GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin (TRX)-domain and three CGFS-active site motif-containing GRX-domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione (GSH)-dependent manner. As a Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, we showed that GRXS17 presents both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induces loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active site cysteines in the corresponding TRX domains. This oxidation leads to a shift of GRXS17 to a high-MW complex and thus, activates its holdase activity. Moreover, we demonstrate that GRXS17 is specifically involved in plant tolerance to moderate high temperature and protects root meristematic cells from heat-induced cell death. Finally, we showed that upon heat stress, GRXS17 changes its client proteins, possibly to protect them from heat injuries. Therefore, we propose that the iron-sulfur cluster enzyme glutaredoxin GRXS17 is an essential guard to protect proteins against moderate heat stress, likely through a redox-dependent chaperone activity. All in all, we reveal the mechanism of an Fe-S cluster-dependent activity shift, turning the holoenzyme GRXS17 into a holdase that prevents damage caused by heat stress.

plant biology↗