Biology subjects

Herrero-Galan, E.

Publications and source records attributed to Herrero-Galan, E..

9 recordsLinked to original sources

bioRxiv2026

Pathological stiffening by crosslinking glycation of titin

Heterogeneous, non-enzymatic glycation chemistry triggered by sugar-derived metabolites is typical of diseases that also entail pathological stiffening of cells, such as diabetes and age-related disorders. However, the mechanisms responsible for cell stiffening and the role of glycated biomolecules remain largely unexplored. Here, we show that glycation of cardiac titin, a giant intracellular protein scaffolding contractile sarcomeres, is increased in diabetes and leads to rigidification of both the protein and cardiomyocytes. Mechanistically, glycation-induced titin stiffening results from decreased contour length and enhanced folding of otherwise structurally intact protein domains following extensive formation of intramolecular crosslinking advanced glycation end products (AGEs). These stiffening effects outweigh softening contributions by competing, non-crosslinking AGEs. In combination, our work overcomes the intrinsic chemical complexity typical of glycation to uncover crosslinking AGEs as a source of pathological stiffening of cells, which we propose contributes to tissue dysfunction in situations of glycative stress.

biophysics↗

bioRxiv2025

Titin cleavage in living cardiomyocytes induces sarcomere disassembly but does not trigger cell proliferation

AimsAdult mammalian hearts have limited regenerative capacity due to the inability of cardiomyocytes to proliferate, a major clinical hurdle in contemporary cardiology. The presence of highly organized, contractile sarcomeres has long been considered an impediment for cardiomyocyte division. Indeed, sarcomere disassembly is a crucial step to complete the cell cycle in the few situations where cardiomyocytes have been observed to proliferate. However, whether sarcomere disassembly can per se trigger cell cycle re-entry remains unknown, a possibility that we have tested here. Methods and resultsWe have engineered a system to induce sarcomere disassembly in living murine cardiomyocytes based on the specific cleavage of the structural protein titin by tobacco etch virus protease (TEVp). Although isolated neonatal cardiomyocytes with disassembled sarcomeres remain viable and retain low-amplitude contractile activity, our results show no evidence of increased cardiomyocyte proliferation in targeted cells, as indicated by analyses of markers of DNA synthesis and cytokinesis. We obtain equivalent results when titin is cleaved in the adult myocardium in vivo. ConclusionThe removal of sarcomere structural barriers is necessary, but not sufficient, for cardiomyocyte proliferation, which implies that additional factors are required for cardiomyocytes to undergo cell division. Translational perspectiveThere is a clinical need to identify therapeutic strategies that promote cardiac regeneration through the proliferation of cardiomyocytes that survive an injury to the heart, for instance after myocardial infarction. Based on the observation that cardiomyocytes require sarcomere disassembly for proliferation, we have examined if the sole disassembly of sarcomeres is enough to promote cell division in cardiomyocytes. Our work demonstrates a strategy to induce specific sarcomere disassembly, which, however does not result in increased proliferative capacity of cardiomyocytes. These results imply that additional factors need to be considered to promote cardiomyocyte proliferation by facilitating sarcomere disassembly.

cell biology↗

bioRxiv2025

Titin cleavage is a driver of cardiomyocyte disengagement and reactive myocardial fibrosis

Myocardial remodeling including cardiomyocyte-death-independent, reactive fibrosis and disconnection of cardiomyocytes is at the basis of prevalent cardiac conditions converging into arrhythmias and heart failure. However, the molecular mechanisms behind these pathogenic responses remain incompletely understood limiting therapeutic opportunities. Here, we find that a molecular event common to unrelated heart diseases, namely the cleavage of the sarcomeric protein titin, is enough to trigger fast myocardial remodeling. Using an engineered system based on the expression of tobacco etch virus protease (TEVp) in mice, we show that 30% mosaic cardiac titin cleavage leads to global cardiomyocyte disengagement, activation of cardiac fibroblasts and interstitial collagen deposition. These effects are concurrent, involve ERK1/2 signaling, and are expected to contribute, at least, to myocardial remodeling in chemotherapy-induced cardiotoxicity and ischemia damage.

physiology↗

bioRxiv2023

Amino acid substitutions hydrophilizing the core of titin domains cause dilated cardiomyopathy

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the increase in hydrophilicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophilizing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

biochemistry↗

bioRxiv2022

Cell response to extracellular matrix energy dissipation outweighs rigidity sensing

The mechanical properties of the extracellular matrix (ECM) determine cell differentiation, proliferation and migration through mechanoresponsive proteins including YAP. However, how different mechanical signals cooperate, synergize or compete to steer cell behavior remains poorly understood. Here, we have examined competition between the two major ECM mechanical cues, i.e. rigidity, which activates cell mechanosensing, and viscous energy dissipation, which reduces stiffness blunting cell mechanotransduction. To trigger competition, we have engineered protein hydrogels allowing concomitant modulation of stiffness and viscosity by mechanisms characteristic of native ECM. Culturing cells on these hydrogels, we have found that substrate energy dissipation attenuates YAP mechanosensing prevailing over stiffness cues. Hampered YAP activation on more dissipative substrates correlates with faster actin flow and smaller focal adhesions. Mechanistically, inhibition of actomyosin contractility reverses the outcome of the competition between rigidity and energy dissipation. Our results highlight the dominating contribution of substrate viscosity to the biology of the cell.

bioengineering↗

bioRxiv2021

Nuclear Translocation of Vitellogenin in the Honey Bee (Apis mellifera)

Vitellogenin (Vg) is a conserved protein used by nearly all oviparous animals to produce eggs. It is also pleiotropic and performs functions in oxidative stress resistance, immunity, and, in honey bees, behavioral development of the worker caste. It has remained enigmatic how Vg affects multiple traits. Here, we asked whether Vg enters the nucleus and acts via DNA-binding. We used immunohistology, cell fractionation and cell culturation to show that a structural subunit of honey bee Vg translocates into cell nuclei. We then demonstrated Vg-DNA binding theoretically and empirically with prediction software and chromatin immunoprecipitation with sequencing (ChIP-seq), finding binding sites at genes influencing immunity and behavior. Finally, we investigated the immunological and enzymatic conditions affecting Vg cleavage and nuclear translocation, and constructed a 3D structural model. Our data are the first to show Vg in the nucleus and suggests a new fundamental regulatory role for this ubiquitous protein.

molecular biology↗

bioRxiv2020

Nanomechanical phenotypes in cMyBP-C mutants that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C mutant domains. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN, and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, non-pathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

biophysics↗

bioRxiv2020

Conserved cysteines in titin sustain the mechanical function of cardiomyocytes

The protein titin determines cardiomyocyte contraction and truncating variants in the titin gene (TTN) are the most common cause of dilated cardiomyopathy (DCM). Different to truncations, missense variants in TTN are currently classified as variants of uncertain significance due to their high frequency in the population and the absence of functional annotation. Here, we report the regulatory role of conserved, mechanically active titin cysteines, which, contrary to current views, we uncover to be reversibly oxidized in basal conditions leading to isoform- and force-dependent modulation of titin stiffness and dynamics. Building on our functional studies, we demonstrate that missense mutations targeting a conserved titin cysteine alter myocyte contractile function and cause DCM in humans. Our findings have a direct impact on genetic counselling in clinical practice. One sentence summaryMutations targeting cysteines key to the mechanoredox control of titin cause human dilated cardiomyopathy

physiology↗

bioRxiv2019

A HaloTag-TEV genetic cassette for mechanical phenotyping of native proteins

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on native molecules in their natural environment remains inaccessible to conventional genetics, biophysics and molecular biology tools. To address these limitations, here we demonstrate a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we have specifically severed the titin filament by digestion with TEV protease, and found that the response of muscle fibers to length changes requires mechanical transduction through titins intact polypeptide chain. HaloTag-based covalent tethering has enabled directed examination of the dynamics of titin under 1-100 pN forces using recently developed magnetic tweezers. At pulling forces lower than 10 pN, titin domains are readily recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Our results support an active role of titin in muscle contraction in coordination with actomyosin motors. Insertion of the HaloTag-TEV cassette in proteins with mechanical roles opens new grounds to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.

biophysics↗