Elías Herrero‐Galán

Spanish National Centre for Cardiovascular Research

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Jorge Alegre‐Cebollada

Spanish National Centre for Cardiovascular Research

Diana Velázquez-Carreras

Spanish National Centre for Cardiovascular Research

María Rosaria Pricolo

Spanish National Centre for Cardiovascular Research

ÁM

Álvaro Martínez‐del‐Pozo

Universidad Complutense de Madrid

Pablo García‐Pavía

Centro de Investigación en Red en Enfermedades Cardiovasculares

José G. Gavilanes

Universidad Complutense de Madrid

Inés Martínez-Martín

Spanish National Centre for Cardiovascular Research

Mercedes Oñaderra

Universidad Complutense de Madrid

Fernándo Domínguez

Hospital Universitario Puerta de Hierro Majadahonda

Giulia Frisso

Ceinge Biotecnologie Avanzate (Italy)

Cooperative Institutions

Spanish National Centre for Cardiovascular Research

Consejo Superior de Investigaciones Científicas

Instituto de Salud Carlos III

Centro de Investigación Biomédica en Red

Universidad Autónoma de Madrid

Centro de Investigación en Red en Enfermedades Cardiovasculares

Medical University of Graz

Universidad Complutense de Madrid

University of Graz

Universitat de Barcelona

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Titin Missense Variants as a Cause of Familial Dilated Cardiomyopathy

Circulation (2023)

Fernándo Domínguez Laura Lalaguna Inés Martínez-Martín Jesús Piqueras‐Flores Torsten B. Rasmussen

Dilated Cardiomyopathy

10.1161/circulationaha.122.062833

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Citations (11)

Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis

Cardiovascular Research (2024)

Miguel López-Unzu María Rosaria Pricolo Roberto Silva‐Rojas Nuno Vicente Manuel Gavilan-Herrera

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live). Background/Introduction Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension. Purpose It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin. Methods For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq. Results Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway. Conclusion Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.

Homeostasis

Myocardial fibrosis

Obscurin

10.1093/cvr/cvae088.138

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The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin

Proteins Structure Function and Bioinformatics (2008)

Elías Herrero‐Galán Javier Lacadena Álvaro Martínez‐del‐Pozo Drion G. Boucias Nieves Olmo

Abstract The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli . Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the α‐fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases. Proteins 2008. © 2008 Wiley‐Liss, Inc.

10.1002/prot.21910

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Citations (46)

A non-cytotoxic but ribonucleolytically specific ribotoxin variant: implication of tryptophan residues in the cytotoxicity of hirsutellin A

Biological Chemistry (2012)

Elías Herrero‐Galán Lucía García‐Ortega Javier Lacadena Álvaro Martínez‐del‐Pozo Nieves Olmo

Abstract Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.

Internalization

Ribosome-inactivating protein

Ricin

10.1515/hsz-2011-0278

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Citations (10)

Abstract Or111: Titin cleavage disrupts sarcomere-adhesion tensional homeostasis triggering cardiomyocyte arrhythmia in vitro

Circulation Research (2024)

María Rosaria Pricolo Miguel López-Unzu Natália Vicente Cristina Morales-Lopez W Franco

The cardiac tissue experiences several forms of mechanical stress, including contraction of cardiomyocytes and forces derived from the stiffness of extracellular matrix (ECM). Preserving homeostatic mechanical forces is crucial for a healthy heart, yet the mechanisms involved are not well-explored. Our study delves into cardiomyocyte responses following the sudden release of mechanical tension across titin, a giant sarcomere protein crucial for setting cardiomyocyte stiffness. Through the expression of virally transduced TEV protease (TEVp) in neonatal mouse cardiomyocytes (NMCMs), we induced the cleavage of mutant titin including the specific TEV protease recognition site (TEVs) within the mechanically active I-band region of the protein. Titin cleavage was validated by 1.8% polyacrylamide SDS-PAGE electrophoresis, revealing a primary band at ~2.2 MDa in TEV-positive NMCMs, corresponding to digested titin fragment A-M. This cleavage, confirmed at 2 days post-infection (dpi) with ~80% efficiency, reached 100% by 5 dpi. Consequently, the Young’s modulus of affected cardiomyocytes dropped by 50%, as determined by Atomic Force Spectroscopy. Sarcomere disarray increased over time, as evidenced by immunofluorescence, yet this effect did not induce cell death or proliferation. Cell viability was confirmed by the fact that affected cardiomyocytes are able to contract spontaneously, although titin cleavage resulted in reduced beating amplitude and induced arrhythmic behavior. Considering this arrhythmogenic situation, we tested cell-cell adhesion through dissociation assays, which revealed significantly reduced intercellular adhesion in affected cardiomyocytes. RNAseq confirmed involvement of pathways related to heart contraction, anchoring junctions, and cell junctions. Specifically, dysregulated genes associated with gap-junction and costamere complexes were observed at 5 dpi. Alterations of adhesion were further confirmed by reductions in connexin 43 and focal adhesion length in immunofluorescence experiments, which also showed mislocalization of important adhesive proteins. In conclusion, titin cleavage in neonatal cardiomyocytes leads to sarcomere disarray, perturbed cell-cell adhesion and arrhythmia. These findings highlight the importance of full mechanical integrity of the titin filament in living cardiomyocytes.

Homeostasis

Myofilament

Cleavage (geology)

10.1161/res.135.suppl_1.or111

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The Therapeutic Potential of Fungal Ribotoxins

Current Pharmaceutical Biotechnology (2008)

Nelson Carreras-Sangrà Elisa Álvarez-García Elías Herrero‐Galán Jaime Tomé‐Amat Javier Lacadena

Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin – ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of high-resolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the mid-term future. Keywords: Asp f 1, fungal allergy, immunotoxin, restrictocin, Rnase, α-sarcin

Phosphodiester bond

Ricin

Mechanism of Action

10.2174/138920108784567335

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Citations (26)

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Redox Biology (2022)

Elías Herrero‐Galán Inés Martínez-Martín Cristina Sánchez-González Natália Vicente Elena Bonzón‐Kulichenko

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.

Immunoglobulin domain

10.1016/j.redox.2022.102306

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Citations (8)

Fungal extracellular ribotoxins as insecticidal agents

Insect Biochemistry and Molecular Biology (2012)

Miriam Olombrada Elías Herrero‐Galán Daniel Tello Mercedes Oñaderra José G. Gavilanes

Fungal pathogen

10.1016/j.ibmb.2012.10.008

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Citations (20)

Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy

Cell Reports (2023)

Inés Martínez-Martín Audrey Crousilles Juan Pablo Ochoa Diana Velázquez-Carreras Simon A. Mortensen

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 reduced hydrophobicity 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 hydrophobicity-reducing 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.

Dilated Cardiomyopathy

Obscurin

10.1016/j.celrep.2023.113490

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Citations (1)

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

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Inés Martínez-Martín Audrey Crousilles Juan Pablo Ochoa Diana Velázquez-Carreras Simon A. Mortensen

ABSTRACT 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.

Dilated Cardiomyopathy

Obscurin

10.1101/2023.06.02.543467

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