Person: Herrero-Galan, Elas
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Elas
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Herrero-Galan
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CNIC
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Publication Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis(Oxford University Press, 2024-05-29) Lopez-Unzu M; Pricolo MR; Silva-Rojas R; Vicente N; Gavilan-Herrera M; Velazquez-Carreras D; Morales-Lopez C; Herrero-Galan, Elas; Alegre-Cebollada, Jorge; Unión Europea. Comisión Europea. European Research Council (ERC)Publication A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.(Springer, 2020-04-28) Rivas-Pardo, Jaime Andrés; Li, Yong; Mártonfalvi, Zsolt; Tapia-Rojo, Rafael; Unger, Andreas; Fernández-Trasancos, Ángel; Herrero-Galan, Elas; Velazquez-Carreras, Diana; Fernández, Julio M; Linke, Wolfgang A; Alegre-Cebollada, Jorge; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España); Instituto de Salud Carlos III; Fundación ProCNIC; Deutsche Forschungsgemeinschaft (Alemania); Hungarian Academy of Sciences; European Molecular Biology Organization; Boehringer Ingelheim FondsSingle-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 proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness. Using our system, we specifically sever titin by digestion with TEV protease, and find that the response of muscle fibers to length changes requires mechanical transduction through titin's intact polypeptide chain. In addition, HaloTag-based covalent tethering enables examination of titin dynamics under force using magnetic tweezers. At pulling forces < 10 pN, titin domains are recruited to the unfolded state, and produce 41.5 zJ mechanical work during refolding. Insertion of the HaloTag-TEV cassette in mechanical proteins opens opportunities to explore the molecular basis of cellular force generation, mechanosensing and mechanotransduction.Publication Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics(Elsevier, 2022-03-28) Herrero-Galan, Elas; Martinez-Martin, Ines; Sanchez-Gonzalez, Cristina; Vicente, Natalia; Bonzon-Kulichenko, Elena; Calvo, Enrique; Suay-Corredera, Carmen; Pricolo, Maria Rosaria; Fernández-Trasancos, Ángel; Velázquez-Carreras, Diana; Badia-Careaga, Claudio; Abdellatif, Mahmoud; Sedej, Simon; Rainer, Peter P.; Giganti, David; Pérez-Jiménez, Raúl; Vazquez, Jesus; Alegre-Cebollada, Jorge; Ministerio de Ciencia e Innovación (España); Comunidad de Madrid (España); Instituto de Salud Carlos III; Fundación La Caixa; FWF Austrian Science Fund; Fundación ProCNIC; Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España)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.Publication Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins(Nature Publishing Group, 2017-08) Manteca, Aitor; Schönfelder, Jörg; Alonso-Caballero, Alvaro; Fertin, Marie J; Barruetabeña, Nerea; Faria, Bruna F; Herrero-Galan, Elas; Alegre-Cebollada, Jorge; De Sancho, David; Perez-Jimenez, Raul; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea; Eusko Jaurlaritza; Fundación ProCNICThe sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this diversity and its evolution has not been proposed. We use phylogenetic analyses and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We resurrect eight-domain fragments of titin corresponding to the common ancestors to mammals, sauropsids, and tetrapods, which lived 105-356 Myr ago, and compare them with titin fragments from some of their modern descendants. We demonstrate that the resurrected titin molecules are rich in disulfide bonds and display high mechanical stability. These mechanochemical elements have changed over time, creating a paleomechanical trend that seems to correlate with animal body size, allowing us to estimate the sizes of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.Publication Protein Thermodynamic Destabilization in the Assessment of Pathogenicity of a Variant of Uncertain Significance in Cardiac Myosin Binding Protein C.(Springer, 2020-10) Pricolo, Maria Rosaria; Herrero-Galan, Elas; Mazzaccara, Cristina; Losi, Maria Angela; Alegre-Cebollada, Jorge; Frisso, Giulia; Ministerio de Ciencia, Innovación y Universidades (España); Comunidad de Madrid (España); Centro Nacional de Investigaciones Cardiovasculares Carlos III (España); Instituto de Salud Carlos III; Ministero dell Istruzione (Italia); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Fundación ProCNICIn the era of next generation sequencing (NGS), genetic testing for inherited disorders identifies an ever-increasing number of variants whose pathogenicity remains unclear. These variants of uncertain significance (VUS) limit the reach of genetic testing in clinical practice. The VUS for hypertrophic cardiomyopathy (HCM), the most common familial heart disease, constitute over 60% of entries for missense variants shown in ClinVar database. We have studied a novel VUS (c.1809T>G-p.I603M) in the most frequently mutated gene in HCM, MYBPC3, which codes for cardiac myosin-binding protein C (cMyBPC). Our determinations of pathogenicity integrate bioinformatics evaluation and functional studies of RNA splicing and protein thermodynamic stability. In silico prediction and mRNA analysis indicated no alteration of RNA splicing induced by the variant. At the protein level, the p.I603M mutation maps to the C4 domain of cMyBPC. Although the mutation does not perturb much the overall structure of the C4 domain, the stability of C4 I603M is severely compromised as detected by circular dichroism and differential scanning calorimetry experiments. Taking into account the highly destabilizing effect of the mutation in the structure of C4, we propose reclassification of variant p.I603M as likely pathogenic. Looking into the future, the workflow described here can be used to refine the assignment of pathogenicity of variants of uncertain significance in MYBPC3.Publication A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.(Cell Press, 2020-10-01) Nicolas-Avila, Jose A.; Lechuga-Vieco, Ana V.; Esteban-Martinez, Lorena; Sanchez-Diaz, Maria; Díaz-García, Elena; Santiago, Demetrio J; Rubio-Ponce, Andrea; Li, Jackson LiangYao; Balachander, Akhila; Quintana, Juan A.; Martínez-de-Mena, Raquel; Castejón-Vega, Beatriz; Pun-Garcia, Andres; Través, Paqui G; Bonzon-Kulichenko, Elena; Garcia-Marques, Fernando; Cusso, Lorena; Alonso-Gonzalez, Noelia; González-Guerra, Andrés; Roche-Molina, Marta; Martin-Salamanca, Sandra; Crainiciuc, Georgiana; Guzman-Martinez, Gabriela; Larrazabal, Jagoba; Herrero-Galan, Elas; Alegre-Cebollada, Jorge; Lemke, Greg; Rothlin, Carla V; Jimenez-Borreguero, Luis J.; Reyes, Guillermo; Castrillo, Antonio; Desco, Manuel; Munoz-Canoves, Pura; Ibáñez, Borja; Torres, Miguel; Ng, Lai Guan; Priori, Silvia G.; Bueno, Hector; Vazquez, Jesus; Cordero, Mario D; Bernal, Juan Antonio; Enriquez, Jose Antonio; Hidalgo, Andres; Ministerio de Ciencia e Innovación (España); Unión Europea. Comisión Europea. European Research Council (ERC); Fondation Leducq; Instituto de Salud Carlos III; Fundación La Caixa; Comunidad de Madrid (España); Fundación La Marató TV3; Howard Hughes Medical Institute; Centro Nacional de Investigaciones Cardiovasculares Carlos III (España); Fundación ProCNICCardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte's autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function. VIDEO ABSTRACT.Publication Redox regulation of protein nanomechanics in health and disease: Lessons from titin(2018-12-12) Herrero-Galan, Elas; Martinez-Martin, Ines; Alegre-Cebollada, Jorge; Ministerio de Ciencia, Innovación y Universidades (España); European Research Area Network on Cardiovascular Diseases; Fundación ProCNICThe nanomechanics of sarcomeric proteins is a key contributor to the mechanical output of muscle. Among them, titin emerges as a main target for the regulation of the stiffness of striated muscle. In the last years, single-molecule experiments by Atomic Force Microscopy (AFM) have demonstrated that redox posttranslational modifications are strong modulators of the mechanical function of titin. Here, we provide an overview of the recent development of the redox mechanobiology of titin, and suggest avenues of research to better understand how the stiffness of molecules, cells and tissues are modulated by redox signaling in health and disease.