USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.

The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.We thank A.P. Ugalde, D. Rodríguez, J.G. Pérez-Silva, Y. Español and F. Rodríguez for helpful comments and advice. We also thank S.A. Miranda, D. Álvarez-Puente and C. Garabaya for excellent technical assistance, as well as the staff of the scientific core facilities from the University of Oviedo (Unidad de Ensayos Biotecnológicos y Biomédicos, Servicios Científico-Técnicos). This work was supported by the Ministerio de Ciencia e Innovación (SAF2017-87655-R, PDI2020-118394RB-100 and PGC2018- 097019-B-I00), “la Caixa” Banking Foundation (project code HR17-00247), and the European Research Council (742067, DeAge). The IUOPA is funded by the Asturian Government and Fundación Cajastur-Liberbank.S

NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome.

The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-β2 (TGFβ2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfβ2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.We thank C. Martí Gómez-Aldaraví for help with graphic representation and critical reading of the manuscript, and S. Bartlett for English editing. RTC is supported by a Foundation La Caixa PhD fellowship (Ref LCF/BQ/ES15/10360023). LLZ is supported by a Ramón y Cajal postdoctoral contract (Ref: RYC-2016-20917). JLdlP is funded by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Ministerio de Ciencia, Innovación y Universidades, and grants from the Fundación BBVA (Ref.: BIO14_298) and Fundación La Marató TV3 (Ref.: 20153431). JV is supported by grants BIO2015-67580-P and CB16/11/00277 (CIBER CV) from the Ministerio de Ciencia, Innovación y Universidades, and Carlos III Institute of Health-Fondo de Investigación Sanitaria (Grant ProteoRed-PRB3-IPT17/0019-ISCIII-SGEFI/ERDF), the Fundación La Marató TV3 (Ref. 122/C/2015) and “La Caixa” Banking Foundation (project code HR17-00247). The cost of this publication was supported in part with funds from the ERDF. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Attitudes of Germans towards distributive issues in the German health system

Social health care systems are inevitably confronted with the scarcity of resources and the resulting distributional challenges. Since prioritization implies distributional effects, decisions on respective rules should take citizens’ preferences into account. Thus, knowledge about citizens’ attitudes and preferences regarding different distributional issues implied by the type of financing health care is necessary to judge the public acceptance of a health system. In this study we concentrate on two distributive issues in the German health system: First, we analyse the acceptance of prioritizing decisions concerning the treatment of certain patient groups, in this case patients who all need a heart operation. Here we focus on the fact that a patient is strong smoker or a non-smoker, the criteria of age or the fact that a patient has or does not have young children. Second, we investigate Germans’ opinions towards income dependent health services. The results reveal strong effects of individuals’ attitudes regarding general aspects of the health system on priorities, e.g. that individuals behaving health demanding should not be preferred. In addition, experiences of limited access to health services are found to have a strong influence on citizens’ attitudes, too. Finally, decisions about different prioritization criteria are found to be not independent.

Comprehensive Quantification of the Modified Proteome Reveals Oxidative Heart Damage in Mitochondrial Heteroplasmy

Post-translational modifications hugely increase the functional diversity of proteomes. Recent algorithms based on ultratolerant database searching are forging a path to unbiased analysis of peptide modifications by shotgun mass spectrometry. However, these approaches identify only one-half of the modified forms potentially detectable and do not map the modified residue. Moreover, tools for the quantitative analysis of peptide modifications are currently lacking. Here, we present a suite of algorithms that allows comprehensive identification of detectable modifications, pinpoints the modified residues, and enables their quantitative analysis through an integrated statistical model. These developments were used to characterize the impact of mitochondrial heteroplasmy on the proteome and on the modified peptidome in several tissues from 12-week-old mice. Our results reveal that heteroplasmy mainly affects cardiac tissue, inducing oxidative damage to proteins of the oxidative phosphorylation system, and provide a molecular mechanism explaining the structural and functional alterations produced in heart mitochondria.We thank Simon Bartlett (CNIC) for English editing. This study was supported by competitive grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (BIO2015-67580-P) through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria (PRB2, IPT13/0001-ISCIII-SGEFI/FEDER; ProteoRed), by Fundacion La Marato TV3, and by FP7-PEOPLE-2013-ITN ``Next-Generation Training in Cardiovascular Research and Innovation-Cardionext.'' N.B. is a FP7-PEOPLE-2013-ITN-Cardionext Fellow. The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO Award SEV-2015-0505).S

Activation of Serine One-Carbon Metabolism by Calcineurin A beta 1 Reduces Myocardial Hypertrophy and Improves Ventricular Function

Background In response to pressure overload, the heart develops ventricular hypertrophy that progressively decompensates and leads to heart failure. This pathological hypertrophy is mediated, among others, by the phosphatase calcineurin and is characterized by metabolic changes that impair energy production by mitochondria. Objectives The authors aimed to determine the role of the calcineurin splicing variant CnAβ1 in the context of cardiac hypertrophy and its mechanism of action. Methods Transgenic mice overexpressing CnAβ1 specifically in cardiomyocytes and mice lacking the unique C-terminal domain in CnAβ1 (CnAβ1Δi12 mice) were used. Pressure overload hypertrophy was induced by transaortic constriction. Cardiac function was measured by echocardiography. Mice were characterized using various molecular analyses. Results In contrast to other calcineurin isoforms, the authors show here that cardiac-specific overexpression of CnAβ1 in transgenic mice reduces cardiac hypertrophy and improves cardiac function. This effect is mediated by activation of serine and one-carbon metabolism, and the production of antioxidant mediators that prevent mitochondrial protein oxidation and preserve ATP production. The induction of enzymes involved in this metabolic pathway by CnAβ1 is dependent on mTOR activity. Inhibition of serine and one-carbon metabolism blocks the beneficial effects of CnAβ1. CnAβ1Δi12 mice show increased cardiac hypertrophy and declined contractility. Conclusions The metabolic reprogramming induced by CnAβ1 redefines the role of calcineurin in the heart and shows for the first time that activation of the serine and one-carbon pathway has beneficial effects on cardiac hypertrophy and function, paving the way for new therapeutic approaches

Activation of amino acid metabolic program in cardiac HIF1-alpha-deficient mice.

HIF1-alpha expression defines metabolic compartments in the developing heart, promoting glycolytic program in the compact myocardium and mitochondrial enrichment in the trabeculae. Nonetheless, its role in cardiogenesis is debated. To assess the importance of HIF1-alpha during heart development and the influence of glycolysis in ventricular chamber formation, herein we generated conditional knockout models of Hif1a in Nkx2.5 cardiac progenitors and cardiomyocytes. Deletion of Hif1a impairs embryonic glycolysis without influencing cardiomyocyte proliferation and results in increased mitochondrial number and transient activation of amino acid catabolism together with HIF2α and ATF4 upregulation by E12.5. Hif1a mutants display normal fatty acid oxidation program and do not show cardiac dysfunction in the adulthood. Our results demonstrate that cardiac HIF1 signaling and glycolysis are dispensable for mouse heart development and reveal the metabolic flexibility of the embryonic myocardium to consume amino acids, raising the potential use of alternative metabolic substrates as therapeutic interventions during ischemic events.This project has been supported by Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain and by grants to S.M.-P. from the European Research Council, European Union, FP7-PEOPLE-2010-RG_276891; Fundación TV3 La Marató, Spain, 201507.30.31; Comunidad de Madrid (CAM); Spain and European Union (EU), B2017/BMD-3875; Instituto de Salud Carlos III, Spain, PI17/01817; Universidad Francisco de Vitoria (UFV), Spain and LeDucq Foundation, France, 17CVD04. I.M.-M. was supported by La Caixa-CNIC and Fundacion Alfonso Martín Escudero fellowships, Spain. T.A.-G. was supported by a predoctoral award granted by CAM/EU and UFV, Spain, PEJD-2018-PRE/SAL-9529 and SM-P by a Contrato de Investigadores Miguel Servet (CPII16/00050) and UFV, Spain.S

Activation of Serine One-Carbon Metabolism by Calcineurin A beta 1 Reduces Myocardial Hypertrophy and Improves Ventricular Function

BACKGROUND In response to pressure overload, the heart develops ventricular hypertrophy that progressively decompensates and leads to heart failure. This pathological hypertrophy is mediated, among others, by the phosphatase calcineurin and is characterized by metabolic changes that impair energy production by mitochondria. OBJECTIVES The authors aimed to determine the role of the calcineurin splicing variant CnA beta 1 in the context of cardiac hypertrophy and its mechanism of action. METHODS Transgenic mice overexpressing CnAb1 specifically in cardiomyocytes and mice lacking the unique C-terminal domain in CnA beta 1 (CnA beta 1(Delta i12) mice) were used. Pressure overload hypertrophy was induced by transaortic constriction. Cardiac function was measured by echocardiography. Mice were characterized using various molecular analyses. RESULTS In contrast to other calcineurin isoforms, the authors show here that cardiac-specific overexpression of CnA beta 1 in transgenic mice reduces cardiac hypertrophy and improves cardiac function. This effect is mediated by activation of serine and one-carbon metabolism, and the production of antioxidant mediators that prevent mitochondrial protein oxidation and preserve ATP production. The induction of enzymes involved in this metabolic pathway by CnAb1 is dependent on mTOR activity. Inhibition of serine and one-carbon metabolism blocks the beneficial effects of CnA beta 1. CnA beta 1(Delta i12) mice show increased cardiac hypertrophy and declined contractility. CONCLUSIONS The metabolic reprogramming induced by CnAb1 redefines the role of calcineurin in the heart and shows for the first time that activation of the serine and one-carbon pathway has beneficial effects on cardiac hypertrophy and function, paving the way for new therapeutic approaches. (J Am Coll Cardiol 2018; 71: 654-67) (C) 2018 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons. org/licenses/by-nc-nd/4.0/).This work was supported by grants from the European Union (CardioNeT-ITN-289600 and CardioNext-608027 to Dr. Lara-Pezzi; Meet-ITN-317433 to Dr. Enriquez; UE0/MCA1108 to Dr. Acin-Perez), from the Spanish Ministry of Economy and Competitiveness (SAF2015-65722-R and SAF2012-31451 to Dr. Lara-Pezzi; SAF2015-71521-REDC, BFU2013-50448, and SAF2012-32776 to Dr. Enriquez; RyC-2011-07826 to Dr. Acin-Perez; BIO2012-37926 and BIO2015-67580-P to Dr. Vazquez), from the Spanish Carlos III Institute of Health (CPII14/00027 to Dr. Lara-Pezzi; RD12/0042/066 to Drs. Garcia-Pavia and Lara-Pezzi), from the Regional Government of Madrid (2010-BMD-2321 ``Fibroteam´´ to Dr. Lara-Pezzi; 2011-BMD-2402 ``Mitolab´´ to Dr. Enriquez) and the FIS-ISCIII (PRB2-IPT13/0001 and RD12/0042/0056-RIC-RETICS to Dr. Vazquez). This work was also supported by the Plan Estatal de IthornDthornI 2013-2016-European Regional Development Fund (FEDER) ``A way of making Europe,´´ Spain. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and by the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Drs. Vazquez and Garcia-Pavia have served as consultants for VL39. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Padron-Barthe, Villalba-Orero, and Gomez-Salinero contributed equally to this work and are joint first authors. Robyn Shaw, MD, PhD, served as Guest Editor for this paper.S

Programmed 'disarming' of the neutrophil proteome reduces the magnitude of inflammation

The antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (NETs). However, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. Here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the NET-forming capacity. This program was driven by the receptor CXCR2 and by regulators of circadian cycles. As a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. Changes in the proteome, granule content and NET formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. Our findings unveil a 'disarming' strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation.We thank members of the Comparative Medicine Unit and Advanced Microscopy Unit at CNIC. This study was supported by Intramural grants from the Severo Ochoa program (IGP-SO), a grant from Fundació La Marató de TV3 (120/C/2015-20153032), grant SAF2015-65607-R from Ministerio de Ciencia, Investigacion y Universidades (MCIU) with cofunding from Fondo Europeo de Desarrollo Regional, grant RTI2018-095497-B-I00 from MCIU and HR17_00527 from Fundación La Caixa (to A.H.), and fellowship BES-2013-065550 from MCIU (to J.M.A.), fellowship from La Caixa Foundation (ID 100010434, code LCF/BQ/DR19/11740022, to A.A.-C.) and fellowship Health-PERIS 2016–2020 (to C.C.) Funds were also obtained from Instituto de Salud Carlos III (FIS PI17/01601, to I.L.) and SAF2015-68632-R from MCIU (to M.A.M.); Wellcome Trust Seed Award in Science (206103/Z/17/Z, to D.R.), SFB1123-A1/A10 from Deutsche Forschungsgemeinschaft and ERC-AdG 692511 (to C.W.); SAF2017-84494-C2-R and Programa Red Guipuzcoana de Ciencia, Tecnología e Información 2018-CIEN-000058-01 (to J.R.-C.). Work at CIC biomaGUNE was performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (MDM-2017-0720). C.W. is a van de Laar professor of atherosclerosis. The CNIC is supported by the MCIU and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).S

ALDH4A1 is an atherosclerosis auto-antigen targeted by protective antibodies

Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies, and there is a connection between atherosclerosis and autoimmunity. However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood. Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD.Ministerio de Economía y Competitividad (SVP-2014-068289); P.D. was supported by an AECC grant (AIO 2012, Ayudas a Investigadores en Oncología 2012); A.S.-B. is a Juan de la Cierva researcher (IJC2018-035279-I); I.M.-F. was a fellow of the research training program funded by Ministerio de Economía y Competitividad (SVP-2014-068216); and A.R.R. and J.V. are supported by Centro Nacional de Investigaciones Cardiovasculares (CNIC). The project leading to these results has received funding from la Caixa Banking Foundation under the project code HR17-00247 and from SAF2016-75511-R and PID2019-106773RB-I00 grants to A.R.R. (Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–201

The chaperonin CCT controls T cell receptor-driven 3D configuration of centrioles

T lymphocyte activation requires the formation of immune synapses (IS) with antigen-presenting cells. The dynamics of membrane receptors, signaling scaffolds, microfilaments, and microtubules at the IS determine the potency of T cell activation and subsequent immune response. Here, we show that the cytosolic chaperonin CCT (chaperonin-containing TCP1) controls the changes in reciprocal orientation of the centrioles and polarization of the tubulin dynamics induced by T cell receptor in T lymphocytes forming an IS. CCT also controls the mitochondrial ultrastructure and the metabolic status of T cells, regulating the de novo synthesis of tubulin as well as posttranslational modifications (poly-glutamylation, acetylation, ?1 and ?2) of ??-tubulin heterodimers, fine-tuning tubulin dynamics. These changes ultimately determine the function and organization of the centrioles, as shown by three-dimensional reconstruction of resting and stimulated primary T cells using cryo-soft x-ray tomography. Through this mechanism, CCT governs T cell activation and polarity.Cryo-SXT work was supported by ALBA Synchrotron standard proposals 2015021148 and 2016021638 to F.J.C., N.B.M.-C., and J.M.V. This study was supported by grants SAF2017-82886-R (to F.S.-M.), PID2019-105872GB-I00/AEI/10.13039/501100011033 (AEI/FEDER, UE), BFU2016-75984 (to J.M.V.), and BIO2015-67580-P and PGC2018-097019-B-I00 (to J.V.) from the Spanish Ministry of Economy and Competitiveness (MINECO), grants INFLAMUNE-S2017/BMD-23671 (to F.S.-M.) and P2018/NMT-4389 (to J.M.V.) from the Comunidad de Madrid, ERC-2011-AdG 294340-GENTRIS (to F.S.-M.), a 2019 grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud” and a 2018 grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (to F.S.-M.), and grants PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF), the Fundació Marató TV3 (grant 122/C/2015), and “La Caixa” Banking Foundation (HR17-00016 to FSM and HR17-00247 to J.V.). D.T. is supported by a PhD fellowship from La Caixa Foundation. Work in the Vernos lab was supported by the grant CSD2006-00023 from the Spanish Ministry of Science and Innovation and grants BFU2012-37163 and BFU2015-68726-P from the Spanish Ministry of Economy and Competitiveness. The CRG acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015- 0505). The Centro Nacional de Biotecnología (CNB) is a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712). Funding agencies have not intervened in the design of the studies, with no copyright over the stud