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Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice. Sí

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism. Sí

Long non-coding RNAs (lncRNAs) have emerged as key regulators in a wide range of biological processes. Here, we identified a mouse miRNA-host gene lncRNA (

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against

A large proportion of end-stage renal disease (ESRD) patients under long-term haemodialysis, have persistent anaemia and require high doses of recombinant human erythropoietin (rhEPO). However, the underlying mechanisms of renal anaemia have not been fully elucidated in these patients. In this study, we will be focusing on anaemia and plasma proteins in ESRD patients on high-flux haemodialysis (HF) and on-line haemodiafiltration (HDF), to investigate using two proteomic approaches if patients undergoing these treatments develop differences in their plasma protein composition and how this could be related to their anaemia. The demographic and biochemical data revealed that HDF patients had lower anaemia and much lower rhEPO requirements than HF patients. Regarding their plasma proteomes, HDF patients had increased levels of a protein highly similar to serotransferrin, trypsin-1 and immunoglobulin heavy constant chain alpha-1, and lower levels of alpha-1 antitrypsin, transthyretin, apolipoproteins E and C-III, and haptoglobin-related protein. Lower transthyretin levels in HDF patients were further confirmed by transthyretin-peptide quantification and western blot detection. Since ESRD patients have increased transthyretin, a protein that can aggregate and inhibit transferrin endocytosis and erythropoiesis, our finding that HDF patients have lower transthyretin and lower anaemia suggests that the decrease in transthyretin plasma levels would allow an increase in transferrin endocytosis, contributing to erythropoiesis. Thus, transthyretin could be a critical actor for anaemia in ESRD patients and a novel player for haemodialysis adequacy. This work was supported by Grants (to A.A.) from the Instituto de Salud Carlos III and cofinanced by the European Development Regional Fund (FEDER) (PI14/00705 and PI18/00255). E.M-A. is a recipient of a research. Sí

El síndrome de progeria de Hutchinson-Gilford (HGPS) es un trastorno fatal raro caracterizado por envejecimiento prematuro y muerte a una mediana de edad de 14,5 años. La causa más común de HGPS (que afecta alrededor del 90% de los pacientes) es una sustitución heterocigótica sinónima de base única de novo (c.1824C>T; p.G608G) en el gen LMNA que resulta en la acumulación de progerina, una forma aberrante de lamina A que, a diferencia de la lámina A madura, permanece permanentemente farnesilada. La proporción de progerina a lámina madura A se correlaciona con la gravedad de la enfermedad en pacientes con HGPS y se puede utilizar para evaluar la eficacia de terapias dirigidas a disminuir el empalme aberrante o la farnesilación de progerina. Recientemente mostramos que el contenido endógeno de lámina A y progerina se puede medir mediante espectrometría de masas (MS), siempre que una alternativa a los métodos inmunológicos, que carecen de la necesaria especificidad y cuantificación precisión. Aquí presentamos el primer método no inmunológico que cuantifica de forma fiable los niveles de lamina A de tipo salvaje y progerina farnesilada en células de pacientes con HGPS Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal disorder characterized by premature aging and death at a median age of 14.5 years. The most common cause of HGPS (affecting circa 90% of patients) is a de novo heterozygous synonymous single-base substitution (c.1824C>T; p.G608G) in the LMNA gene that results in the accumulation of progerin, an aberrant form of lamin A that, unlike mature lamin A, remains permanently farnesylated. The ratio of progerin to mature laminA correlates with disease severity in HGPS patients, and can be used to assess the effectiveness of therapies aimed at lessening aberrant splicing or progerin farnesylation. We recently showed that the endogenous content of lamin A and progerin can be measured by mass spectrometry (MS), providing an alternative to immunological methods, which lack the necessary specificity and quantitative accuracy. Here, we present the first non-immunological method that reliably quantifies the levels of wild-type lamin A and farnesylated progerin in cells from HGPS patients. This method, which is based on a targeted MS approach and the use of isotope-labeled internal standards, could be applied in ongoing clinical trials evaluating the efficacy of drugs that inhibit progerin farnesylation Spanish Ministry of Science, Innovation and Universities (PGC2018-097019-B-I00 and PID2021-122348NB-I00) Instituto de Salud Carlos III (PT17/0019/0003- ISCIII-SGEFI/ERDF, ProteoRed) Progeria Research Foundation, Asociación Progeria Alexandra Peraut, and “la Caixa” Banking Foundation (HR17-00247, HR22-00253) CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033) 6.208 JCR (2021) Q1, 69/297 Biochemistry & Molecular Biology 1.176 SJR (2021) Q1, 128/729 Computer Science Applications No data IDR 2021 UEM

Supplemental Digital Content is available in the text., Background: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. Methods: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type–specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. Results: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P

Supplemental Digital Content is available in the text., Rationale: RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. Objective: To investigate the role of SRSF3 in cardiac function. Methods and Results: Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. Conclusions: We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools.

Background The mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. Objectives This study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. Methods Using inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. Results Under hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. Conclusions Increased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins. Background: The mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. Objectives: This study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. Methods: Using inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. Results: Under hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. Conclusions: Increased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins.

Mutations in desmosomal Plakophilin-2 (PKP2) are the most prevalent drivers of arrhythmogenic cardiomyopathy (ACM) and a common cause of sudden cardiac death in young athletes. However, partner proteins that elucidate PKP2 cellular mechanism to understand cardiac dysfunction in ACM are mostly unknown. Here we identify the actin-based motor proteins Myh9 and Myh10 as key PKP2 interactors, and demonstrate that the expression of the ACM-related PKP2 mutant R735X alters actin fiber organization and cell mechanical stiffness. We also show that SARS-CoV-2 Nsp1 protein acts similarly to this known pathogenic R735X mutant, altering the actomyosin component distribution on cardiac cells. Our data reveal that the viral Nsp1 hijacks PKP2 into the cytoplasm and mimics the effect of delocalized R735X mutant. These results demonstrate that cytoplasmic PKP2, wildtype or mutant, induces the collapse of the actomyosin network, since shRNA-PKP2 knockdown maintains the cell structure, validating a critical role of PKP2 localization in the regulation of actomyosin architecture. The fact that Nsp1 and PKP2 mutant R735X share similar phenotypes also suggests that direct SARS-CoV-2 heart infection could induce a transient ACM-like disease in COVID-19 patients, which may contribute to right ventricle dysfunction, observed in patients with poor survival prognosis., The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). This study was supported by MCIU grant BFU2016-75144-R. The study was also partially supported by the “Ayudas a la Investigación Cátedra Real Madrid-Universidad Europea” (2017/RM01). C.M-L. and S.S. hold MCIU predoctoral contracts BES-2017- 079715, and BES-2017-079707 respectively. RG acknowledges funding from the European Research Council under grant ERC-AG-340177 (3DNanoMech) and from the MCIU under grant MAT2016-76507-R.

Changes in the oxidation state of protein Cys residues are involved in cell signalling and play a key role in a variety of pathophysiological states. We had previously developed GELSILOX, an in-gel method that enables the large-scale, parallel analysis of dynamic alterations to the redox state of Cys sites and protein abundance changes. Here we present FASILOX, a further development of the GELSILOX approach featuring: i) significantly increased peptide recovery, ii) enhanced sensitivity for the detection of Cys oxidative alterations, and iii) streamlined workflow that results in shortened assay duration. In mitochondria isolated from the adipose tissue of obese, diabetic patients, FASILOX revealed a sexually dimorphic trait of Cys oxidation involving mainly mitochondrial oxidative phosphorylation complexes. These results provide the first evidence for a decreased efficiency in the antioxidant response of men as compared to women., This study was supported by competitive grants from the Spanish Ministry of Science, Innovation and Universities (BIO2015-67580-P, PGC2018-097019-B-I00 and RTI2018-093864-B-100), the Ministry of Economy, Industry and Competitiveness (BFU2016-80006-P) through the Carlos III Institute of Health-Fondo de Investigación Sanitaria grant PRB3 (IPT17/0019 - ISCIII-SGEFI / ERDF, ProteoRed), the Fundació MaratóTV3 (grant 122/C/2015) and “la Caixa” Banking Foundation (project code HR17-00247). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

Aging is the main risk factor for cardiovascular and metabolic diseases, which have become a global concern as the world population ages. These diseases and the aging process are exacerbated in Hutchinson–Gilford progeria syndrome (HGPS or progeria). Here, we evaluated the cardiometabolic disease in animal models of premature and normal aging with the aim of identifying alterations that are shared or specific to each condition. Despite differences in body composition and metabolic markers, prematurely and normally aging mice developed heart failure and similar cardiac electrical abnormalities. High‐throughput proteomics of the hearts of progeric and normally aged mice revealed altered protein oxidation and glycation, as well as dysregulated pathways regulating energy metabolism, proteostasis, gene expression, and cardiac muscle contraction. These results were corroborated in the hearts of progeric pigs, underscoring the translational potential of our findings, which could help in the design of strategies to prevent or slow age‐related cardiometabolic disease., We have evaluated cardiac and metabolic disease in animal models of aging. Despite differences in body composition, progeric and normally aged mice developed heart failure and similar cardiac electrical alterations. High‐throughput proteomics of the hearts of these animals revealed dysregulation of energy metabolism, proteostasis, gene expression, and cardiac contraction pathways. These results were corroborated in progeric pigs, underscoring the translational potential of our findings.

16 p.-5 fig.-3 tab.-1 il. cent., BACKGROUND: The mechanisms underlying early atherosclerotic plaque formation are not completely understood. Moreover, plasma biomarkers of subclinical atherosclerosis are lacking., OBJECTIVES: The purpose of this study was to analyze the temporal and topologically resolved protein changes taking place in human aortas with early atherosclerosis to find new potential diagnostic and/or therapeutic targets., METHODS: The protein composition of healthy aortas (media layer) or with early atheroma (fatty streak and fibrolipidic, media and intima layers) was analyzed by deep quantitative multiplexed proteomics. Further analysis was performed by Western blot, immunohistochemistry, real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Plasma levels of complement C5 were analyzed in relation to the presence of generalized (>2 plaques) or incipient (0 to 2 plaques) subclinical atherosclerosis in 2 independent clinical cohorts (PESA [Progression of Early Subclinical Atherosclerosis] [n=360] and NEFRONA [National Observatory of Atherosclerosis in Nephrology] [n=394])., RESULTS: Proteins involved in lipid transport, complement system, immunoglobulin superfamily, and hemostasis are increased in early plaques. Components from the complement activation pathway were predominantly increased in the intima of fibrolipidic plaques. Among them, increased C5 protein levels were further confirmed by Western blot, enzyme-linked immunosorbent assay and immunohistochemistry, and associated with in situ complement activation. Plasma C5 was significantly increased in individuals with generalized subclinical atherosclerosis in both PESA and NEFRONA cohorts, independently of risk factors. Moreover, in the PESA study, C5 plasma levels positively correlated with global plaque volume and coronary calcification., CONCLUSIONS: Activation of the complement system is a major alteration in early atherosclerotic plaques and is reflected by increased C5 plasma levels, which have promising value as a novel circulating biomarker of subclinicalatherosclerosis., This study was funded by the Spanish MINECO(SAF2016-80843-R, BIO2015-67580-P, and PGC2018-097019-B-I00), CAM (Complemento II-CM, S2017/BMD-3673),Fondo de Investigaciones Sanitarias ISCiii-FEDER (Biobancos RD09/0076/00101) and PRB3 (IPT17/0019, ProteoRed), and FEDER “Una manera de hacer Europa.” CIBERCV is an Instituto de Salud Carlos III project. The work leading to these results has also received funding from “la Caixa” Banking Foundation under the project code HR17-00247. The PESA study is cofunded equally by the Centro Nacional de Investigaciones Cardiovasculares (CNIC), and Banco Santander, Madrid, Spain. The study also receives funding from the Instituto de Salud Carlos III (PI15/02019) and the European Regional Development Fund (ERDF) “Una manera de hacer Europa.”

Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount.

The coordinated behavior of proteins is central to systems biology. However, the underlying mechanisms are poorly known and methods to analyze coordination by conventional quantitative proteomics are still lacking. We present the Systems Biology Triangle (SBT), a new algorithm that allows the study of protein coordination by pairwise quantitative proteomics. The Systems Biology Triangle detected statistically significant coordination in diverse biological models of very different nature and subjected to different kinds of perturbations. The Systems Biology Triangle also revealed with unprecedented molecular detail an array of coordinated, early protein responses in vascular smooth muscle cells treated at different times with angiotensin-II. These responses included activation of protein synthesis, folding, turnover, and muscle contraction – consistent with a differentiated phenotype—as well as the induction of migration and the repression of cell proliferation and secretion. Remarkably, the majority of the altered functional categories were protein complexes, interaction networks, or metabolic pathways. These changes could not be detected by other algorithms widely used by the proteomics community, and the vast majority of proteins involved have not been described before to be regulated by AngII. The unique capabilities of The Systems Biology Triangle to detect functional protein alterations produced by the coordinated action of proteins in pairwise quantitative proteomics experiments make this algorithm an attractive choice for the biological interpretation of results on a routine basis.

Review article., Mitochondria are highly dynamic and regulated organelles that historically have been defined based on their crucial role in cell metabolism. However, they are implicated in a variety of other important functions, making mitochondrial dysfunction an important axis in several pathological contexts. Despite that conventional biochemical and molecular biology approaches have provided significant insight into mitochondrial functionality, innovative techniques that provide a global view of the mitochondrion are still necessary. Proteomics fulfils this need by enabling accurate, systems-wide quantitative analysis of protein abundance. More importantly, redox proteomics approaches offer unique opportunities to tackle oxidative stress, a phenomenon that is intimately linked to aging, cardiovascular disease, and cancer. In addition, cutting-edge proteomics approaches reveal how proteins exert their functions in complex interaction networks where even subtle alterations stemming from early pathological states can be monitored. Here, we describe the proteomics approaches that will help to deepen the role of mitochondria in health and disease by assessing not only changes to mitochondrial protein composition but also alterations to their redox state and how protein interaction networks regulate mitochondrial function and dynamics. This review is aimed at showing the reader how the application of proteomics approaches during the last 20 years has revealed crucial mitochondrial roles in the context of aging, neurodegenerative disorders, metabolic disease, and cancer., This work was supported by grant SAF2012-33014 from the Ministry of Economy, Industry and Competitiveness (MEIC), Spain, and partially financed with FEDER funds (to B.P.). 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).

Vitellogenins (Vtg) are glycolipophosphoproteins synthesized by oviparous vertebrates as yolk proteins precursors. These proteins have been studied for their role in reproduction and in endocrine disruption. In this work, we report the first proteomic study towards the characterization of Vtg from Pseudoplatystoma fasciatum , Piaractus brachypomus and Colossoma macropomum . Male specimens of each of the three fish species were estradiol-induced (experimental) and non-induced (control). The initial Vtg characterization was made by 2D protein gel electrophoresis of both groups The identification of the high molecular weight spots, presumed to be Vtgs, was assessed by MALDI-TOF mass spectrometry analysis. A post-translational modification study was performed by differential staining of 2D gels in order to visualize phosphoproteins and glycoproteins. Plasma samples from the three species, induced with estrogen, showed three high molecular weight spots with variable isoelectric points. Post-translational modifications showed that Vtgs from P. brachypomus and C. macropomum presented a phosphorylated and glycosylated subunit, while the same subunit in P. fasciatum was only glycosylated. This characterization will help in the development of reliable immunoassays, which could be used for studies of endocrine disruption or for the improvement of artificial spawning, by uncovering the time of fish maturation or sex determination.

Matrix metalloproteinases are involved in vascular remodeling. Little is known about their immune regulatory role in atherosclerosis. Here we show that mice deficient for MT4-MMP have increased adherence of macrophages to inflamed peritonea, and larger lipid deposits and macrophage burden in atherosclerotic plaques. We also demonstrate that MT4-MMP deficiency results in higher numbers of patrolling monocytes crawling and adhered to inflamed endothelia, and the accumulation of Mafb+ apoptosis inhibitor of macrophage (AIM)+ macrophages at incipient atherosclerotic lesions in mice. Functionally, MT4-MMP-null Mafb+AIM+ peritoneal macrophages express higher AIM and scavenger receptor CD36, are more resistant to apoptosis, and bind acLDL avidly, all of which contribute to atherosclerosis. CCR5 inhibition alleviates these effects by hindering the enhanced recruitment of MT4-MMP-null patrolling monocytes to early atherosclerotic lesions, thus blocking Mafb+AIM+ macrophage accumulation and atherosclerosis acceleration. Our results suggest that MT4-MMP targeting may constitute a novel strategy to boost patrolling monocyte activity in early inflammation., This study was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (MEIC; RD12/0042/0023 [FEDER cofunded] and SAF2014-52050R and SAF2017-83229R to A.G.A., SAF2015-64287R and SAF2015-71878-REDT to M.R., RD12/0042/0053 [FEDER cofunded] and SAF2015-64767-R to J.M-G., and SAF2016-79490-R and RD12/0042/0028 [FEDER cofunded] to V.A.) and from La Marató TV3 Foundation. C.C., M.M-A., and L.A-H. were funded by fellowships from the Spanish Ministry of Education, MEIC, and La Caixa-CNIC, respectively. C.R. was funded by a competitive postdoctoral contract grant FPDI-2013-17423 from MEIC. The CNIC is supported by the Spanish MEIC and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).

Reperfusion alters post-myocardial infarction (MI) healing; however, very few systematic studies report the early molecular changes following ischemia/reperfusion (I/R). Alterations in the remote myocardium have also been neglected, disregarding its contribution to post-MI heart failure (HF) development. This study characterizes protein dynamics and contractile abnormalities in the ischemic and remote myocardium during one week after MI. Closed-chest 40 min I/R was performed in 20 pigs sacrificed at 120 min, 24 hours, 4days, and 7days after reperfusion (n = 5 per group). Myocardial contractility was followed up by cardiac magnetic resonance (CMR) and tissue samples were analyzed by multiplexed quantitative proteomics. At early reperfusion (120 min), the ischemic area showed a coordinated upregulation of inflammatory processes, whereas interstitial proteins, angiogenesis and cardio-renal signaling processes increased at later reperfusion (day 4 and 7). Remote myocardium showed decreased contractility at 120 min- and 24 h-CMR accompanied by transient alterations in contractile and mitochondrial proteins. Subsequent recovery of regional contractility was associated with edema formation on CMR and increases in inflammation and wound healing proteins on post-MI day 7. Our results establish for the first time the altered protein signatures in the ischemic and remote myocardium early after I/R and might have implications for new therapeutic targets to improve early post-MI remodeling. 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 Investigación Sanitaria (PI13/01979 and PRB2 (IPT13/0001 - ISCIII-SGEFI/FEDER, ProteoRed)), the Fondo Europeo de Desarrollo Regional (FEDER, RD: SAF2013-49663EXP), the Fundación La Marato TV3 and in part by the FP7-PEOPLE-2013-ITN Next generation training in cardiovascular research and innovation-Cardionext. Borja Ibanez and Jesús Vázquez are supported by the Red de Investigación Cardiovascular (RIC) of the Spanish Ministry of Health (RD 12/0042/0054 and RD12/0042/0056). Rodrigo Fernández-Jiménez is a recipient of non-overlapping grants from the Ministry of Economy and Competitiveness through the Instituto de Salud Carlos III (Rio Hortega fellowship); and the Fundació Jesús Serra, the Fundación Interhospitalaria de Investigación Cardiovascular (FIC), and the CNIC (FICNIC Fellowship). Jaume Agüero, Aleksandra Binek, and Navratan Bagwan are FP7-PEOPLE-2013-ITN-Cardionext fellows. This work has been co-funded by FEDER funds. This study forms part of a Master Research Agreement (MRA) between CNIC and Philips Healthcare. 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). Sí

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6 -/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6 +/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6 -/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6 +/+ counterparts, Hdac6 -/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Moreover, Hdac6 -/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6 -/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection., This study was supported by the following grants to FSM: SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/ BMD-2332 from the Comunidad de Madrid, CIBER CARDIOVASCULAR and grant PIE13/00041 from the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III with co-funding from the Fondo Europeo de Desarrollo Regional; FEDER), and ERC-2011-AdG Desarrollo Regional; FEDER), and ERC-2011-AdG 294340- GENTRIS and COST-Action BM1202 from the European Comission. 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). OMG was supported by the fellowship FPU programme was supported by the fellowship FPU programme supported by the fellowship FPI programme (Spanish Ministry of Economy).

Cadherin-based intercellular adhesions are essential players in epithelial homeostasis, but their dynamic regulation during tissue morphogenesis and remodeling remain largely undefined. Herein, we characterize an unexpected role for the membrane-anchored metalloproteinase MT2-MMP in regulating epithelial cell quiescence. Following coimmunoprecipitation and mass spectrometry, the MT2-MMP cytosolic tail was found to interact with the zonula occludens protein-1 (ZO-1) at the apical junctions of polarized epithelial cells. Functionally, MT2-MMP localizes in the apical domain of epithelial cells where it cleaves E-cadherin and promotes epithelial cell accumulation, a phenotype observed in 2D polarized cells as well as 3D cysts. MT2-MMP-mediated cleavage subsequently disrupts apical E-cadherin-mediated cell quiescence resulting in; i) relaxed apical cortical tension favoring cell extrusion and ii) re-sorting of Src kinase activity to junctional complexes, thereby promoting proliferation. Physiologically, MT2-MMP lossof- function alters E-cadherin distribution leading to impaired 3D organoid formation by mouse colonic epithelial cells ex vivo and reduction of cell proliferation within intestinal crypts in vivo. Taken together, these studies identify an MT2-MMP/E-cadherin axis that functions as a novel regulator of epithelial cell homeostasis in vivo. © 2017. Published by The Company of Biologists Ltd. Locked until 13.10.2018 due to copyright restrictions.

Recent findings support potential roles for HDL in cardiovascular pathophysiology not related to lipid metabolism. We address whether HDL proteome is dynamically altered in atheroma plaque rupture. We used immunoaffinity purification of HDL samples from coronary artery disease patients before and after percutaneous transluminal coronary angioplasty (PTCA), a model of atheroma plaque disruption. Samples were analyzed by quantitative proteomics using stable isotope labeling and results were subjected to statistical analysis of protein variance using a novel algorithm. We observed high protein variability in HDL composition between individuals, indicating that HDL protein composition is highly patient-specific. However, intra-individual protein variances remained at low levels, confirming the reproducibility of the method used for HDL isolation and protein quantification. A systems biology analysis of HDL protein alterations induced by PTCA revealed an increase in two protein clusters that included several apolipoproteins, fibrinogen-like protein 1 and other intracellular proteins, and a decrease in antithrombin-III, annexin A1 and several immunoglobulins. Our results support the concept of HDL as dynamic platforms that donate and receive a variety of molecules and provide an improved methodology to use HDL proteome for the systematic analysis of differences among individuals and the search for cardiovascular biomarkers. Biological significance The HDL proteome is an interesting model of clinical relevance and has been previously described to be dynamically altered in response to pathophysiological conditions and cardiovascular diseases. Our study suggests that interindividual variability of HDL proteome is higher than previously thought and provided the detection of a set of proteins that changed their abundance in response to plaque rupture, supporting the concept of HDL as dynamic platforms that donate and receive a variety of molecules.

Multiple epidemiological and prospective studies have established that high-density lipoprotein cholesterol (HDLc) is an independent risk factor for cardiovascular disease (CVD). However, pharmacological treatments focused on HDLc increase have failed to improve vascular protection, suggesting that high-density lipoprotein (HDL) functions other than cholesterol transport and metabolism could play a key role in CVD. In this regard, the study of the HDL proteome provides an improved characterization of the particle and unveils differences among individuals that could explain divergences in HDL functionality.

Human age-related diseases, including obesity and type 2 diabetes (T2DM), have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS) subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly) pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM., Graphical abstract fx1, Highlights • First high-throughput redox proteomic analysis of human adipocyte mitochondria. • Thiol protein oxidation is inversely correlated to protein levels in adipocyte mitochondria. • OXPHOS Mitochondrial- vs. nuclear-encoded protein modules are altered in T2DM. • Redox proteomics highlights defects in protein import through the MIA pathway in aging and T2DM. • Complex IV emerges as a common target of oxidative modifications connecting aging and T2DM.

High-density lipoproteins (HDLs) are complex protein and lipid assemblies whose composition is known to change in diverse pathological situations. Analysis of the HDL proteome can thus provide insight into the main mechanisms underlying abdominal aortic aneurysm (AAA) and potentially detect novel systemic biomarkers. We performed a multiplexed quantitative proteomics analysis of HDLs isolated from plasma of AAA patients (N = 14) and control study participants (N = 7). Validation was performed by western-blot (HDL), immunohistochemistry (tissue), and ELISA (plasma). HDL from AAA patients showed elevated expression of peroxiredoxin-6 (PRDX6), HLA class I histocompatibility antigen (HLA-I), retinol-binding protein 4, and paraoxonase/arylesterase 1 (PON1), whereas alpha-2 macroglobulin and C4b-binding protein were decreased. The main pathways associated with HDL alterations in AAA were oxidative stress and immune-inflammatory responses. In AAA tissue, PRDX6 colocalized with neutrophils, vascular smooth muscle cells, and lipid oxidation. Moreover, plasma PRDX6 was higher in AAA (N = 47) than in controls (N = 27), reflecting increased systemic oxidative stress. Finally, a positive correlation was recorded between PRDX6 and AAA diameter. The analysis of the HDL proteome demonstrates that redox imbalance is a major mechanism in AAA, identifying the antioxidant PRDX6 as a novel systemic biomarker of AAA. We thank Simon Bartlett for language and scientific editing. This study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2016-80843-R, BIO2012-37926 and BIO2015-67580-P), Fondo de Investigaciones Sanitarias ISCiii-FEDER (PRB2) (IPT13/0001, ProteoRed, Redes RIC RD12/0042/00038 and RD12/0042/0056, Biobancos RD09/0076/00101 and CA12/00371), Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas (CIBERDEM), and FRIAT. The 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). Sí

This work is licensed under a Creative Commons Attribution 4.0 International License.-- et al., Obesity is a main global health issue and an outstanding cause of morbidity and mortality predisposing to type 2 diabetes (T2DM) and cardiovascular diseases. Huge research efforts focused on gene expression, cellular signalling and metabolism in obesity have improved our understanding of these disorders; nevertheless, to bridge the gap between the regulation of gene expression and changes in signalling/metabolism, protein levels must be assessed. We have extensively analysed visceral adipose tissue from age-, T2DM- and gender-matched obese patients using high-throughput proteomics and systems biology methods to identify new biomarkers for the onset of T2DM in obesity, as well as to gain insight into the influence of aging and gender in these disorders. About 250 proteins showed significant abundance differences in the age, T2DM and gender comparisons. In diabetic patients, remarkable gender-specific hallmarks were discovered regarding redox status, immune response and adipose tissue accumulation. Both aging and T2DM processes were associated with mitochondrial remodelling, albeit through well-differentiated proteome changes. Systems biology analysis highlighted mitochondrial proteins that could play a key role in the age-dependent pathophysiology of T2DM. Our findings could serve as a framework for future research in Translational Medicine directed at improving the quality of life of obese patients., This work was supported by grant SAF2012-33014 from MINECO (B.P.), FPI-UAM Program (M.G.S.) and Pro CNIC Foundation (E.C., J.A.L. and J.V).

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3¿-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal., Spanish Ministry of Economy and Competitiveness, NDISNET-S2011/BMD-2332 from the Comunidad de Madrid ERC-2011-AdG 294340-GENTRIS and ERC-2013-AdG 334763-NOVARIPP. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation, and the Pro-CNIC Foundation

Abdominal aortic aneurysm (AAA) is an important health problem, both because of AAA rupture and death and because of increased cardiovascular mortality. Identification of new biomarkers of AAA may suggest novel pathological mechanisms and targets for new medical treatments to slow AAA progression. Metabolic changes in AAA patients were mainly related to carbohydrate and lipid metabolism and many of these changes can be associated with a situation of insulin resistance (which can be related to metabolic syndrome) together with altered amino acid metabolism. For the first time, metabolites that can be associated with differential metabolism by the gut microflora of AAA patients have also been found. Moreover, aminomalonic acid in plasma has been shown to be the metabolite with the biggest difference between patients suffering from large aneurysm (>5 cm) and controls.

Diabetes with or without the presence of hypertension damages the heart. However, there is currently a lack of information about these associated pathologies and the alteration of linked proteins. For these reasons, we were interested in the potential synergistic interaction of diabetes and hypertension in the heart, focusing on the proteome characterization of the pathological phenotypes and the associated hypertrophic response. We treated normotensive and spontaneously hypertensive (SHR) rats with either streptozotocin or vehicle. After 22weeks, type-I diabetic (DM1), SHR, SHR/DM1 and control left-ventricles were studied using proteomic approaches. Proteomics revealed that long-term DM1, SHR and SHR/DM1 rats exhibited 24, 53 and 53 altered proteins in the myocardia, respectively. DM1 myocardium showed over-expression of apoptotic and cytoskeleton proteins, and down-regulation of anti-apoptotic and mitochondrial metabolic enzymes. In both SHR and SHR/DM1 these changes were exacerbated and free fatty-acid (FFA) s-oxidation enzymes were additionally decreased. Furthermore, SHR/DM1 hearts exhibited a misbalance of specific pro-hypertrophic, anti-apoptotic and mitochondrial ATP-carrier factors, which could cause additional damage. Differential proteins were validated and then clustered into different biological pathways using bioinformatics. These studies suggested the implication of FFA-nuclear receptors and hypertrophic factors in these pathologies. Although key s-oxidation enzymes were not stimulated in DM1 and hypertensive hearts, peroxisome proliferator-activated receptors-α (PPARα) were potentially activated for other responses. In this regard, PPARα stimulation reduced hypertrophy and pro-hypertrophic factors such as annexin-V in high-glucose and angiotensin-II induced cardiomyocytes. Thus, activation of PPARα could reflect a compensatory response to the metabolic-shifted, apoptotic and hypertrophic status of the hypertensive-diabetic cardiomyopathy.