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.

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics.

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.

Defective dimerization of FoF1-ATP synthase secondary to glycation favors mitochondrial energy deficiency in cardiomyocytes during aging.

Aged cardiomyocytes develop a mismatch between energy demand and supply, the severity of which determines the onset of heart failure, and become prone to undergo cell death. The FoF1-ATP synthase is the molecular machine that provides >90% of the ATP consumed by healthy cardiomyocytes and is proposed to form the mitochondrial permeability transition pore (mPTP), an energy-dissipating channel involved in cell death. We investigated whether aging alters FoF1-ATP synthase self-assembly, a fundamental biological process involved in mitochondrial cristae morphology and energy efficiency, and the functional consequences this may have. Purified heart mitochondria and cardiomyocytes from aging mice displayed an impaired dimerization of FoF1-ATP synthase (blue native and proximity ligation assay), associated with abnormal mitochondrial cristae tip curvature (TEM). Defective dimerization did not modify the in vitro hydrolase activity of FoF1-ATP synthase but reduced the efficiency of oxidative phosphorylation in intact mitochondria (in which membrane architecture plays a fundamental role) and increased cardiomyocytes' susceptibility to undergo energy collapse by mPTP. High throughput proteomics and fluorescence immunolabeling identified glycation of 5 subunits of FoF1-ATP synthase as the causative mechanism of the altered dimerization. In vitro induction of FoF1-ATP synthase glycation in H9c2 myoblasts recapitulated the age-related defective FoF1-ATP synthase assembly, reduced the relative contribution of oxidative phosphorylation to cell energy metabolism, and increased mPTP susceptibility. These results identify altered dimerization of FoF1-ATP synthase secondary to enzyme glycation as a novel pathophysiological mechanism involved in mitochondrial cristae remodeling, energy deficiency, and increased vulnerability of cardiomyocytes to undergo mitochondrial failure during aging.

Malondialdehyde-modified HDL particles elicit a specific IgG response in abdominal aortic aneurysm.

High Density Lipoprotein (HDL) plays a protective role in abdominal aortic aneurysm (AAA); however, recent findings suggest that oxidative modifications could lead to dysfunctional HDL in AAA. This study aimed at testing the effect of oxidized HDL on aortic lesions and humoral immune responses in a mouse model of AAA induced by elastase, and evaluating whether antibodies against modified HDL can be found in AAA patients. HDL particles were oxidized with malondialdehyde (HDL-MDA) and the changes were studied by biochemical and proteomics approaches. Experimental AAA was induced in mice by elastase perfusion and then mice were treated with HDL-MDA, HDL or vehicle for 14 days. Aortic lesions were studied by histomorphometric analysis. Levels of anti-HDL-MDA IgG antibodies were measured by an in-house immunoassay in the mouse model, in human tissue-supernatants and in plasma samples from the VIVA cohort. HDL oxidation with MDA was confirmed by enhanced susceptibility to diene formation. Proteomics demonstrated the presence of MDA adducts on Lysine residues of HDL proteins, mainly ApoA-I. MDA-modification of HDL abrogated the protective effect of HDL on cultured endothelial cells as well as on AAA dilation in mice. Exposure to HDL-MDA elicited an anti-HDL-MDA IgG response in mice. Anti-HDL-MDA were also detected in tissue-conditioned media from AAA patients, mainly in intraluminal thrombus. Higher plasma levels of anti-HDL-MDA IgG antibodies were found in AAA patients compared to controls. Anti-HDL-MDA levels were associated with smoking and were independent predictors of overall mortality in AAA patients. Overall, MDA-oxidized HDL trigger a specific humoral immune response in mice. Besides, antibodies against HDL-MDA can be detected in tissue and plasma of AAA patients, suggesting its potential use as surrogate stable biomarkers of oxidative stress in AAA.

A Network of Macrophages Supports Mitochondrial Homeostasis in the Heart.

Cardiomyocytes 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.

Improved integrative analysis of the thiol redox proteome using filter-aided sample preparation.

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.

Generation of a lentiviral vector system to efficiently express bioactive recombinant human prolactin hormones.

The contribution of the pleiotropic hormone Prolactin (PRL) to several physiological and pathological processes is still unknown. To clarify the role of PRL in these processes during the last decade, different human PRL antagonists have been produced to either partially or fully block the wild type hormone activity. In this work, we have cloned these wild type and antagonist sequences in lentivectors (LV) to express them as recombinant self-processing polypeptides by employing a P2A sequence (hPRL-P2A-GFP). We show that these LVs can efficiently transduce and express the hPRL proteins in different cell types and that the P2A sequence does not affect their activities. Additionally, we have tested their activities in paracrine and autocrine cell culture experiments. Our results demonstrate that these recombinant hPRL-P2A proteins are bioactive in both paracrine and autocrine modes, highlighting the potential usefulness of these hPRL-containing LVs for determining the contribution of hPRL to different biological processes.

Severe Cardiac Dysfunction and Death Caused by Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Are Improved by Inhibition of Glycogen Synthase Kinase-3ß.

BACKGROUND: Arrhythmogenic cardiomyopathy/arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium, resulting in heart failure and sudden cardiac death. The most aggressive arrhythmogenic cardiomyopathy/ARVC subtype is ARVC type 5 (ARVC5), caused by a p.S358L mutation in TMEM43 (transmembrane protein 43). The function and localization of TMEM43 are unknown, as is the mechanism by which the p.S358L mutation causes the disease. Here, we report the characterization of the first transgenic mouse model of ARVC5. METHODS: We generated transgenic mice overexpressing TMEM43 in either its wild-type or p.S358L mutant (TMEM43-S358L) form in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. RESULTS: We found that mice expressing TMEM43-S358L recapitulate the human disease and die at a young age. Mutant TMEM43 causes cardiomyocyte death and severe fibrofatty replacement. We also demonstrate that TMEM43 localizes at the nuclear membrane and interacts with emerin and ß-actin. TMEM43-S358L shows partial delocalization to the cytoplasm, reduced interaction with emerin and ß-actin, and activation of glycogen synthase kinase-3ß (GSK3ß). Furthermore, we show that targeting cardiac fibrosis has no beneficial effect, whereas overexpression of the calcineurin splice variant calcineurin Aß1 results in GSK3ß inhibition and improved cardiac function and survival. Similarly, treatment of TMEM43 mutant mice with a GSK3ß inhibitor improves cardiac function. Finally, human induced pluripotent stem cells bearing the p.S358L mutation also showed contractile dysfunction that was partially restored after GSK3ß inhibition. CONCLUSIONS: Our data provide evidence that TMEM43-S358L leads to sustained cardiomyocyte death and fibrofatty replacement. Overexpression of calcineurin Aß1 in TMEM43 mutant mice or chemical GSK3ß inhibition improves cardiac function and increases mice life span. Our results pave the way toward new therapeutic approaches for ARVC5.

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-ß2 (TGFß2)-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 (Tgfß2, 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.

SanXoT: a modular and versatile package for the quantitative analysis of high-throughput proteomics experiments.

SUMMARY: Mass spectrometry-based proteomics has had a formidable development in recent years, increasing the amount of data handled and the complexity of the statistical resources needed. Here we present SanXoT, an open-source, standalone software package for the statistical analysis of high-throughput, quantitative proteomics experiments. SanXoT is based on our previously developed weighted spectrum, peptide and protein statistical model and has been specifically designed to be modular, scalable and user-configurable. SanXoT allows limitless workflows that adapt to most experimental setups, including quantitative protein analysis in multiple experiments, systems biology, quantification of post-translational modifications and comparison and merging of experimental data from technical or biological replicates. AVAILABILITY AND IMPLEMENTATION: Download links for the SanXoT Software Package, source code and documentation are available at https://wikis.cnic.es/proteomica/index.php/SSP. CONTACT: jvazquez@cnic.es or ebonzon@cnic.es. SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

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.

Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling.

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

Revisiting peptide identification by high-accuracy mass spectrometry: problems associated with the use of narrow mass precursor windows.

Peptide identification is increasingly achieved through database searches in which mass precursor tolerance is set in the ppm range. This trend is driven by the high resolution and accuracy of modern mass spectrometers and the belief that the quality of peptide identification is fully controlled by estimating the false discovery rate (FDR) using the decoy-target approach. However, narrowing mass tolerance decreases the number of sequence candidates, and several authors have raised concerns that these search conditions can introduce inaccuracies. Here, we demonstrate that when scores that only depend on one sequence candidate are used, decoy-based estimates of the number of false positive identifications are accurate even with an average number of candidates of just 200, to the point that remarkably accurate FDR predictions can be made in completely different search conditions. However, when scores that are constructed taking information from additional sequence candidates are used together with low precursor mass tolerances, the proportion of peptides incorrectly identified may become significantly higher than the FDR estimated by the target-decoy approach. Our results suggest that with this kind of score the high mass accuracy of modern mass spectrometers should be exploited by using wide mass windows followed by postscoring mass filtering algorithms.

The human HDL proteome displays high inter-individual variability and is altered dynamically in response to angioplasty-induced atheroma plaque rupture.

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.

Quantitative in-depth analysis of the dynamic secretome of activated Jurkat T-cells.

Proteins secreted by cells are of the highest biomedical relevance since they play a significant role in the progression of numerous diseases. However, characterization of the proteins specifically secreted in response to precise stimuli is challenging, since these proteins are contaminated by cellular byproducts. Here we present a method to characterize a dynamic secretome and demonstrate its utility by performing the deepest quantitative analysis to date of proteins secreted by lymphoid Jurkat T-cells upon activation. Cell-free supernatant proteins were analyzed by using an optimized protocol for differential (18)O/(16)O-labeling and LC-MS/MS, followed by statistical analysis using a random-effects model. More than 4000 unique peptides belonging to 1288 proteins were identified and a large proportion could be quantified. To determine the proteins whose secretion was up-regulated upon T-cell activation, protein variance of the null hypothesis was estimated after protein classification in terms of secretion and ontology using bioinformatic tools. 62 proteins showed a statistically significant change in abundance upon cell activation and most of them (49 proteins) were up-regulated. These proteins were functionally involved mainly in inflammatory response, signal transduction, cell growth and differentiation and cell redox homeostasis. Our approach provides a promising technology for the high-throughput quantitative study of dynamic secretomes.

A robust method for quantitative high-throughput analysis of proteomes by 18O labeling.

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of (18)O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide (18)O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with (18)O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by (18)O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.

MTPA: a crustacean metallothionein that affects hepatopancreatic mitochondrial functions.

Metallothioneins are cysteine-rich proteins, with a high capacity to bind metallic ions, and for which a precise biological role has not been established. Here we investigated the effects of MTPA, a metallothionein from the lobster Panulirus argus, on mitochondrial oxygen consumption and ROS production. An HPLC-RP-ESI-MS analysis of recombinant MTPA showed that despite its extra Cys, MTPA binds 6 Zn2+ per molecule akin to other crustacean metallothioneins with 18 Cys. The extra Cys is not involved in zinc binding, since its side-chain would be oriented to the outside of the molecule according to a preliminary model of the tridimensional structure of MTPA. MTPA-Zn2+(6) is imported into the hepatopancreatic mitochondria intermembrane space and inhibits mitochondrial oxygen consumption, increasing thereby ROS production. Nevertheless, the stimulation of ROS production by MT-bound Zn2+ is weaker compared to equivalent amounts of free Zn2+, suggesting that MTPA protects against oxidative stress. This constitutes the first report on metallothioneins effects on mitochondrial function in invertebrates and agrees with the results described for mammals, suggesting a connection between metallothioneins and energy metabolism.

Cloning, tissue expression and metal inducibility of an ubiquitous metallothionein from Panulirus argus.

Invertebrate metallothioneins (MT) have mainly been reported in digestive tissues, but data about the existence of a ubiquitous isoform expressed also in nervous tissue, are not available. Here we report the identification of a new metallothionein gene (MTPA) from the lobster Panulirus argus, putatively encoding a 59 residue polypeptide including 19 Cys. Tissue specific analysis indicated that MTPA is ubiquitously expressed in the hepatopancreas, intestine, nervous tissue and muscle, with the highest levels in the hepatopancreas and the lowest in muscle and nervous tissue. In addition, our data showed that MTPA is differentially regulated by metals: tissue explants exposed to Cd exhibited increased MTPA mRNA levels in all cases, except in muscle, with the highest effects in the nervous tissue, while Zn was effective only in the hepatopancreas. Interestingly, Cu showed no effects in any of the analyzed tissues. Taken together, these results suggest that MTPA in the hepatopancreas likely plays an important role in Cd detoxification and Zn homeostasis. The potent Cd-inducibility of MTPA in the nervous tissue might suggest a key function of this protein in protecting this highly sensitive tissue from cadmium-induced neurotoxicity.