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Heat alters biology from molecular to ecological levels, but may also have unknown indirect effects. This includes the concept that animals exposed to abiotic stress can induce stress in naive receivers. Here, we provide a comprehensive picture of the molecular signatures of this process, by integrating multiomic and phenotypic data. In individual zebrafish embryos, repeated heat peaks elicited both a molecular response and a burst of accelerated growth followed by a growth slowdown in concert with reduced responses to novel stimuli. Metabolomes of the media of heat treated vs. untreated embryos revealed candidate stress metabolites including sulfur-containing compounds and lipids. These stress metabolites elicited transcriptomic changes in naive receivers related to immune response, extracellular signaling, glycosaminoglycan/keratan sulfate, and lipid metabolism. Consequently, non-heat-exposed receivers (exposed to stress metabolites only) experienced accelerated catch-up growth in concert with reduced swimming performance. The combination of heat and stress metabolites accelerated development the most, mediated by apelin signaling. Our results prove the concept of indirect heat-induced stress propagation toward naive receivers, inducing phenotypes comparable with those resulting from direct heat exposure, but utilizing distinct molecular pathways. Group-exposing a nonlaboratory zebrafish line, we independently confirm that the glycosaminoglycan biosynthesis-related gene chs1 and the mucus glycoprotein gene prg4a, functionally connected to the candidate stress metabolite classes sugars and phosphocholine, are differentially expressed in receivers. This hints at the production of Schreckstoff-like cues in receivers, leading to further stress propagation within groups, which may have ecological and animal welfare implications for aquatic populations in a changing climate.

Treatment of human brain tumour biopsies, maintained on-chip, with type I PRMT inhibitors leads to apoptosis through changes in gene expression and RNA processing, mediated by cross-talk with type II PRMT.

Understanding the genomic basis of adaptation to different abiotic environments is important in the context of climate change and resulting short-term environmental fluctuations. Using functional and comparative genomics approaches, we here investigated whether signatures of genomic adaptation to a set of environmental parameters are concentrated in specific subsets of genes and functions in lacertid lizards and other vertebrates. We first identify 200 genes with signatures of positive diversifying selection from transcriptomes of 24 species of lacertid lizards and demonstrate their involvement in physiological and morphological adaptations to climate. To understand how functionally similar these genes are to previously predicted candidate functions for climate adaptation and to compare them with other vertebrate species, we then performed a meta-analysis of 1, 100 genes under selection obtained from -omics studies in vertebrate species adapted to different abiotic factors. We found that the vertebrate gene set formed a tightly connected interactome, which was to 23% enriched in previously predicted functions of adaptation to climate, and to a large part (18%) involved in organismal stress response. We found a much higher degree of identical genes being repeatedly selected among different animal groups (43.6%), and of functional similarity and post-translational modifications than expected by chance, and no clear functional division between genes used for ectotherm and endotherm physiological strategies. In total, 171 out of 200 genes of Lacertidae were part of this network. These results highlight an important role of a comparatively small set of genes and their functions in environmental adaptation and narrow the set of candidate pathways and markers to be used in future research on adaptation and stress response related to climate change.

[Image: see text] Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to arginine residues in proteins. PRMT inhibitors are novel, promising drugs against cancer that are currently in clinical trials, which include oral administration of the drugs. However, off-target activities of systemically available PRMT inhibitors have not yet been investigated. In this work, we study the relevance of arginine methylation in platelets and investigate the effect of PRMT inhibitors on platelet function and on the expression of relevant platelet receptors. We show that (1) key platelet proteins are modified by arginine methylation; (2) incubation of human platelets with PRMT inhibitors for 4 h results in impaired capacity of platelets to aggregate in response to thrombin and collagen, with IC(50) values in the μM range; and (3) treatment with PRMT inhibitors leads to decreased membrane expression and reduced activation of the critical platelet integrin α(IIb)β(3). Our contribution opens new avenues for research on arginine methylation in platelets, including the repurposing of arginine methylation inhibitors as novel antiplatelet drugs. We also recommend that current and future clinical trials with PRMT inhibitors consider any adverse effects associated with platelet inhibition of these emerging anticancer drugs.

Heat alters biology from molecular to ecological levels, but may also have unknown indirect effects. This includes the novel concept that animals exposed to abiotic stress can induce stress in naive receivers. Here, we provide a comprehensive picture of the molecular signatures of this process, by integrating multi-omic and phenotypic data. In individual zebrafish embryos, repeated heat peaks elicited both a molecular response and a burst of accelerated growth followed by a growth slow-down in concert with reduced responses to novel stimuli. Metabolomes of the media of heat treated vs. untreated embryos revealed candidate stress metabolites including sulphur-containing compounds and lipids. These stress metabolites elicited transcriptomic changes in naive receivers related to immune response, extracellular signalling, glycosaminoglycan/keratan sulphate, and lipid metabolism. Consequently, non heat-exposed receivers (exposed to stress metabolites only) experienced accelerated catch-up growth in concert with reduced swimming performance. The combination of heat and stress metabolites accelerated development the most, mediated by apelin signalling. Our results prove the concept of indirect heat-induced stress propagation towards naive receivers, inducing phenotypes comparable to those resulting from direct heat exposure, but utilising distinct molecular pathways. Group-exposing a non-laboratory zebrafish line, we independently confirm that the glycosaminoglycan biosynthesis-related gene chs1, and the mucus glycoprotein gene prg4a, functionally connected to the candidate stress metabolite classes sugars and phosphocholine, are differentially expressed in receivers. This hints at production of Schreckstoff-like cues in receivers, leading to further stress propagation within groups, which may have ecological and animal welfare implications for aquatic populations in a changing climate.Significance StatementAquatic animals utilise chemicals to mediate adaptive behaviours. For instance, predated fish release chemical cues that elicit antipredatory responses in naive receivers. But whether abiotic factors such as heat likewise alter chemical communication has received little focus. Here, we uncover a novel dimension of chemical communication — heat-stressed donors can induce stress in naive receivers. We show that heat activates molecular stress responses, leading to the release of distinct stress metabolite classes into the environment. These stress metabolites alter the transcriptome of receivers, resulting in faster development and hypoactivity. Heat combined with stress metabolites had the largest effect, highlighting that abiotic stress, experienced both directly and indirectly, can alter chemical communication and affect embryonic development.Graphical AbstractHighlightsWe elucidate the mechanism for a novel dimension of the heat stress response — chemical communication from heat-stressed donors that induces stress in naive receivers — constituting a positive feedback loopRepeated heat stress induces a cellular and cortisol stress response and alters the phenotype of zebrafish embryosHeat-stressed embryos release stress metabolites enriched in lipids and sulphur-containing organo-oxygen compoundsIn combination, heat and stress metabolites induced 47% distinct differentially expressed genes, with many related to organ developmentThese stress metabolites alter the transcriptome and induce both faster development and hypoactivity in naive receivers, a similar response to that of heat stress itself

Despite intense research efforts, our pharmaceutical repertoire against high-grade brain tumours has not been able to increase patient survival for a decade and life expectancy remains at less than 16 months after diagnosis, on average. Inhibitors of protein arginine methyltransferases (PRMTs) have been developed and investigated over the past 15 years and have now entered oncology clinical trials, including for brain tumours. This review collates recent advances in the understanding of the role of PRMTs and arginine methylation in brain tumours. We provide an up-to-date literature review on the mechanisms for PRMT regulation. These include endogenous modulators such as alternative splicing, miRNA, post-translational modifications and PRMT–protein interactions, and synthetic inhibitors. We discuss the relevance of PRMTs in brain tumours with a particular focus on PRMT1, -2, -5 and -8. Finally, we include a future perspective where we discuss possible routes for further research on arginine methylation and on the use of PRMT inhibitors in the context of brain tumours.

Chromobox 2 (CBX2) is a chromatin-binding component of polycomb repressive complex 1, which causes gene silencing. CBX2 expression is elevated in triple-negative breast cancer (TNBC), for which there are few therapeutic options. Here, we aimed to investigate the functional role of CBX2 in TNBC. CBX2 knockdown in TNBC models reduced cell numbers, which was rescued by ectopic expression of wild-type CBX2 but not a chromatin binding-deficient mutant. Blocking CBX2 chromatin interactions using the inhibitor SW2_152F also reduced cell growth, suggesting CBX2 chromatin binding is crucial for TNBC progression. RNA sequencing and gene set enrichment analysis of CBX2-depleted cells identified downregulation of oncogenic signalling pathways, including mTORC1 and E2F signalling. Subsequent analysis identified that CBX2 represses the expression of mTORC1 inhibitors and the tumour suppressor RBL2. RBL2 repression, in turn, inhibits DREAM complex activity. The DREAM complex inhibits E2F signalling, causing cell senescence; therefore, inhibition of the DREAM complex via CBX2 may be a key oncogenic driver. We observed similar effects in oestrogen receptor-positive breast cancer, and analysis of patient datasets suggested CBX2 inhibits RBL2 activity in other cancer types. Therapeutic inhibition of CBX2 could therefore repress mTORC1 activation and promote DREAM complex-mediated senescence in TNBC and could have similar effects in other cancer types.

Aquatic organisms must cope with both rising and rapidly changing temperatures. These environmental changes can affect numerous traits, from molecular to ecological scales. Biotic stressors can induce the release of chemical cues which trigger behavioural responses in other individuals. In this study, we infer whether abiotic stressors, such as fluctuating temperature, may similarly propagate stress responses between individuals in fish not directly exposed to the stressor. To test this hypothesis, zebrafish (Danio rerio) embryos were exposed for 24 hours to fluctuating thermal stress, to medium in which another embryo was thermally stressed before (“stress medium”), and to a combination of these. Growth, behaviour, and expression of a panel of genes were used to characterise the thermal stress response and its propagation between embryos. Both high temperatures and stress medium significantly accelerated development and altered embryonic behaviour. Thermal stress significantly decreased the expression of the antioxidant gene SOD1, eight hours after the end of exposure. Of note, we found that the expression of sulfide:quinone oxidoreductase (SQOR), likewise a part of the antioxidant metabolism relevant in vertebrate stress response, and of interleukin-1β (IL-1β), involved in the immune response, were significantly altered by stress medium. This study illustrates the existence of positive thermal stress feedback loops in zebrafish embryos that induce stress in conspecifics. This evidence that thermal stress due to fluctuating, high temperatures can be propagated may be relevant for species found in high densities, either in aquaculture or in the natural environment, in a context of global change.

Cardiovascular disease is the major cause of death worldwide. Extensive cardiovascular biomarkers are available using blood tests but very few, if any, investigations have described non-invasive tests for cardiovascular biomarkers based on readily available hair samples. Here we show, first, that human hair proteins are post-translationally modified by arginine methylation (ArgMe). Using western blot, proteomic data mining and mass spectrometry, we identify several ArgMe events in hair proteins and we show that keratin-83 is extensively modified by ArgMe in the human hair. Second, using a preliminary cohort (n = 18) of heterogenous healthy donors, we show that the levels of protein ArgMe in hair correlate with serum concentrations of a well-established cardiovascular biomarker, asymmetric dimethylarginine (ADMA). Compared to blood collection, hair sampling is cheaper, simpler, requires minimal training and carries less health and safety and ethical risks. For these reasons, developing the potential of hair protein ArgMe as clinically useful cardiovascular biomarkers through further research could be useful in future prevention and diagnosis of cardiovascular disease.

Aquatic organisms must cope with both rising and rapidly changing temperatures. These thermal changes can affect numerous traits, from molecular to ecological scales. Biotic stressors are already known to induce the release of chemical cues which trigger behavioural responses in other individuals. In this study, we infer whether fluctuating temperature, as an abiotic stressor, may similarly induce stress-like responses in individuals not directly exposed to the stressor. To test this hypothesis, zebrafish (Danio rerio) embryos were exposed for 24 h to fluctuating thermal stress, to medium in which another embryo was thermally stressed before (“stress medium”), and to a combination of these. Growth, behaviour, expression of molecular markers, and of whole-embryo cortisol were used to characterise the thermal stress response and its propagation between embryos. Both fluctuating high temperature and stress medium significantly accelerated development, by shifting stressed embryos from segmentation to pharyngula stages, and altered embryonic activity. Importantly, we found that the expression of sulfide:quinone oxidoreductase (SQOR), the antioxidant gene SOD1, and of interleukin-1β (IL-1β) were significantly altered by stress medium. This study illustrates the existence of positive thermal stress feedback loops in zebrafish embryos where heat stress can induce stress-like responses in conspecifics, but which might operate via different molecular pathways. If similar effects also occur under less severe heat stress regimes, this mechanism may be relevant in natural settings as well.

Aberrant expression of the sodium channel gene (SCN5A) has been proposed to disrupt cardiac action potential and cause human cardiac arrhythmias, but the mechanisms of SCN5A gene regulation and dysregulation still remain largely unexplored. To gain insight into the transcriptional regulatory networks of SCN5A, we surveyed the promoter and first intronic regions of the SCN5A gene, predicting the presence of several binding sites for GATA transcription factors (TFs). Consistent with this prediction, chromatin immunoprecipitation (ChIP) and sequential ChIP (Re-ChIP) assays show co-occupancy of cardiac GATA TFs GATA4 and GATA5 on promoter and intron 1 SCN5A regions in fresh-frozen human left ventricle samples. Gene reporter experiments show GATA4 and GATA5 synergism in the activation of the SCN5A promoter, and its dependence on predicted GATA binding sites. GATA4 and GATA6 mRNAs are robustly expressed in fresh-frozen human left ventricle samples as measured by highly sensitive droplet digital PCR (ddPCR). GATA5 mRNA is marginally but still clearly detected in the same samples. Importantly, GATA4 mRNA levels are strongly and positively correlated with SCN5A transcript levels in the human heart. Together, our findings uncover a novel mechanism of GATA TFs in the regulation of the SCN5A gene in human heart tissue. Our studies suggest that GATA5 but especially GATA4 are main contributors to SCN5A gene expression, thus providing a new paradigm of SCN5A expression regulation that may shed new light into the understanding of cardiac disease.

Background The inhibitory subunit of cardiac troponin (cTnI) is a gold standard cardiac biomarker and also an essential protein in cardiomyocyte excitation-contraction coupling. The interactions of cTnI with other proteins are fine-tuned by post-translational modification of cTnI. Mutations in cTnI can lead to hypertrophic cardiomyopathy. Methods and results Here we report, for the first time, that cTnI is modified by arginine methylation in human myocardium. Using Western blot, we observed reduced levels of cTnI arginine methylation in human hypertrophic cardiomyopathy compared to dilated cardiomyopathy biopsies. Similarly, using a rat model of cardiac hypertrophy we observed reduced levels of cTnI arginine methylation compared to sham controls. Using mass spectrometry, we identified cTnI methylation sites at R74/R79 and R146/R148 in human cardiac samples. R146 and R148 lie at the boundary between the critical cTnI inhibitory and switch peptides; PRMT1 methylated an extended inhibitory peptide at R146 and R148 in vitro. Mutations at R145 that have been associated with hypertrophic cardiomyopathy hampered R146/R148 methylation by PRMT1 in vitro. H9c2 cardiac-like cells transfected with plasmids encoding for a methylation-deficient R146A/R148A cTnI protein developed cell hypertrophy, with a 32% increase in cell size after 72 h, compared to control cells. Discussion Our results provide evidence for a novel and significant cTnI post-translational modification. Our work opens the door to translational investigations of cTnI arginine methylation as a biomarker of disease, which can include e.g. cardiomyopathies, myocardial infarction and heart failure, and offers a novel way to investigate the effect of cTnI mutations in the inhibitory/switch peptides.

Obesity leading to hyperlipidaemia and atherosclerosis is recognised to induce\ud morphological and metabolic changes in many tissues. However, both hyperlipidaemia and\ud atherosclerosis can occur in the absence of obesity. The impact of the latter scenario on\ud skeletal muscle and liver is not understood sufficiently. In this regard, we used the\ud Apolipoprotein E-deficient (ApoE-/-) mouse model, an established model of hyperlipidaemia\ud and atherosclerosis, that does not become obese when subjected to a high-fat diet, to\ud determine the impact of Western-type diet (WD) and ApoE deficiency on skeletal muscle\ud morphological, metabolic and biochemical properties. To establish the potential of\ud therapeutic targets, we further examined the impact of Nox2 pharmacological inhibition on\ud skeletal muscle redox biology. We found ectopic lipid accumulation in skeletal muscle and\ud the liver, and altered skeletal muscle morphology and intramuscular triacylglycerol fatty acid\ud composition. WD and ApoE deficiency had a detrimental impact in muscle metabolome,\ud followed by perturbed gene expression for fatty acid uptake and oxidation. Importantly, there\ud was enhanced oxidative stress in the skeletal muscle and development of liver steatosis,\ud inflammation and oxidative protein modifications. Pharmacological inhibition of Nox2\ud decreased reactive oxygen species production and protein oxidative modifications in the\ud muscle of ApoE-/- mice subjected to a Western-type diet. This study provides key evidence to\ud better understand the pathophysiology of skeletal muscle in the context of hyperlipidaemia\ud and atherosclerosis and identifies Nox2 as a potential target for attenuating oxidative stress\ud in skeletal muscle in a mouse model of obesity-independent hyperlipidaemia.

Glioblastomas (GBM) are the most common grade 4 brain tumours; patients have very poor prognosis with an average survival of 15 months after diagnosis. Novel research lines have begun to explore aberrant protein arginine methylation (ArgMe) as a possible therapeutic target in GBM and ArgMe inhibitors are currently in clinical trials. Enzymes known as protein arginine methyltransferases (PRMT1-9) can lead to mono- or di-ArgMe, and in the latter case symmetric or asymmetric dimethylation (SDMA and ADMA, respectively). Using the most common GBM cell line, we have profiled the expression of PRMTs, used ArgMe inhibitors as tools to investigate post-translational modifications cross-talk and measured the effect of ArgMe inhibitors on cell viability. We have identified novel SDMA events upon inhibition of ADMA in GBM cells and spheroids. We have observed cross-talk between ADMA and lysine acetylation in GBM cells and platelets. Treatment of GBM cells with furamidine, a PRMT1 inhibitor, reduces cell viability in 2D and 3D models. These data provide new molecular understanding of a disease with unmet clinical needs.

Background Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, including 36 SCDs during youth. Objectives We sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation. Methods Genetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with β-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload–induced calcium release activity of the mutant channel in HEK293 cells. Results We identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload–induced calcium release activity under conditions that mimic catecholaminergic stress. Conclusion Our study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on β-adrenergic stimulation.

Background Glioblastoma is a deadly disease with a median survival of 15 months after treatment. While maximal safe surgical resection and adjuvant chemoradiotherapy continues to be the mainstay of treatment, glioblastoma demostrates a remarkably heterogeneous molecular profile, and there is a drive to discover further chemotherapeutic targets that can effectively augment current multimodal therapy. Introduction Post-translational modification of proteins plays a key role in maintenance of regulatory cell networks, and protein arginine methyltransferases (PRMT) are one of the enzymes involved in symmetric and asymmetric methylation of various proteins. We aim to demonstrate the entire range of PRMT proteins expressed in two different glioblastoma cell lines (U87MG and U251) in order to elucidate a consistency in expression of these proteins across cell lines. Methods Cells from 2 different glioblastoma cell lines (U87MG and U251) were cultured and lysed using standard aseptic techniques. Protein profiling was done using SDS-PAGE electrophoresis with a molecular weight marker as the reference and all primary antibodies to various PRMTs (1–10) and respective secondary antibodies. Membranes were visualised with a chemiluminescent protocol. Experiments were repeated in order to reduce bias. Conclusion It was clearly seen that in both cell lines there is a strong tendency for PRMT5 expression and relative under-expresssion of PRMT 9/10. In addition, however, there are varying expressions of other PRMTs as well. We aim to further explore this to improve the strength of this correlation.

Voltage-gated sodium channels are essential proteins in brain physiology, as they generate the sodium currents that initiate neuronal action potentials. Voltage-gated sodium channels expression, localisation and function are regulated by a range of transcriptional and post-translational mechanisms. Here, we review our understanding of regulation of brain voltage-gated sodium channels, in particular SCN1A (NaV1.1), SCN2A (NaV1.2), SCN3A (NaV1.3) and SCN8A (NaV1.6), by transcription factors, by alternative splicing, and by post-translational modifications. Our focus is strongly centred on recent research lines, and newly generated knowledge.

The cardiac voltage-gated sodium channel (gene: SCN5A, protein: NaV1.5) is responsible for the sodium current that initiates the cardiomyocyte action potential. Research into the mechanisms of SCN5A gene expression has gained momentum over the last few years. We have recently described the transcriptional regulation of SCN5A by GATA4 transcription factor. In this addendum to our study, we report our observations that 1) the linker between domains I and II (LDI-DII) of NaV1.5 contains a nuclear localization signal (residues 474–481) that is necessary to localize LDI-DII into the nucleus, and 2) nuclear LDI-DII activates the SCN5A promoter in gene reporter assays using cardiac-like H9c2 cells. Given that voltage-gated sodium channels are known targets of proteases such as calpain, we speculate that NaV1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of LDI-DII and subsequent stimulation of the SCN5A promoter.

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%-25% of BrS patients carry genetic defects that cause loss-of-function of the voltage-gated cardiac sodium channel. Thus, 70%-75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa ) from cells coexpressing Nav 1.5 and wild-type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav 1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav 1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav 1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and beta-ketoacyl-ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo-ACP synthase (ACPS) let to production of NSAS holo-ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self-malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo-ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo-ACP and NSAS AT domain-catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo-ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo-ACP. The NSAS holo-ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.

Arginine methylation is a novel post-translational modification within the voltage-gated ion channel superfamily, including the cardiac sodium channel, NaV1.5. We show that NaV1.5 R513 methylation decreases S516 phosphorylation rate by 4 orders of magnitude, the first evidence of protein kinase A inhibition by arginine methylation. Reciprocally, S516 phosphorylation blocks R513 methylation. NaV1.5 p.G514C, associated to cardiac conduction disease, abrogates R513 methylation, while leaving S516 phosphorylation rate unchanged. This is the first report of methylation-phosphorylation cross-talk of a cardiac ion channel.

The α subunit of the cardiac voltage-gated sodium channel, NaV1.5, provides the rapid sodium inward current that initiates cardiomyocyte action potentials. Here, we analyzed for the first time the post-translational modifications of NaV1.5 purified from end-stage heart failure human cardiac tissue. We identified R526 methylation as the major post-translational modification of any NaV1.5 arginine or lysine residue. Unexpectedly, we found that the N terminus of NaV1.5 was: 1) devoid of the initiation methionine, and 2) acetylated at the resulting initial alanine residue. This is the first evidence for N-terminal acetylation in any member of the voltage-gated ion channel superfamily. Our results open the door to explore NaV1.5 N-terminal acetylation and arginine methylation levels as drivers or markers of end-stage heart failure.

The α-subunit of the cardiac voltage-gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.

Purpose Arginine methylation (ArgMe) is one of the most ubiquitous post-translational modifications, and hundreds of proteins undergo ArgMe in e.g. brain. However, the scope of ArgMe in many tissues, including the heart, is currently under explored. Here, we aimed to 1) identify proteins undergoing ArgMe in human organs, and 2) expose the relevance of ArgMe in cardiac disease. Experimental design We used publicly available proteomic data to search for ArgMe in 13 human tissues. We used glucose to induce H9c2 cardiac-like cell hypertrophy. Results Our results show that ArgMe is mainly tissue-specific; nevertheless, we suggest an embryonic origin of core ArgMe events. In the heart, we found 103 mostly novel ArgMe sites in 58 non-histone proteins. We provide compelling evidence that cardiac protein ArgMe is relevant to cardiomyocyte ontology, and important for proper cardiac function. This is highlighted by the fact that genetic mutations affecting methylated arginine positions are often associated with cardiac disease, including hypertrophic cardiomyopathy. We provide pilot experimental data suggesting significant changes in ArgMe profiles of H9c2 cells upon induction of cell hypertrophy using glucose. Conclusions and clinical relevance Our work calls for in-depth investigation of ArgMe in normal and diseased tissues, using methods including clinical proteomics.

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.

15 páginas, 8 figuras, 1 tabla, Protein–metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein–lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein–lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids, This work is partially funded by Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN) to ACG (BMBF NGNF IG-Cellular Systems Genomics, 01GS0865). OG is a fellow of the Ministerio de ciencia e innovación, Spain. KM is a fellow of the Danish Natural Science Research Council (09-064986/FNU). The protein interactions from this publication have been submitted to the IntAct database (pmid: 19850723) and assigned the identifier EBI-2933237

Sudden cardiac death is a major contributor to mortality in industrialized nations; in fact, it is the cause of more deaths than acquired immune deficiency syndrome, lung and breast cancer, and stroke together. Frequently, the autopsy becomes the principal diagnostic tool because macroscopic and microscopic analyses reveal the underlying cause of death. However, a significant number of sudden cardiac deaths remain unexplained. These cases are referred to as "natural" or arrhythmogenic. In the young, in up to 50% of sudden cardiac death cases, sudden death is the first and only clinical manifestation of an inherited cardiac disease that had remained undetected by conventional clinical investigations. To improve diagnosis, genetic testing has recently been added to these clinical tools. During the last two decades, there has been considerable progress in the understanding about genetics of sudden cardiac death. With that new information, the probands and their family members can make an informed decision regarding their care and know whether and to what extent they are at risk of suffering from the disease. Thus, genetic technology and expertise have become essential for the diagnosis of some forms of inherited cardiac diseases and to provide a basis for subsequent prevention strategies. This review focuses on recent advances in the understanding of cardiopathies owing to genetic investigations.

Interactions between the acyl carrier protein (ACP) and ketoacylsynthase (KS) components of the actinorhodin polyketide synthase have been investigated using kinetic assays. These indicate that for three different quantifiable interactions (acceleration of self-malonylation, initiation and extension) mutations of E47 and E53 residues located on ACP helix II have different effects. Initiation clearly involves interaction between KS(beta) and ACP helix II, but self-malonylation acceleration and extension by KS(alpha) appear not to be affected strongly by the same mutations.

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k approximately 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.

Long QT Syndrome (LQTS) is a rare inherited cardiac disorder with a high risk of sudden cardiac death in a structurally normal heart. To date, pathogenic mutations have been described as responsible for approximately 70-75% of LQTS patients, mainly in ion channel genes. Mutations in genes encoding the sodium channel α subunit or other regulatory proteins, affecting cardiac sodium current, have been previously related to LQTS. Five sodium channel β subunits have been identified, which are encoded by four genes (SCN1B-4B). Pathogenic mutations in the SCN4B gene, but not in other β subunits, have been reported in LQTS. We tested whether mutations in SCN1B-4B could be responsible for LQTS in patients without mutations in the common LQTS-related genes. We screened for mutations in SCN1B-4B genes in 30 non-related patients clinically diagnosed with LQTS carrying no mutations in the major LQTS-related genes. The screening revealed a novel mutation in the SCN1B gene in an 8-year-old boy. The base change resulted in an amino acid variation from proline to threonine in the alternative C-terminus of the sodium channel β1 subunit (β1b). Using the patch clamp technique, we measured sodium current density, and Nav1.5 gaiting properties, in HEK cells transiently transfected with Nav1.5 and β1b subunits. Our electrophysiological analysis revealed that the mutant β1b altered Nav1.5 function by shifting the window current to negative potentials, increasing recovery from inactivation, decreasing slow inactivation, and increasing late sodium current. In addition, we recorded action potentials from mouse atrial cardiomyocytes, HL-1 cells, transfected with β1b subunits. These experiments revealed that the action potential duration significantly increased when the mutant β1b was overexpressed compared to β1bWT. These findings suggest that the mutation in β1b could explain the LQTS in our patient, revealing SCN1Bb as a new susceptibility gene for LQTS. Our results confirm the importance of sodium channel β subunits in the modulation of cardiac sodium channel. In addition, they highlight the need for further investigation to detect new candidate genes underlying the LQTS patients that currently remain without genetic diagnosis.

Brugada Syndrome (BrS) is a life-threating arrhythmogenic disease associated with a high risk of Sudden Cardiac Death. Genetic alterations in SCN5A, which encodes the alpha subunit of the cardiac voltage-gated sodium channel (Nav1.5), are the most common cause of BrS. Nav1.5 channel is responsible for the rapid influx of sodium ions that initiate the propagation of the action potential in cardiomyocytes. However, mutations in SCN5A gene only explain 20-25% of the cases with BrS. Our working hypothesis is that a deregulation of SCN5A expression could cause BrS and explain, at least in part, some cases of BrS with no mutation identified. Nevertheless, little is known regarding the regulation of SCN5A expression. Therefore, it is the goal of this study to gain insight into the molecular mechanisms that regulate SCN5A expression at the transcriptional level, and how alterations of these mechanisms contribute to cardiac arrhythmias. The human SCN5A promoter contains putative binding sites for the zinc finger transcription factor GATA-4. We performed GATA-4 overexpression and knockdown studies in cardiac H9c2 cells, and analyzed the effect on the SCN5A promoter by luciferase reporter experiments and real-time PCR. Our data showed that GATA-4 is a novel transcriptional activator of the SCN5A promoter. We also analyzed the effect of other GATA family members, GATA-5 and -6. Whereas GATA-6 did not have any effect on the SCN5A promoter, GATA-5 increased SCN5A transcriptional activity although to a less extent than GATA-4. Moreover, we observed that GATA-4 significantly synergizes with GATA-5 on SCN5A transcriptional activation. Chromatin Immunoprecipitation (ChIP) experiments from human cardiac tissue showed that GATA-4 and GATA-5 bind to the SCN5A proximal promoter region, confirming a critical role of these factors in regulating SCN5A expression in vivo. Our current experiments are also focused on studying the interaction between GATA-4 and GATA-5 transcription factors. We performed co-transfection experiments of GATA-4 with p300 acetyltransferase and observed a further increase on SCN5A promoter activity. These results suggest that GATA-4 transcriptional activity on the SCN5A promoter is modulated by acetylation. We are now investigating which histones acetyltransferases (HDACs) could participate in the activity of GATA-4 on the SCN5A promoter. Our findings provide novel insights on the regulation of Nav1.5 expression, which will ultimately contribute to further understanding unexplored causes of BrS and other type of cardiac arrhythmias.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare cardiac disease characterized by fibrofatty replacement of the right ventricular myocardium, which may cause ventricular arrhythmias and sudden cardiac death (SCD). Mutations in genes encoding desmosome proteins have been associated with ARVC: plakophilin-2 (PKP2), desmoplakin (DSP), desmocolin-2 (DSC2), desmoglein-2 (DSG2) and plakoglobin (PG). Plakophilin-2 (PKP2) is the most prevalent gene associated with ARVC, especially stop-gained mutations, which lead to truncated PKP2 (PKP2TR). Our recent genetic studies have shown that PKP2TR is associated to a later age of ARVC onset (37 years old) compared to missense carriers (27 years old). This raised an alternative hypothesis as to the pathogenicity of missense and truncated proteins, and their role in phenotype. We hypothesize that PKP2TR is associated with trafficking to the plasma membrane, which can be compensated by the normal allele; while missense variations may act through a dominant negative effect, disrupting the function of the wild type protein since they are mostly incorporated to desmosome. To explore this hypothesis, we performed an in vitro study with the three following aims: (1) to examine the subcellular location of PKP2TR forms in the in HL-1 cardiac-like cells, (2) how PKP2TR length affect to their proper traffic to plasma membrane and (3) PKP2TR effect on desmosome assembly. Based on mutations associated to ARVC in our genetic study, we selected four PKP2TR versions (at amino acids 91, 412, 637 and 734), which were transiently co-transfected in HL-1 cells with major desmosome proteins (DSP,DSC2, DSG2 and PG). We observed that these PKP2TR forms are not localized efficiently to the plasma membrane; PKP2TR-91 remained diffuse in the cytoplasm; PKP2TR-412 was retained in the endoplasmic reticulum displaying a dotted pattern. In contrast PKP2TR-637 and PKP2TR-734 were only partially located to the plasma membrane with a major fraction still cytosolic. We also observed that all PKP2TR caused deficient localization of other desmosome proteins, especially DSP, which requires PKP2 for its proper trafficking to the plasma membrane. Thus, these data suggest that the degree of the truncation in PKP2 does not correlate with a clear effect in desmosome assembly. Results clearly show that PKP2TR forms are not efficiently incorporated to the plasma membrane, and they affect desmosome assembly. These data favour the idea that PKP2TR may be associated to trafficking defect, while missense mutations may act in a dominant negative way disrupting function of the wild-type protein.