Interplay between R513 methylation and S516 phosphorylation of the cardiac voltage-gated sodium channel

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

Estudi de la interpretació entre els factors de transcripció de Drosophila GAGA i Tramtrack

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls factors de transcripció de Drosophila GAGA i Tramtrack (TTK) tenen en comú la presència d'un domini N-terminal de tipus POZ/BTB, un motiu estructural altament conservat present en factors de transcripció, proteïnes «kelch» i proteïnes de poxvirus que media interaccions proteïna-proteïna específiques. En algunes proteïnes, el domini POZ/BTB s'ha observat que participa en la formació d'homooligòmers i, basant-se en experiments de co-expressió in vitro, s'ha proposat que els dominis POZ/BTB també poden formar heterooligòmers. A més a més, en alguns factors de transcripció, el domini POZ/BTB s'ha demostrat que està involucrat en interaccions amb varis co-repressors i desacetilases d'histones. A la primera part d'aquest treball s'ha pogut demostrar que les isoformes GAGA519 i TTK69 interaccionen tant in vitro com in vivo, en llevats i en cèl·lules de Drosophila Schneider SL2, i que els seus dominis POZ/BTB són necessaris i suficients perquè aquesta interacció tingui lloc. Com molts promotors de Drosophila, la regió reguladora even-skipped (eve) stripe 2 conté alguns llocs d'unió per GAGA i TTK els quals estan relativament propers, suggerint que la interacció GAGA-TTK descrita podria participar en la seva regulació funcional. GAGA i TTK semblaria que tenen efectes oposats en la transcripció: l'expressió de molts gens de Drosophila està regulada positivament per GAGA i a més s'ha demostrat que GAGA actua com un activador transcripcional en condicions in vitro. Per altra banda, durant el desenvolupament embrionari primerenc, TTK actua com un repressor transcripcional d'alguns gens pair-rule, incloent-hi eve. Mitjançant experiments de transfecció transitòria en cèl·lules SL2 hem pogut demostrar que GAGA activa la transcripció del promotor eve stripe 2 i que TTK inhibeix aquesta activació mediada per GAGA. La repressió per TTK del promotor eve requereix de l'activació per GAGA i depèn de la presència d'ambdós dominis POZ de TTK i GAGA, suggerint que aquesta repressió és una conseqüència de la interacció GAGA-TTK. Interessantment, el domini POZ de TTK és capaç de reprimir per sí sol l'activació mediada per GAGA, mentre que la regió C-terminal de TTK, que conté la seqüència consens P-DLS d'unió al co-repressor dCtBP, no és necessària per una repressió eficient. Consistent amb això, la substitució del domini POZ de GAGA pel domini POZ de TTK aboleix per complet la capacitat transactivadora de GAGA i converteix la proteïna de fusió POZTTKDPOZGAGA en un repressor de l'activació de GAGA, un resultat que dóna suport a la consideració del domini POZ de TTK com un motiu repressor. Com els dominis POZ dels factors de transcripció PLZF i Bcl-6 reprimeixen la transcripció per un reclutament de desacetilases, es va testar aquesta possibilitat pel domini POZ de TTK. Per aquesta raó, es varen realitzar assajos transcripcionals a cèl·lules SL2 en presència de TSA, un inhibidor de les desacetilases de classe I i II. Els resultats obtinguts indiquen que la repressió de TTK sobre l'activació mediada per GAGA no involucra un reclutament de desacetilases al promotor, ja que la presència de TSA no té cap efecte sobre l'activitat de TTK. No obstant, no es pot descartar la possibilitat que la repressió per TTK pogués estar mediada per desacetilases insensibles a TSA. Utilitzant delecions del promotor eve stripe 2, s'ha observat que la repressió per TTK pot tenir lloc en absència de llocs d'unió per TTK al promotor, indicant que aquesta repressió no requereix unió directa de TTK al DNA. Amb aquesta observació ens vàrem plantejar si TTK pot interferir en la unió de GAGA al DNA, per la qual cosa vàrem realitzar assajos in vitro EMSA i de footprinting amb DNasa I utilitzant un fragment eve sense llocs d'unió per TTK emprant proteïnes recombinants GAGA i TTK. Els resultats obtinguts mostren que TTK no interfereix en la unió de GAGA al DNA.; contràriament, els complexes GAGA-TTK semblen tenir una major afinitat d'unió al DNA. En resum, aquestes observacions suggereixen un model en el qual la repressió per TTK de l'activació mediada per GAGA implica una interacció directa GAGA-TTK la qual facilita el reclutament de TTK al promotor. No obstant, els nostres resultats no permeten diferenciar si la repressió per TTK és conseqüència d'una interferència en l'activació mediada per GAGA o a un reclutament actiu de co-repressors.The Drosophila transcription factors GAGA and TTK share in common the presence of a N-terminal POZ/BTB-domain, a highly conserved structural motif present in transcription factors, kelch proteins and poxviruses proteins that mediates specific protein-protein interactions. In several proteins, the POZ/BTB-domain has been found to participate in the formation of homo-oligomers and, based on in vitro co-expression experiments, it has been proposed that POZ/BTB-domains can also form hetero-oligomers. In addition, in several transcription factors, the POZ/BTB-domain has been shown to be involved in interactions with various co-repressor proteins and HDACs. In the first part of this work, we have demonstrated that GAGA519 and TTK69 isoforms interact in vitro as well as in vivo, both in yeast and Schneider S2 cells, and that their POZ/BTB-domains are necessary and sufficient for this interaction to occur. As many Drosophila promoters, the even-skipped (eve) stripe 2 regulatory region contains several binding sites for GAGA and TTK which are in relatively close proximity, suggesting that the GAGA-TTK interaction described might participate in its functional regulation. GAGA and TTK appear to have opposite effects on transcription: the expression of many genes in Drosophila is positively regulated by GAGA and it has been shown that GAGA acts as a transcription activator in vitro. On the other hand, during early embryo development, TTK was shown to function as a transcription repressor of several pair-rule genes, including eve. Based on transient expression experiments in SL2 cells we have shown that GAGA activates transcription from the eve stripe 2 promoter and that TTK inhibits this GAGA-dependent activation. Repression by TTK of the eve promoter requires its activation by GAGA and depends on the presence of the POZ/BTB-domains of TTK and GAGA, suggesting that this repression is a consequence of GAGA-TTK interaction. Interestingly, the POZ domain of TTK appears to be able to repress GAGA-mediated activation, while the C-terminal region of TTK, that contains the P-DLS consensus sequence for dCtBP corepressor binding, is not necessary for an efficient repression. Consistent with this, the substitution of the POZ domain of GAGA by the POZ domain of TTK fully abolishes the transactivation activity of GAGA and converts the fusion protein POZTTKDPOZGAGA in a repressor of GAGA-activation. This result argues in favour of the repression activity of the POZ domain of TTK. As the POZ domains of PLZF and Bcl-6 transcription factors repress transcription by recruiting HDACs to the promoter, we tested this possibility for the POZ domain of TTK. For this reason, we have performed transfection assays in the presence of TSA, an inhibitor of HDACs class I and II. The results obtained indicated that TTK repression of GAGA-mediated activation doesn't involve recruitment of HDACs to the promoter, since the presence of TSA hasn't any effect in TTK activity. However, we can't discard the possibility that TTK repression should be mediated by HDACs insensitive to TSA. Using deletions of the eve stripe 2 promoter, we have observed that TTK repression could take place in the absence of TTK-binding sites in the promoter, indicating that TTK repression doesn't require direct binding of TTK to DNA. Then, we asked if TTK could interfere in GAGA-binding to DNA, so we performed in vitro EMSA and DNase I footprinting assays using an eve fragment without TTK binding sites and recombinant GAGA and TTK. The results obtained have shown that TTK doesn't interfere in the binding of GAGA to DNA.; on the contrary, GAGA-TTK complexes appear to have a higher affinity to bind DNA. In summary, these observations suggest a model in which TTK repression of GAGA-mediated activation involves a direct interaction of GAGA and TTK that facilitates TTK recruitment to the promoter. However, our results don't allow to differentiate if TTK repression is a consequence of an interference of GAGA-dependent activation or an active recruitment of co-repressors

Identification of N-terminal protein acetylation and arginine methylation of the voltage-gated sodium channel in end-stage heart failure human heart

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

Do sodium channel proteolytic fragments regulate sodium channel expression?

© 2017 Taylor & Francis The cardiac voltage-gated sodium channel (gene: SCN5A, protein: Na V 1.5) 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 (L DI-DII ) of Na V 1.5 contains a nuclear localization signal (residues 474–481) that is necessary to localize L DI-DII into the nucleus, and 2) nuclear L DI-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 Na V 1.5 degradation is signaled to the cell transcriptional machinery via nuclear localization of L DI-DII and subsequent stimulation of the SCN5A promoter

Transcriptional regulation of the sodium channel gene (SCN5A) by GATA4 in human heart

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 freshfrozen 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

The HIV-1 Tat Protein is Monomethylated at Lysine 71 by the Lysine Methyltransferase KMT7

The HIV-1 transactivator protein Tat is a critical regulator of HIV transcription primarily enabling efficient elongation of viral transcripts. Its interactions with RNA and various host factors are regulated by ordered, transient post-translational modifications. Here, we report a novel Tat modification, monomethylation at lysine 71 (K71). We found that Lys-71 monomethylation (K71me) is catalyzed by KMT7, a methyltransferase that also targets lysine 51 (K51) in Tat. Using mass spectrometry, in vitro enzymology, and modification-specific antibodies, we found that KMT7 monomethylates both Lys-71 and Lys-51 in Tat. K71me is important for full Tat transactivation, as KMT7 knockdown impaired the transcriptional activity of wild type (WT) Tat but not a Tat K71R mutant. These findings underscore the role of KMT7 as an important monomethyltransferase regulating HIV transcription through Tat

Community-acquired methicillin-resistant Staphylococcus aureus: what do we need to know?

AbstractCommunity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a matter of concern worldwide, in particular in the USA. For the analysis of emergence and spread, clear definitions based on epidemiological origin are needed for discrimination between CA-MRSA, healthcare-associated community MRSA, and healthcare-associated MRSA (HA-MRSA). Although its role in pathogenesis is currently under debate, the capability for Panton–Valentine leukocidin formation is associated with the majority of CA-MRSA isolates from North America and from Europe. Most CA-MRSA isolates are attributed to clonal lineages different from HA-MRSA; there are, however, clonal lineages from which both HA-MRSA and CA-MRSA have been reported (e.g. ST1, ST5, ST8, and ST22); CA-MRSA ST8 (USA300), which is most frequent in the USA, has meanwhile been reported from Europe. CA-MRSA ST80 is widely disseminated in Europe; because of its pronounced oxacillin heteroresistance phenotype, cefoxitin-based assays are advisable for reliable detection. So far, CA-MRSA infections seem to be much less frequent in Europe than in the USA, where patients with particular predispositions and low social status are at especial risk

Large Genomic Imbalances in Brugada Syndrome

Purpose Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. Methods 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). Results The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. Conclusion CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes

SIRT1 Regulates HIV Transcription via Tat Deacetylation

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation

Famílies botàniques de plantes medicinals

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica