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1.
Sci Rep ; 10(1): 6878, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327677

RESUMO

Alterations in connexins and specifically in 43 isoform (Cx43) in the heart have been associated with a high incidence of arrhythmogenesis and sudden death in several cardiac diseases. We propose to determine salutary effect of Cx43 mimetic peptide Gap27 in the progression of heart failure. High-output heart failure was induced by volume overload using the arterio-venous fistula model (AV-Shunt) in adult male rats. Four weeks after AV-Shunt surgery, the Cx43 mimetic peptide Gap27 or scrambled peptide, were administered via osmotic minipumps (AV-ShuntGap27 or AV-ShuntScr) for 4 weeks. Cardiac volumes, arrhythmias, function and remodeling were determined at 8 weeks after AV-Shunt surgeries. At 8th week, AV-ShuntGap27 showed a marked decrease in the progression of cardiac deterioration and showed a significant improvement in cardiac functions measured by intraventricular pressure-volume loops. Furthermore, AV-ShuntGap27 showed less cardiac arrhythmogenesis and cardiac hypertrophy index compared to AV-ShuntScr. Gap27 treatment results in no change Cx43 expression in the heart of AV-Shunt rats. Our results strongly suggest that Cx43 play a pivotal role in the progression of cardiac dysfunction and arrhythmogenesis in high-output heart failure; furthermore, support the use of Cx43 mimetic peptide Gap27 as an effective therapeutic tool to reduce the progression of cardiac dysfunction in high-output heart failure.


Assuntos
Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Conexina 43/química , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Peptídeos/uso terapêutico , Remodelação Ventricular/efeitos dos fármacos , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/diagnóstico por imagem , Derivação Arteriovenosa Cirúrgica , Cardiomegalia/complicações , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/fisiopatologia , Fibrose , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/diagnóstico por imagem , Ventrículos do Coração/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Masculino , Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacos
2.
Mater Sci Eng C Mater Biol Appl ; 108: 110191, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923988

RESUMO

Effective therapeutic delivery of peptide and protein drugs is challenged by short in vivo half-lives due to rapid degradation. Sustained release formulations of αCT1, a 25 amino acid peptide drug, would afford lower dosing frequency in indications that require long term treatment, such as chronic wounds and cancers. In this study, rhodamine B (RhB) was used as a model drug to develop and optimize a double emulsion-solvent evaporation method of poly(lactic-co-glycolic acid) (PLGA) nanoparticle synthesis. Encapsulation of αCT1 in these nanoparticles (NPs) resulted in a sustained in vitro release profile over three weeks, characterized by an initial burst release of approximately 50% of total encapsulated drug over the first three days followed by sustained release over the remaining two and a half weeks. NP uptake by glioblastoma stem cells was through endocytosis and RhB and αCT1 were observed in cells after at least 4 days.


Assuntos
Materiais Biomiméticos , Conexina 43 , Glioblastoma , Nanopartículas , Peptídeos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Nanopartículas/química , Nanopartículas/uso terapêutico , Peptídeos/química , Peptídeos/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia
3.
J Biol Chem ; 294(45): 16789-16803, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31554662

RESUMO

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Fenômenos Eletrofisiológicos , Simulação de Dinâmica Molecular , Permeabilidade , Porosidade , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Especificidade por Substrato
4.
Int J Oncol ; 55(4): 845-859, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485592

RESUMO

Reports have highlighted an association between connexins (CXs) or gap junction proteins and non­small cell lung cancer (NSCLC). In the present study, it was aimed to elucidate the regulatory mechanism of CX26 and CX43 under hypoxic conditions in NSCLC. Clinical samples were collected for analysis of CX26 and CX43 expression and clinical cancerization followed by quantification of CX26 and CX43 expression. Following the establishment of an in vitro hypoxia model, P53/murine double minute­2 (MDM2) signaling pathway­, proliferation­ and epithelial­mesenchymal transition (EMT)­related genes were quantified to evaluate the influence of CX26 and CX43 on the biological functions of pulmonary epithelial cells in NSCLC. In addition, the proliferation and tumorigenicity of cancer cells were assessed by EdU staining and xenograft tumors, respectively. Decreased expression of CX26 and CX43 was found in cancer tissues compared with surrounding normal tissue. Hypoxia was shown to activate the P53/MDM2 axis and stimulate the downregulation, ubiquitination and degradation of CX26 and CX43, which were translocated from the membrane to the cytoplasm. Low levels of CX26 and CX43 were demonstrated to further promote EMT and the induction of the proliferation and tumorigenicity of cancer cells. These results were reflected by decreased E­cadherin expression and increased N­cadherin expression, along with increased cell migration, promoted cell proliferation ability and elevated relative protein expression of Oct4 and Nanog, and accelerated tumor growth, accompanied by a higher number of metastatic nodes. Taken together, the key observations of the present study demonstrate that the internalization of CX26 and CX43 promoted proliferation, EMT and migration and thus induced NSCLC via aberrant activation of the P53/MDM2 signaling pathway under hypoxic conditions.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Conexina 43/metabolismo , Conexinas/metabolismo , Células Epiteliais/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Hipóxia Celular , Proliferação de Células , Conexina 43/química , Conexina 43/genética , Conexinas/química , Conexinas/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação
5.
Acta Biomater ; 96: 517-536, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31284098

RESUMO

Exosomes are naturally secreted nanovesicles that have emerged as a promising therapeutic nanodelivery platform, due to their specific composition and biological properties. However, challenges like considerable complexity, low isolation yield, drug payload, and potential safety concerns substantially reduce their pharmaceutical acceptability. Given that the nano-bio-interface is a crucial factor for nanocarrier behavior and function, modification of synthetic nanoparticles with the intrinsic hallmarks of exosomes' membrane to create exosome mimetics could allow for siRNA delivery in a safer and more efficient manner. Herein, connexin 43 (Cx43)-embedded, exosome-mimicking lipid bilayers coated chitosan nanoparticles (Cx43/L/CS NPs) were constructed by using cell-free (CF) synthesis systems with plasmids encoding Cx43 in the presence of lipid-coated CS NPs (L/CS NPs). The integration of de novo synthesized Cx43 into the lipid bilayers of L/CS NPs occurred cotranslationally during one-pot reaction and, more importantly, the integrated Cx43 was functionally active in transport. In addition to considerably lower cytotoxicity (

Assuntos
Materiais Biomiméticos , Conexina 43 , Sistemas de Liberação de Medicamentos , Exossomos/química , Nanopartículas/química , RNA Interferente Pequeno , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacocinética , Materiais Biomiméticos/farmacologia , Sistema Livre de Células , Conexina 43/biossíntese , Conexina 43/química , Conexina 43/farmacocinética , Conexina 43/farmacologia , Células HEK293 , Humanos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/farmacologia
6.
Biochem Biophys Res Commun ; 513(3): 567-572, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30981509

RESUMO

Connexin 43 (Cx43) phosphorylation plays a pivotal role in cardiac electrical and contractile performance. In a previous study we have found that Cx43 phosphorylation at serine 282 (pS282) regulates cardiomyocyte survival. Considering that both sites are altered simultaneously in many studies, we designed this study to identify the status of S279 phosphorylation upon pS282 manipulation. In heterozygous mice with S282 gene substituted with alanine (S282A), we found ventricular arrhythmias with inhibition of Cx43 phosphorylation at both S282 and S279 in the hearts. In cultured neonatal rat ventricular myocytes (NRVMs), transfection of virus carrying S282A mutant also blocked Cx43 phosphorylation at both S279/282 and gap junction coupling, while expression of wild-type Cx43 or S279A did not. Further, NRVMs transfected with S282 phospho-mimicking mutant substituted with aspartate or treated with ATP exhibited promotions of Cx43 phosphorylation at S279/282 and intercellular communication. Therefore, this study demonstrated a regulatory role of Cx43-S282 on S279 phosphorylation in cardiomyocytes, and suggested an involvement of S279 in the Cx43-S282 mediated cardiomyocyte homeostasis.


Assuntos
Conexina 43/metabolismo , Miócitos Cardíacos/metabolismo , Serina/metabolismo , Animais , Comunicação Celular , Células Cultivadas , Conexina 43/química , Conexina 43/genética , Doxorrubicina/farmacologia , Junções Comunicantes/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley
7.
Biochem J ; 476(7): 1137-1148, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30910801

RESUMO

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca2+ has been questioned for decades. To assess the ability of Ca2+ ions to modulate Cx43 junctional conductance (g j) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full-length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminus (CT) truncated version of Cx43 lacking pH-sensitivity. The addition of 1 µM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 g j to zero within 15 min of perfusion. This response was prevented by Ca2+-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca2+/ionomycin-induced decrease in Cx43 g j, while 100 µM Gap19 peptide had minimal effect. The investigation of the transjunctional voltage (V j) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 g j, but also noted the abolishment of the previously reported facilitated recovery of g j from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the V j-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca2+/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca2+/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Substituição de Aminoácidos , Animais , Sinalização do Cálcio , Linhagem Celular , Conexina 43/química , Conexina 43/genética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ativação do Canal Iônico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/metabolismo , Domínios Proteicos
8.
Cancer Lett ; 442: 439-444, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30472182

RESUMO

Gap junctions are membrane channels found in all cells of the human body that are essential to cellular physiology. Gap junctions are formed from connexin proteins and are responsible for transfer of biologically active molecules, metabolites, and salts between neighboring cells or cells and their extracellular environment. Over the last few years, aberrant connexin 43 (Cx43) expression has been associated with cancer recurrence, metastatic spread, and poor survival. Here we provide an overview of the general structure and function of gap junctions and review their roles in different cancer types. We discuss new therapeutic approaches targeting Cx43 and potential new ways of exploiting gap junction transfer for drug delivery and anti-cancer treatment. The permeability of Cx43 channels to small molecules and macromolecules makes them highly attractive targets for delivering drugs directly into the cytoplasm. Cancer cells overexpressing Cx43 may be more permeable and sensitive to chemotherapeutics. Because Cx43 can either act as a tumor suppressor or oncogene, biomarker analysis and a better understanding of how Cx43 contextually mediates cancer phenotypes will be required to develop clinically viable Cx43-based therapies.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Neoplasias/metabolismo , Animais , Antineoplásicos/administração & dosagem , Comunicação Celular , Conexina 43/antagonistas & inibidores , Conexina 43/química , Conexina 43/genética , Sistemas de Liberação de Medicamentos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/genética , Junções Comunicantes/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Permeabilidade , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
9.
J Mol Cell Cardiol ; 126: 36-49, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30448479

RESUMO

Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Neuropeptídeos/metabolismo , Fosfotirosina/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos , Conexina 43/química , Células HeLa , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Fosforilação , Ligação Proteica , Ratos
10.
Int J Mol Sci ; 20(1)2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30583492

RESUMO

It is becoming clear that in addition to gap junctions playing a role in cell⁻cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures-annular gap junctions-were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation.


Assuntos
Vesículas Citoplasmáticas/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias/metabolismo , Imagem Óptica , Comunicação Celular , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Vesículas Citoplasmáticas/química , Junções Comunicantes/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Imuno-Histoquímica , Citometria de Varredura a Laser , Lisossomos/química , Lisossomos/metabolismo , Mitocôndrias/química
11.
Curr Protein Pept Sci ; 19(11): 1049-1057, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29984651

RESUMO

Connexins are a family of gap junction proteins widely distributed in human organs and tissues. Gap junctions are organized systems of intercellular protein channels that allow the exchange of ions, chemical signals, and energy substrates between two adjacent cells. Connexin43 (Cx43) is the most abundant isoform of connexins in the heart which play an important role in myocardium disease. Numerous studies have shown that Cx43 was involved in tumor migration and invasion by mediating gap junctions between tumor cells and normal cells. Changes in the expression and distribution of Cx43 contribute to heart disease and cancer. This review discusses current knowledge on the functional and structural abnormalities in Cx43 associated with heart disease and cancer, aiming to highlight the importance of this connexin as an emerging therapeutic target. Here, the current knowledge on the pharmacology of Gap Junction Channels and Hemichannels were also summarized. Finally, we propose that these knowledges can be exploited to identify new diagnostic and effective therapeutic approaches for ischemic heart disease and cancer.


Assuntos
Conexina 43/metabolismo , Cardiopatias/metabolismo , Neoplasias/metabolismo , Animais , Biomarcadores Farmacológicos/metabolismo , Conexina 43/química , Junções Comunicantes/metabolismo , Humanos , Conformação Proteica
12.
J Cell Sci ; 131(15)2018 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-30054380

RESUMO

Gap junctions (GJs) assembled from connexin (Cx) proteins allow direct cell-cell communication. While phosphorylation is known to regulate multiple GJ functions, much less is known about the role of ubiquitin in these processes. Using ubiquitylation-type-specific antibodies and Cx43 lysine-to-arginine mutants we show that ∼8% of a GJ, localized in central plaque domains, is K63-polyubiquitylated on K264 and K303. Levels and localization of ubiquitylation correlated well with: (1) the short turnover rate of Cxs and GJs; (2) removal of older channels from the plaque center; and (3) the fact that not all Cxs in an internalizing GJ channel need to be ubiquitylated. Connexins mutated at these two sites assembled significantly larger GJs, exhibited much longer protein half-lives and were internalization impaired. Interestingly, these ubiquitin-deficient Cx43 mutants accumulated as hyper-phosphorylated polypeptides in the plasma membrane, suggesting that K63-polyubiquitylation is triggered by phosphorylation. Phospho-specific anti-Cx43 antibodies revealed that upregulated phosphorylation affected serines 368, 279/282 and 255, which are well-known regulatory PKC and MAPK sites. Together, these novel findings suggest that the internalizing portion of channels in a GJ is K63-polyubiquitylated, ubiquitylation is critical for GJ internalization and that phosphorylation induces Cx K63-polyubiquitylation.This article has an associated First Person interview with the first author of the paper.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Animais , Anticorpos , Arginina/química , Arginina/genética , Membrana Celular/metabolismo , Conexina 43/genética , Cães , Endocitose/genética , Endocitose/fisiologia , Células HeLa , Humanos , Lisina/química , Lisina/genética , Células Madin Darby de Rim Canino , Peso Molecular , Fosforilação/genética , Fosforilação/fisiologia , Ubiquitinação/genética , Ubiquitinação/fisiologia
13.
Int J Mol Sci ; 19(6)2018 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-29867029

RESUMO

Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes' lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels' function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.


Assuntos
Conexina 43/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Animais , Arritmias Cardíacas/metabolismo , Caseína Quinase I/metabolismo , Linhagem Celular Tumoral , Conexina 43/química , Conexina 43/genética , Conexina 43/fisiologia , Mutação , Fosforilação , Ratos
14.
Int J Mol Sci ; 19(6)2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29914066

RESUMO

The most ubiquitous gap junction protein within the body, connexin 43 (Cx43), is a target of interest for modulating the dermal wound healing response. Observational studies found associations between Cx43 at the wound edge and poor healing response, and subsequent studies utilizing local knockdown of Cx43 found improvements in wound closure rate and final scar appearance. Further preclinical work conducted using Cx43-based peptide therapeutics, including alpha connexin carboxyl terminus 1 (αCT1), a peptide mimetic of the Cx43 carboxyl terminus, reported similar improvements in wound healing and scar formation. Clinical trials and further study into the mode of action have since been conducted on αCT1, and Phase III testing for treatment of diabetic foot ulcers is currently underway. Therapeutics targeting connexin activity show promise in beneficially modulating the human body's natural healing response for improved patient outcomes across a variety of injuries.


Assuntos
Cicatriz/metabolismo , Conexina 43/metabolismo , Pé Diabético/tratamento farmacológico , Pele/metabolismo , Animais , Cicatriz/tratamento farmacológico , Conexina 43/química , Conexina 43/genética , Pé Diabético/metabolismo , Humanos , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Pele/efeitos dos fármacos
15.
Int J Mol Sci ; 19(6)2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29882937

RESUMO

Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of ß-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, ß-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether ß-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct protein⁻protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that ß-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ratos , Ressonância de Plasmônio de Superfície , beta Catenina/química
16.
Int J Mol Sci ; 19(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748463

RESUMO

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.


Assuntos
Conexina 43/genética , Citoesqueleto/genética , Junções Comunicantes/genética , Mapas de Interação de Proteínas/genética , Fenômenos Biofísicos , Comunicação Celular/genética , Conexina 43/química , Citoesqueleto/química , Junções Comunicantes/química , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética
17.
Biomaterials ; 168: 10-23, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29597134

RESUMO

Recent studies have shown that Connexin43 mimetic peptide (Cx43 MP) can prevent secondary damage following retinal ischaemic and inflammatory disorders by blocking uncontrolled Cx43 hemichannel opening. However, limitations in peptide stability and the presence of various intraocular barriers limit efficient retinal delivery in the clinical setting. The present study aimed to achieve targeted and sustained peptide delivery to the retina by encapsulating Cx43 MP into hyaluronic acid (HA) coated albumin nanoparticles (NPs). Intraocular biodistribution, particle retention, retinal targeting, and therapeutic efficacy of intravitreally injected NPs encapsulating Cx43 MP were evaluated in a rat model of retinal ischaemia-reperfusion injury. NPs rapidly diffused through the vitreous and specifically targeted CD44-expressing retinal cells. NPs remained at the target site for extended periods enabling sustained peptide release and thus prolonged therapeutic action. Compared to free Cx43 MP, Cx43 MP loaded NPs enabled enhanced therapeutic efficacy preventing thinning of retinal layers and disruption of retinal blood vessels. Immunohistochemical results confirm that Cx43 MP loaded NPs efficiently reduced Cx43 expression, thereby suppressing ongoing inflammation and preventing the loss of retinal ganglion cells. Overall, HA coated NPs could have great potential as a peptide delivery platform in the treatment of chronic retinal degenerative and inflammatory disorders.


Assuntos
Albuminas , Portadores de Fármacos , Ácido Hialurônico , Nanopartículas , Peptídeos/uso terapêutico , Doenças Retinianas/tratamento farmacológico , Animais , Linhagem Celular , Conexina 43/química , Conexina 43/metabolismo , Conexina 43/uso terapêutico , Isquemia/tratamento farmacológico , Masculino , Ratos , Ratos Wistar , Doenças Retinianas/metabolismo
18.
Biochem Biophys Res Commun ; 497(2): 734-741, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29462621

RESUMO

Propofol is widely used as a general anesthetic and is generally considered to exert its action by regulating neuronal firing via facilitation of GABAA receptors. However, accumulating evidence suggests that propofol also acts on astrocytes, including inhibitory effects on gap junctional coupling, but the underlying molecular mechanisms remain largely unknown. Here, using acute cortical brain slices prepared from mice, we characterize propofol-induced molecular changes in astrocytic gap junction protein connexin 43 (Cx43). Propofol does not change the protein expression level of Cx43 or its incorporation into gap junctional plaques, according to biochemical and immunohistochemical analyses. However, propofol alters migration pattern of Cx43 on western blot, suggesting changes in its posttranslational modifications. Indeed, this change is accompanied by an increase in the phosphorylation of Cx43 at serine 368, which is known to reduce permeability of Cx43 gap junctions. Finally, we show that this change occurs in the absence of neuronal firing or glutamatergic transmissions. Overall, these results show that propofol induces posttranslational modification of Cx43 directly on astrocytes at the site of gap junctional plaques, exerting direct pharmacological action on astrocytes in parallel with its action on neurons.


Assuntos
Anestésicos Gerais/farmacologia , Astrócitos/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Conexina 43/metabolismo , Propofol/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Conexina 43/química , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos
19.
Sci Rep ; 8(1): 166, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29317708

RESUMO

Cytoplasmic membrane-bound connexin 43 (Cx43) proteins oligomerize into hexameric channels (hemichannels) that can sometimes dock with hemichannels on adjacent cells to form gap junctional (GJ) channels. However, the possible role of Cx43 hemichannels in sterile and infectious inflammatory diseases has not been adequately defined due to the lack of selective interventions. Here we report that a proinflammatory mediator, the serum amyloid A (SAA), resembled bacterial endotoxin by stimulating macrophages to up-regulate Cx43 expression and double-stranded RNA-activated protein kinase R (PKR) phosphorylation in a TLR4-dependent fashion. Two well-known Cx43 mimetic peptides, the GAP26 and TAT-GAP19, divergently affected macrophage hemichannel activities in vitro, and differentially altered the outcome of lethal sepsis in vivo. By screening a panel of Cx43 mimetic peptides, we discovered that one cysteine-containing peptide, P5 (ENVCYD), effectively attenuated hemichannel activities, and significantly suppressed endotoxin-induced release of ATP and HMGB1 in vitro. In vivo, the P5 peptide conferred a significant protection against hepatic ischemia/reperfusion injury and lethal microbial infection. Collectively, these findings have suggested a pathogenic role of Cx43 hemichannels in sterile injurious as well as infectious inflammatory diseases possibly through facilitating extracellular ATP efflux to trigger PKR phosphorylation/activation.


Assuntos
Conexina 43/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Conexina 43/antagonistas & inibidores , Conexina 43/química , Endotoxinas/metabolismo , Humanos , Inflamação/etiologia , Inflamação/mortalidade , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peptídeos/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Sepse/etiologia , Sepse/metabolismo , Sepse/mortalidade , eIF-2 Quinase/metabolismo
20.
Biochim Biophys Acta Biomembr ; 1860(1): 48-64, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28526583

RESUMO

Connexins are chordate gap junction channel proteins that, by enabling direct communication between the cytosols of adjacent cells, create a unique cell signalling network. Gap junctional intercellular communication (GJIC) has important roles in controlling cell growth and differentiation and in tissue development and homeostasis. Moreover, several non-canonical connexin functions unrelated to GJIC have been discovered. Of the 21 members of the human connexin family, connexin 43 (Cx43) is the most widely expressed and studied. The long cytosolic C-terminus (CT) of Cx43 is subject to extensive post-translational modifications that modulate its intracellular trafficking and gap junction channel gating. Moreover, the Cx43 CT contains multiple domains involved in protein interactions that permit crosstalk between Cx43 and cytoskeletal and regulatory proteins. These domains endow Cx43 with the capacity to affect cell growth and differentiation independently of GJIC. Here, we review the current understanding of the regulation and unique functions of the Cx43 CT, both as an essential component of full-length Cx43 and as an independent signalling hub. We highlight the complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. We discuss these data in relation to the recent discovery of the direct translation of specific truncated forms of Cx43. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.


Assuntos
Conexina 43 , Junções Comunicantes , Ativação do Canal Iônico , Canais Iônicos , Processamento de Proteína Pós-Traducional , Animais , Conexina 43/química , Conexina 43/genética , Conexina 43/metabolismo , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Junções Comunicantes/química , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Domínios Proteicos , Transporte Proteico , Relação Estrutura-Atividade
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