Steve Catarino

Eps15 interacts with ubiquitinated Cx43 and mediates its internalization.

Gap junctions (GJ) are specialized cell-cell contacts that provide direct intercellular communication (IC) between eukaryotic cells. Regulation of GJIC by degradation of Cx43 has been a matter of debate over the last two decades and both the proteasome and the lysosome have been implicated. However, the underlying mechanism and molecular players involved remain elusive. In this paper we demonstrate, for the first time, that the ubiquitin ligase Nedd4 is involved in Cx43 ubiquitination. Indeed, depletion of Nedd4 with siRNA resulted in a decrease of the amount of ubiquitin attached to Cx43. Ubiquitinated membrane proteins are often recognized and targeted by endocytic adaptors containing ubiquitin-binding domains, such as Eps15. By coimmunoprecipitation and immunofluorescence we show interaction of Cx43 with Eps15 and colocalization of these proteins mainly at the plasma membrane. Moreover, depletion of Eps15 results in an accumulation of Cx43 at the plasma membrane. Furthermore, the interaction of Eps15 with Cx43 requires the ubiquitin-interacting motif of Eps15 suggesting that the interaction occurs through the ubiquitin attached to Cx43. Data presented in this manuscript are consistent with a new molecular model in which Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15, through its ubiquitin-interacting motif, and targets ubiquitinated Cx43 to the endocytic pathway. This provides the basis for future studies aiming at identifying the molecular players and mechanisms involved in Cx43 internalization and degradation.

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal

Gap junctions are specialized cell-cell contacts that provide direct intercellular communication between eukaryotic cells. The tyrosine-sorting signal (YXXØ), present at amino acids 286-289 of Cx43 (connexin43), has been implicated in the internalization of the protein. In recent years, ubiquitination of Cx43 has also been proposed to regulate gap junction intercellular communication; however, the underlying mechanism and molecular players involved remain elusive. In the present study, we demonstrate that ubiquitinated Cx43 is internalized through a mechanism that is independent of the YXXØ signal. Indeed, expression of a Cx43-Ub (ubiquitin) chimaera was shown to drive the internalization of a mutant Cx43 in which the YXXØ motif was eliminated. Immunofluorescence, cycloheximide-chase and cell-surface-protein biotinylation experiments demonstrate that oligomerization of Cx43-Ub into hemichannels containing wild-type Cx43 or mutant Cx43Y286A is sufficient to drive the internalization of the protein. Furthermore, the internalization of Cx43 induced by Cx43-Ub was shown to depend on its interaction with epidermal growth factor receptor substrate 15.

AMSH-mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions

Gap junctions (GJs) are specialized cell‐cell contacts formed by connexins (Cxs), which provide direct intercellular communication between eukaryotic cells. Although Cx43 has long been known to be a substrate for ubiquitination, the reversal of this modification by deubiquitylases (DUBs) has never been described. Here we report that the DUB‐associated molecule with the SH3 domain of STAM (AMSH) interacts with Cx43 and mediates its deubiquitination. In this study, we demonstrate that Cx43 is modified with lysine 63‐linked polyubiquitin chains and that these increase the interaction between Cx43 and AMSH. We also show that AMSH is recruited to GJ plaque sites at the plasma membrane, where it mediates the deubiquitination of Cx43. Using siRNA depletion or overexpression of a catalytically inactive mutant of AMSH, we show that by decreasing Cx43 deubiquitination, both the internalization and degradation rate of Cx43 are increased. Overall, these data strongly suggest that AMSH‐mediated deubiquitination of Cx43 protects GJs from degradation.—Ribeiro‐Rodrigues, T. M., Catarino, S., Marques, C., Ferreira, J. V., Martins‐Marques, T., Pereira, P., Girão, H., AMSH‐mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions. FASEB J. 28, 4629–4641 (2014). www.fasebj.org

7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

BackgroundConnexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC).ResultsPrimary cultures of lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH) or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake.ConclusionsResults demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication, that is most likely due to an increased stability of protein at the plasma membrane and to increased abundance of Cx43 assembled in gap junction plaques.

Molecular control of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective form of autophagy in which cytosolic proteins bearing a pentapeptide motif biochemically related to the KFERQ sequence, are recognized by the heat shock protein family A member 8 (HSPA8) chaperone, delivered to the lysomal membrane, and directly translocated across the lysosomal membrane by a protein complex containing lysosomal associated membrane protein 2a (Lamp2a). Since its discovery over two decades ago, the importance of this pathway in cell proteostasis has been made increasingly apparent. Deregulation of this pathway has been implicated in a variety of diseases and conditions, including lysosomal storage diseases, cancer, neurodegeneration and even aging. Here, we describe the main molecular features of the pathway, its regulation, cross-talk with other degradation pathways and importance in disease.

To beat or not to beat: degradation of Cx43 imposes the heart rhythm

The main function of the heart is to pump blood to the different parts of the organism, a task that is efficiently accomplished through proper electric and metabolic coupling between cardiac cells, ensured by gap junctions (GJ). Cardiomyocytes are the major cell population in the heart, and as cells with low mitotic activity, are highly dependent upon mechanisms of protein degradation. In the heart, both the ubiquitin-proteasome system (UPS) and autophagy participate in the fine-tune regulation of cardiac remodelling and function, either in physiological or pathological conditions. Indeed, besides controlling cardiac signalling pathways, UPS and autophagy have been implicated in the turnover of several myocardial proteins. Degradation of Cx43, the major ventricular GJ protein, has been associated to up-regulation of autophagy at the onset of heart ischemia and ischemia/reperfusion (I/R), which can have profound implications upon cardiac function. In this review, we present recent studies devoted to the involvement of autophagy and UPS in heart homoeostasis, with a particular focus on GJ.

MicroRNA-424(322) as a new marker of disease progression in pulmonary arterial hypertension and its role in right ventricular hypertrophy by targeting SMURF1.

Aims MicroRNAs (miRNAs) have been implicated in the pathogenesis of pulmonary hypertension (PH), a multifactorial and progressive condition associated with an increased afterload of the right ventricle leading to heart failure and death. The main aim of this study was to correlate the levels of miR-424(322) with the severity and prognosis of PH and with right ventricle hypertrophy progression. Additionally, we intended to evaluate the mechanisms and signalling pathways whereby miR-424(322) secreted by pulmonary arterial endothelial cells (PAECs) impacts cardiomyocytes. Methods and results Using quantitative real-time PCR, we showed that the levels of circulating miR-424(322) are higher in PH patients when compared with healthy subjects. Moreover, we found that miR-424(322) levels correlated with more severe symptoms and haemodynamics. In the subgroup of Eisenmenger syndrome patients, miR-424(322) displayed independent prognostic value. Furthermore, we demonstrated that miR-424(322) targets SMURF1, through which it sustains bone morphogenetic protein receptor 2 signalling. Moreover, we showed that hypoxia induces the secretion of miR-424(322) by PAECs, which after being taken up by cardiomyocytes leads to down-regulation of SMURF1. In the monocrotaline rat model of PH, we found an association between circulating miR-424(322) levels and the stage of right ventricle hypertrophy, as well as an inverse correlation between miR-424(322) and SMURF1 levels in the hypertrophied right ventricle. Conclusions This study shows that miR-424(322) has diagnostic and prognostic value in PH patients, correlating with markers of disease severity. Additionally, miR-424(322) can target proteins with a direct effect on heart function, suggesting that this miRNA can act as a messenger linking pulmonary vascular disease and right ventricle hypertrophy.

Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive

Connexins (Cxs) are transmembrane proteins that form channels which allow direct intercellular communication (IC) between neighbouring cells via gap junctions. Mechanisms that modulate the amount of channels at the plasma membrane have emerged as important regulators of IC and their de-regulation has been associated with various diseases. Although Cx-mediated IC can be modulated by different mechanisms, ubiquitination has been described as one of the major post-translational modifications involved in Cx regulation and consequently IC. In this review, we focus on the role of ubiquitin and its effect on gap junction intercellular communication.

Regulation of the expression of interleukin‐8 induced by 25‐hydroxycholesterol in retinal pigment epithelium cells

Purpose:  This study aimed at elucidating the molecular mechanisms involved in the regulation of IL‐8 production by several oxysterols in retinal pigment epithelium (RPE) cells.