Inhibition of Pyk2 Improves Cx43 Intercalated Disc Localization, Infarct Size, and Cardiac Function in Rats With Heart Failure.

Background: Heart failure causes changes in Cx43 (Connexin43) regulation that are associated with arrhythmic heart disease. Pyk2 (proline-rich tyrosine kinase 2) is activated in cardiomyopathies and phosphorylates Cx43 to decrease intercellular communication. This study was designed to determine if Pyk2 inhibition improves cardiac function in a myocardial infarction (MI)-induced heart failure model in rats.

Regulation of Cx43 Gap Junction Intercellular Communication by Bruton's Tyrosine Kinase and Interleukin-2-Inducible T-Cell Kinase.

T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43.

In situ delivery of a curcumin-loaded dynamic hydrogel for the treatment of chronic peripheral neuropathy.

Patients with peripheral nerve injuries would highly likely suffer from chronic neuropathic pain even after surgical intervention. The primary reasons for this involve sustained neuroinflammatory and dysfunctional changes in the nervous system after the nerve injury. We previously reported an injectable boronic ester-based hydrogel with inherent antioxidative and nerve protective properties.

Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease.

Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313.

Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome.

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome.

Phosphorylation of Cx43 residue Y313 by Src contributes to blocking the interaction with Drebrin and disassembling gap junctions.

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.

Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase.

Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners.

Connexin43 Carboxyl-Terminal Domain Directly Interacts with β-Catenin.

Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of β-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, β-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.

Protein⁻Protein Interactions with Connexin 43: Regulation and Function.

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.

Regulation of Cx37 channel and growth-suppressive properties by phosphorylation.

Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown.

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization.

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes.

Chemical shift assignments of the connexin37 carboxyl terminal domain.

Connexin37 (Cx37) is a gap junction protein involved in cell-to-cell communication in the vasculature and other tissues. Cx37 suppresses proliferation of vascular cells involved in tissue development and repair in vivo, as well as tumor cells. Global deletion of Cx37 in mice leads to enhanced vasculogenesis in development, as well as collateralgenesis and angiogenesis in response to injury, which together support improved tissue remodeling and recovery following ischemic injury.

Connexin43 Forms Supramolecular Complexes through Non-Overlapping Binding Sites for Drebrin, Tubulin, and ZO-1.

Gap junctions are membrane specialization domains identified in most tissue types where cells abut each other. The connexin channels found in these membrane domains are conduits for direct cell-to-cell transfer of ions and molecules. Connexin43 (Cx43) is the most ubiquitous connexin, with critical roles in heart, skin, and brain.

Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2.

Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane.

Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) was the first ubiquitin protein ligase identified to interact with connexin43 (Cx43), and its suppressed expression results in accumulation of gap junction plaques at the plasma membrane. Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15 and target Cx43 to the endocytic pathway. Although the Cx43 residues that undergo ubiquitination are still unknown, in this study we address other unresolved questions pertaining to the molecular mechanisms mediating the direct interaction between Nedd4 (WW1-3 domains) and Cx43 (carboxyl terminus (CT)).

Biophysical characterization and modeling of human Ecdysoneless (ECD) protein supports a scaffolding function.

The human homolog of Drosophila ecdysoneless protein (ECD) is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse is early embryonic lethal and deletion delays G-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb.

Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain.

Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75).

Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 [connexin43 (Cx43)-interacting protein of 75 kDa], a protein involved in proteasomal degradation, as a novel Cx43-interacting protein.

Characterization of the connexin45 carboxyl-terminal domain structure and interactions with molecular partners.

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication.

Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane.

Structural order in Pannexin 1 cytoplasmic domains.

Pannexin 1 forms ion and metabolite permeable hexameric channels with abundant expression in the central nervous system and elsewhere. Although pannexin 1 does not form intercellular channels, a common channel topology and oligomerization state, as well as involvement of the intracellular carboxyl terminal (CT) domain in channel gating, is shared with connexins. In this study, we characterized the secondary structure of the mouse pannexin 1 cytoplasmic domains to complement structural studies of the transmembrane segments and compare with similar domains from connexins.