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. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

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. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.

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. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.

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. 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.

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.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.

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

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.

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. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43.

EHD3 Protein Is Required for Tubular Recycling Endosome Stabilization, and an Asparagine-Glutamic Acid Residue Pair within Its Eps15 Homology (EH) Domain Dictates Its Selective Binding to NPF Peptides.

An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively.

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)). All three WW domains display a similar three antiparallel ß-strand structure and interact with the same Cx43CT(283)PPXY(286)sequence. Although Tyr(286)is essential for the interaction, MAPK phosphorylation of the preceding serine residues (Ser(P)(279)and Ser(P)(282)) increases the binding affinity by 2-fold for the WW domains (WW2 > WW3 ≫ WW1). The structure of the WW2·Cx43CT(276-289)(Ser(P)(279), Ser(P)(282)) complex reveals that coordination of Ser(P)(282)with the end of ß-strand 3 enables Ser(P)(279)to interact with the back face of ß-strand 3 (Tyr(286)is on the front face) and loop 2, forming a horseshoe-shaped arrangement. The close sequence identity of WW2 with WW1 and WW3 residues that interact with the Cx43CT PPXY motif and Ser(P)(279)/Ser(P)(282)strongly suggests that the significantly lower binding affinity of WW1 is the result of a more rigid structure. This study presents the first structure illustrating how phosphorylation of the Cx43CT domain helps mediate the interaction with a molecular partner involved in gap junction regulation.

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. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health.

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. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function.

Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain.

Phosphorylation of the connexin43 C-terminal (Cx43CT) domain regulates gap junction intercellular communication. However, an understanding of the mechanisms by which phosphorylation exerts its effects is lacking. Here, we test the hypothesis that phosphorylation regulates Cx43 gap junction intercellular communication by mediating structural changes in the C-terminal domain. Circular dichroism and nuclear magnetic resonance were used to characterize the effects of phosphorylation on the secondary structure and backbone dynamics of soluble and membrane-tethered Cx43CT domains. Cx43CT phospho-mimetic isoforms, which have Asp substitutions at specific Ser/Tyr sites, revealed phosphorylation alters the α-helical content of the Cx43CT domain only when attached to the membrane. The changes in secondary structure are due to variations in the conformational preference and backbone flexibility of residues adjacent and distal to the site(s) of modification. In addition to the known direct effects of phosphorylation on molecular partner interactions, the data presented here suggest phosphorylation may also indirectly regulate binding affinity by altering the conformational preference of the Cx43CT domain.

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. METHODS: MI (ligation of left anterior descending artery) rats were treated with the Pyk2 inhibitor PF4618433. Hemodynamic and structural parameters were monitored in Sham (n=5), MI-vehicle (n=5), and MI-PF4618433 (n=8) groups. Heart tissues were collected after 6 weeks to assess Pyk2 and Cx43 protein level and localization. RESULTS: PF4618433 produced no observed adverse effects and inhibited ventricular Pyk2. PF4618433 reduced the MI infarct size from 34% to 17% (P=0.007). PF4618433 improved stroke volume (P=0.031) and cardiac output (P=0.009) in comparison to MI-vehicle with values similar to the Sham group. PF4618433 also led to an increase in the ejection fraction (P=0.002) and fractional shortening (P=0.006) when compared with the MI-vehicle (32% and 35% improvement, respectively) yet were lower in comparison with the Sham group. Pyk2 inhibition decreased Cx43 tyrosine phosphorylation (P=0.043) and maintained Cx43 at the intercalated disc in the distal ventricle 6 weeks post-MI. CONCLUSIONS: Unlike other attempts to decrease Cx43 remodeling after MI-induced heart failure, inhibition of Pyk2 activity maintained Cx43 at the intercalated disc. This may have aided in the reduced infarct size (acute time frame) and improved cardiac function (chronic time frame). Additionally, we provide evidence that Pyk2 is activated following MI in human left ventricle, implicating a novel potential target for therapy in patients with heart failure.

Cardiomyocyte ATP release through pannexin 1 aids in early fibroblast activation.

Fibrosis following myocardial infarction is associated with increases in arrhythmias and sudden cardiac death. Initial steps in the development of fibrosis are not clear; however, it is likely that cardiac fibroblasts play an important role. In immune cells, ATP release from pannexin 1 (Panx1) channels acts as a paracrine signal initiating activation of innate immunity. ATP has been shown in noncardiac systems to initiate fibroblast activation. Therefore, we propose that ATP release through Panx1 channels and subsequent fibroblast activation in the heart drives the development of fibrosis in the heart following myocardial infarction. We identified for the first time that Panx1 is localized within sarcolemmal membranes of canine cardiac myocytes where it directly interacts with the postsynaptic density 95/Drosophila disk large/zonula occludens-1-containing scaffolding protein synapse-associated protein 97 via its carboxyl terminal domain (amino acids 300-357). Induced ischemia rapidly increased glycosylation of Panx1, resulting in increased trafficking to the plasma membrane as well as increased interaction with synapse-associated protein 97. Cellular stress enhanced ATP release from myocyte Panx1 channels, which, in turn, causes fibroblast transformation to the activated myofibroblast phenotype via activation of the MAPK and p53 pathways, both of which are involved in the development of cardiac fibrosis. ATP release through Panx1 channels in cardiac myocytes during ischemia may be an early paracrine event leading to profibrotic responses to ischemic cardiac injury.

Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides.

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Characterization of the structure and intermolecular interactions between the connexin40 and connexin43 carboxyl-terminal and cytoplasmic loop domains.

Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the "particle-receptor" model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

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. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Amphiphilic polyanhydride-based recombinant MUC4ß-nanovaccine activates dendritic cells.

Mucin 4 (MUC4) is a high molecular weight glycoprotein that is differentially overexpressed in pancreatic cancer (PC), functionally contributes to disease progression, and correlates with poor survival. Further, due to its aberrant glycosylation and extensive splicing, MUC4 is a potential target for cancer immunotherapy. Our previous studies have demonstrated the utility of amphiphilic polyanhydride nanoparticles as a useful platform for the development of protein-based prophylactic and therapeutic vaccines. In the present study, we encapsulated purified recombinant human MUC4-beta (MUC4ß) protein in polyanhydride (20:80 CPTEG:CPH) nanoparticles (MUC4ß-nanovaccine) and evaluated its ability to activate dendritic cells and induce adaptive immunity. Immature dendritic cells when pulsed with MUC4ß-nanovaccine exhibited significant increase in the surface expressions of MHC I and MHC II and costimulatory molecules (CD80 and CD86), as well as, secretion of pro-inflammatory cytokines (IFN-γ, IL-6, and IL-12) as compared to cells exposed to MUC4ß alone or MUC4ß mixed with blank nanoparticles (MUC4ß+NP). Following immunization, as compared to the other formulations, MUC4ß-nanovaccine elicited higher IgG2b to IgG1 ratio of anti-MUC4ß-antibodies suggesting a predominantly Th1-like class switching. Thus, our findings demonstrate MUC4ß-nanovaccine as a novel platform for PC immunotherapy.

Impact of electron-electron spin interaction on electron spin relaxation of nitroxide diradicals and tetraradical in glassy solvents between 10 and 300 k.

To determine the impact of electron-electron spin-spin interactions on electron spin relaxation rates, 1/T1 and 1/Tm were measured for nitroxide monoradical, diradical, and tetraradical derivatives of 1,3-alternate calix[4]arenes, for two pegylated high-spin nitroxide diradicals, and for an azine-linked nitroxide diradical. The synthesis and characterization by SQUID (superconducting quantum interference device) magnetometry of one of the high-spin diradicals, in which nitroxides are conformationally constrained to be coplanar with the m-phenylene unit, is reported. The interspin distances ranged from about 5-9 A, and the magnitude of the exchange interaction ranged from >150 to >0.1 K. 1/T1 and 1/Tm were measured by long-pulse saturation recovery, three-pulse inversion recovery, and two-pulse echo decay at X-band (9.5 GHz) and Q-band (35 GHz). For a diradical with interspin distance about 9 A, relaxation rates were only slightly faster than for a monoradical with analogous structure. For interspin distances of about 5-6 A, relaxation rates in glassy solvents up to 300 K increased in the order monoradical < diradical < tetraradical. Modulation of electron-electron interaction enhanced relaxation via the direct, Raman, and local mode processes. The largest differences in 1/T1 were observed below 10 K, where the direct process dominates. For the three diradicals with comparable magnitude of dipolar interaction, 1/Tm and 1/T1 were faster for the molecules with more flexible structures. Relaxation rates were faster in the less rigid low-polarity sucrose octaacetate glass than in the more rigid 4:1 toluene/chloroform or in hydrogen-bonded glycerol glasses, which highlights the impact of motion on relaxation.