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.

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.

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. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 and Cx45. Reduction in CIP75 by shRNA in HeLa cells expressing Cx40 or Cx45 resulted in increased levels of the connexins. Furthermore, treatment with trafficking inhibitors confirmed that both connexins undergo endoplasmic reticulum-associated degradation (ERAD), and that CIP75 preferentially interacts with the connexin proteins bound for proteasomal degradation from the ER. In addition, we have also discovered that CIP75 interacts with ER-localized Cx32 in a process that is likely mediated by Cx32 ubiquitination. Thus, we have identified novel interacting connexin proteins of CIP75, indicating a role for CIP75 in regulating the levels of connexins in general, through proteasomal degradation.

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. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.

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.

Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations.

Connexin45 (Cx45) is a gap junction protein involved in cell-to-cell communication in the heart and other tissues. Here we report the (1)H, (15)N, and (13)C resonance assignments for the monomer and dimer conformations of the Cx45 carboxyl terminal (Cx45CT) domain and provide evidence of dimerization using diffusion ordered spectroscopy. The predicted secondary structure of the Cx45CT domain based on the chemical shifts identified one region of α-helical structure, which corresponds to the residues that broadened beyond detection in the dimer confirmation. Previous biophysical studies from our laboratory characterizing the CT domain from the other major cardiac connexins, Cx40 and Cx43, suggest that the amount of α-helical content may translate into the ability of a protein to dimerize. Even though the CT domain is thought to be the main regulatory domain of most connexins, the physiological role of CT dimerization is currently unknown. Therefore, these assignments will be useful for determining the intermolecular interactions that mediate Cx45CT dimerization, information that will be used to characterize dimerization in functional channels, as well as characterizing the binding sites for molecular partners involved in Cx45 regulation.

An Escherichia coli strain for expression of the connexin45 carboxyl terminus attached to the 4th transmembrane domain.

A major problem for structural characterization of membrane proteins, such as connexins, by nuclear magnetic resonance (NMR) occurs at the initial step of the process, the production of sufficient amounts of protein. This occurs because proteins must be expressed in minimal based media. Here, we describe an expression system for membrane proteins that significantly improves yield by addressing two common problems, cell toxicity caused by protein translation and codon bias between genomes. This work provides researchers with a cost-effective tool for NMR and other biophysical studies, to use when faced with little-to-no expression of eukaryotic membrane proteins in Escherichia coli expression systems.

Well-defined cross-linked antioxidant nanozymes for treatment of ischemic brain injury.

Development of well-defined nanomedicines is critical for their successful clinical translation. A simple synthesis and purification procedure is established for chemically cross-linked polyion complexes of Cu/Zn superoxide dismutase (SOD1) or catalase with a cationic block copolymer, methoxy-poly(ethylene glycol)-block-poly(L-lysine hydrochloride) (PEG-pLL50). Such complexes, termed cross-linked nanozymes (cl-nanozymes) retain catalytic activity and have narrow size distribution. Moreover, their cytotoxicity is decreased compared to non-cross-linked complexes due to suppression of release of the free block copolymer. SOD1 cl-nanozymes exhibit prolonged ability to scavenge experimentally induced reactive oxygen species (ROS) in cultured brain microvessel endothelial cells and central neurons. In vivo they decrease ischemia/reperfusion-induced tissue injury and improve sensorimotor functions in a rat middle cerebral artery occlusion (MCAO) model after a single intravenous (i.v.) injection. Altogether, well-defined cl-nanozymes are promising modalities for attenuation of oxidative stress after brain injury.

Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer.

Oligomerization of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II.