Harnessing the Therapeutic Potential of Antibodies Targeting Connexin Hemichannels

Connexin hemichannels have been implicated in pathology-promoting conditions, including inflammation, numerous widespread human diseases, including cancer and diabetes, and several rare diseases linked to pathological point mutations

Cues to opening mechanisms from in silico electric field excitation of cx26 hemichannel and in vitro mutagenesis studies in HeLa transfectans

Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies

Coarse Grained Density Functional Theories for Metallic Alloys: Generalized Coherent Potential Approximations and Charge Excess Functional Theory

The class of the Generalized Coherent Potential Approximations (GCPA) to the Density Functional Theory (DFT) is introduced within the Multiple Scattering Theory formalism for dealing with, ordered or disordered, metallic alloys. All GCPA theories are based on a common ansatz for the kinetic part of the Hohenberg-Kohn functional and each theory of the class is specified by an external model concerning the potential reconstruction. The GCPA density functional consists of marginally coupled local contributions, does not depend on the details of the charge density and can be exactly rewritten as a function of the appropriate charge multipole moments associated with each lattice site. A general procedure based on the integration of the 'qV' laws is described that allows for the explicit construction the same function. The coarse grained nature of the GCPA density functional implies great computational advantages and is connected with the O(N) scalability of GCPA algorithms. Moreover, it is shown that a convenient truncated series expansion of the GCPA functional leads to the Charge Excess Functional (CEF) theory [E. Bruno, L. Zingales and Y. Wang, Phys. Rev. Lett. {\bf 91}, 166401 (2003)] which here is offered in a generalized version that includes multipolar interactions. CEF and the GCPA numerical results are compared with status of art LAPW full-potential density functional calculations for 62, bcc- and fcc-based, ordered CuZn alloys, in all the range of concentrations. These extensive tests show that the discrepancies between GCPA and CEF are always within the numerical accuracy of the calculations, both for the site charges and the total energies. Furthermore, GCPA and CEF very carefully reproduce the LAPW site charges and the total energy trends.Comment: 19 pages, 11 figure

The p.Cys169Tyr variant of connexin 26 is not a polymorphism

Mutations in the GJB2 gene, which encodes the gap junction protein connexin 26 (Cx26), are the primary cause of hereditary prelingual hearing impairment. Here, the p.Cys169Tyr missense mutation of Cx26 (Cx26C169Y), previously classified as a polymorphism, has been identified as causative of severe hearing loss in two Qatari families. We have analyzed the effect of this mutation using a combination of confocal immunofluorescence microscopy and molecular dynamics simulations. At the cellular level, our results show that the mutant protein fails to form junctional channels in HeLa transfectants despite being correctly targeted to the plasma membrane. At the molecular level, this effect can be accounted for by disruption of the disulfide bridge that Cys169 forms with Cys64 in the wild-type structure (Cx26WT). The lack of the disulfide bridge in the Cx26C169Y protein causes a spatial rearrangement of two important residues, Asn176 and Thr177. In the Cx26WT protein, these residues play a crucial role in the intra-molecular interactions that permit the formation of an intercellular channel by the head-to-head docking of two opposing hemichannels resident in the plasma membrane of adjacent cells. Our results elucidate the molecular pathogenesis of hereditary hearing loss due to the connexin mutation and facilitate the understanding of its role in both healthy and affected individuals

Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1−/− mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1−/− mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1−/− cochleae. Hearing sensitivity, outer hair cell-based “cochlear amplifier” and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/− and Panx1−/− mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function

Coarse grained Density Functional theory of order-disorder phase transitions in metallic alloys

The technological performances of metallic compounds are largely influenced by atomic ordering. Although there is a general consensus that successful theories of metallic systems should account for the quantum nature of the electronic glue, existing non-perturbative high-temperature treatments are based on effective classical atomic Hamiltonians. We propose a solution for the above paradox and offer a fully quantum mechanical, though approximate, theory that on equal footing deals with both electrons and ions. By taking advantage of a coarse grained formulation of the density functional theory [Bruno et al., Phys. Rev. B 77, 155108 (2008)] we develop a MonteCarlo technique, based on an ab initio Hamiltonian, that allows for the efficient evaluation of finite temperature statistical averages. Calculations of the relevant thermodynamic quantities and of the electronic structures for CuZn and Ni$_3$V support that our theory provides an appropriate description of order-disorder phase transitions.Comment: 4 pages, 4 figure

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT

Protein pathway activation mapping of colorectal metastatic progression reveals metastasis-specific network alterations

The mechanism by which tissue microecology influences invasion and metastasis is largely unknown. Recent studies have indicated differences in the molecular architecture of the metastatic lesion compared to the primary tumor, however, systemic analysis of the alterations within the activated protein signaling network has not been described. Using laser capture microdissection, protein microarray technology, and a unique specimen collection of 34 matched primary colorectal cancers (CRC) and synchronous hepatic metastasis, the quantitative measurement of the total and activated/phosphorylated levels of 86 key signaling proteins was performed. Activation of the EGFR-PDGFR-cKIT network, in addition to PI3K/AKT pathway, was found uniquely activated in the hepatic metastatic lesions compared to the matched primary tumors. If validated in larger study sets, these findings may have potential clinical relevance since many of these activated signaling proteins are current targets for molecularly targeted therapeutics. Thus, these findings could lead to liver metastasis specific molecular therapies for CRC. \uc2\ua9 2012 Springer Science+Business Media Dordrecht

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity.Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells.Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action.Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies