Examinando por Autor "Retamal, Mauricio A."
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Ítem Connexin46 in the nucleus of cancer cells: a possible role as transcription modulator(BioMed Central Ltd, 2025-12) Fernández-Olivares, Ainoa; Orellana, Viviana P; Llanquinao, Jesús; Nuñez, Gonzalo; Pérez-Moreno, Pablo; Contreras-Riquelme, Sebastián; Martin, Alberto JM; Mammano, Fabio; Alfaro, Ivan E; Calderón, Juan F; Stehberg, Jimmy; Sáez, Mauricio A; Retamal, Mauricio A.Background Oncogenes drive cancer progression, but few are active exclusively in tumor cells. Connexins (Cxs), traditionally recognized as ion channel proteins, can localize to the nucleus and regulate gene expression, playing key roles in both physiological and pathological processes. Cx46, once thought to be restricted to the eye lens, has been implicated in tumor growth, though its underlying mechanisms remain unclear. This study investigates the nuclear presence of Cx46 in cancer cells and its potential role as a transcriptional modulator. Methods We employed ChIP-Seq, confocal immunofuorescence, and nuclear protein purifcation to assess Cx46 localization and DNA interactions. Functional assays were conducted to evaluate its efects on invasion, division, spheroid formation, and mesenchymal marker expression. Single-point mutations and molecular dynamics simulations were used to explore potential Cx46-DNA interactions. Results Cx46 mRNA upregulation was found in a variety of tumors compared to adjacent healthy tissue. In HeLa cells, which do not express Cx46, its transfection promoted proliferation, invasion and self-renewal capacity, cancer stem cell traits and mesenchymal features. Consistently, in Sk-Mel-2, which naturally express Cx46, reduced Cx46 expression led to a decrease in the similar parameters. In HeLa cells, nuclear Cx46 was detected in two forms, full length 46 kDa and a 30 kDa fragment (GJA3-30 k), ChIP-Seq experiments revealed that Cx46 binds to the DNA at intergenic and promoter regions, leading to the activation of oncogenic pathways. Molecular dynamics simulations suggest that GJA3-30 k dimerizes in a RAD50-like structure, forming stable DNA complexes. Cx46 and in some cases GJA3-30 k were detected in the nuclei of multiple cancer cell lines, including prostate, breast and skin cancers. Conclusions Our fndings reveal a novel nuclear role for Cx46 in cancer, demonstrating its function as a transcriptional regulator and its potential as a therapeutic targetÍtem Extracellular Cysteines Are Critical to Form Functional Cx46 Hemichannels(MDPI, 2022-07-01) Fernández Olivares, Ainoa; Durán Jara, Eduardo; Verdugo, Daniel A.; Fiori, Mariana C.; Altenberg, Guillermo A.; Stehberg, Jimmy; Alfaro, Iván; Calderón, Juan Francisco; Retamal, Mauricio A.Connexin (Cxs) hemichannels participate in several physiological and pathological pro-cesses, but the molecular mechanisms that control their gating remain elusive. We aimed at deter-mining the role of extracellular cysteines (Cys) in the gating and function of Cx46 hemichannels. We studied Cx46 and mutated all of its extracellular Cys to alanine (Ala) (one at a time) and studied the effects of the Cys mutations on Cx46 expression, localization, and hemichannel activity. Wild-type Cx46 and Cys mutants were expressed at comparable levels, with similar cellular localization. However, functional experiments showed that hemichannels formed by the Cys mutants did not open either in response to membrane depolarization or removal of extracellular divalent cations. Molecular-dynamics simulations showed that Cys mutants may show a possible alteration in the electrostatic potential of the hemichannel pore and an altered disposition of important residues that could contribute to the selectivity and voltage dependency in the hemichannels. Replacement of extracellular Cys resulted in “permanently closed hemichannels”, which is congruent with the inhibition of the Cx46 hemichannel by lipid peroxides, through the oxidation of extracellular Cys. These results point to the modification of extracellular Cys as potential targets for the treatment of Cx46-hemichannel associated pathologies, such as cataracts and cancer, and may shed light into the gating mechanisms of other Cx hemichannels. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Ítem KI04 an Aminoglycosides-Derived Molecule Acts as an Inhibitor of Human Connexin46 Hemichannels Expressed in HeLa Cells(MDPI, 2023-02) Chang, Cheng-Wei T.; Poudyal, Naveena; Verdugo, Daniel A.; Peña, Francisca; Stehberg, Jimmy; Retamal, Mauricio A.Background: Connexins (Cxs) are proteins that help cells to communicate with the extracellular media and with the cytoplasm of neighboring cells. Despite their importance in several human physiological and pathological conditions, their pharmacology is very poor. In the last decade, some molecules derived from aminoglycosides have been developed as inhibitors of Cxs hemichannels. However, these studies have been performed in E. coli, which is a very simple model. Therefore, our main goal is to test whether these molecules have similar effects in mammalian cells. Methods: We transfected HeLa cells with the human Cx46tGFP and characterized the effect of a kanamycin-derived molecule (KI04) on Cx46 hemichannel activity by time-lapse recordings, changes in phosphorylation by Western blot, localization by epifluorescence, and possible binding sites by molecular dynamics (MD). Results: We observed that kanamycin and KI04 were the most potent inhibitors of Cx46 hemichannels among several aminoglycosides, presenting an IC50 close to 10 μM. The inhibitory effect was not associated with changes in Cx46 electrophoretic mobility or its intracellular localization. Interestingly, 5 mM DTT did not reverse KI04 inhibition, but the KI04 effect completely disappeared after washing out KI04 from the recording media. MD analysis revealed two putative binding sites of KI04 in the Cx46 hemichannel. Results: These results demonstrate that KI04 could be used as a Cx46 inhibitor and could help to develop future selective Cx46 inhibitors. © 2023 by the authors.Ítem Opening of pannexin- and connexin-based channels increases the excitability of nodose ganglion sensory neurons(Frontiers Media S.A., 2014-06) Retamal, Mauricio A.; Alcayaga, Julio; Verdugo, Christian A.; Bultynck, Geert; Leybaert, Luc; Sáez, Pablo J.; Fernández, Ricardo; León, Luis E.; Sáez, Juan C.Satellite glial cells (SGCs) are the main glia in sensory ganglia. They surround neuronal bodies and form a cap that prevents the formation of chemical or electrical synapses between neighboring neurons. SGCs have been suggested to establish bidirectional paracrine communication with sensory neurons. However, the molecular mechanism involved in this cellular communication is unknown. In the central nervous system (CNS), astrocytes present connexin43 (Cx43) hemichannels and pannexin1 (Panx1) channels, and the opening of these channels allows the release of signal molecules, such as ATP and glutamate. We propose that these channels could play a role in glia-neuron communication in sensory ganglia. Therefore, we studied the expression and function of Cx43 and Panx1 in rat and mouse nodose-petrosal-jugular complexes (NPJcs) using confocal immunofluorescence, molecular and electrophysiological techniques. Cx43 and Panx1 were detected in SGCs and in sensory neurons, respectively. In the rat and mouse, the electrical activity of vagal nerve increased significantly after nodose neurons were exposed to a Ca2+/Mg2+-free solution, a condition that increases the open probability of Cx hemichannels. This response was partially mimicked by a cell-permeable peptide corresponding to the last 10 amino acids of Cx43 (TAT-Cx43CT). Enhanced neuronal activity was reduced by Cx hemichannel, Panx1 channel and P2X7 receptor blockers. Moreover, the role of Panx1 was confirmed in NPJc, because in those from Panx1 knockout mice showed a reduced increase of neuronal activity induced by Ca2+/Mg2+-free extracellular conditions. The data suggest that Cx hemichannels and Panx channels serve as paracrine communication pathways between SGCs and neurons by modulating the excitability of sensory neurons.Ítem Over-activated hemichannels: A possible therapeutic target for human diseases(Elsevier B.V., 2021-11-01) Retamal, Mauricio A.; Fernandez-Olivares, Ainoa; Stehberg, JimmyIn our body, all the cells are constantly sharing chemical and electrical information with other cells. This intercellular communication allows them to respond in a concerted way to changes in the extracellular milieu. Connexins are transmembrane proteins that have the particularity of forming two types of channels; hemichannels and gap junction channels. Under normal conditions, hemichannels allow the controlled release of signaling molecules to the extracellular milieu. However, under certain pathological conditions, over-activated hemichannels can induce and/or exacerbate symptoms. In the last decade, great efforts have been put into developing new tools that can modulate these over-activated hemichannels. Small molecules, antibodies and mimetic peptides have shown a potential for the treatment of human diseases. In this review, we summarize recent findings in the field of hemichannel modulation via specific tools, and how these tools could improve patient outcome in certain pathological conditions. © 2021 Elsevier B.V.