Uncovering a novel mechanism: Butyrate induces estrogen receptor alpha activation independent of estrogen stimulation in MCF-7 breast cancer cells.

Butyrate is a promising candidate for an antitumoral drug, as it promotes cancer cell apoptosis and reduces hormone receptor activity, while promoting differentiation and proliferation in normal cells. However, the effects of low-dose butyrate on breast cancer cell cultures are unclear. We explored the impact of sub-therapeutic doses of butyrate on estrogen receptor alpha (ERα) transcriptional activity in MCF-7 cells, using RT-qPCR, Western blot, wound-healing assays, and chromatin immunoprecipitation. Our results showed that sub-therapeutic doses of sodium butyrate (0.1 - 0.2 mM) increased the transcription of ESR1, TFF1, and CSTD genes, but did not affect ERα protein levels. Moreover, we observed an increase in cell migration in wound-healing assays. ChIP assays revealed that treatment with 0.1 mM of sodium butyrate resulted in estrogen-independent recruitment of ERα at the pS2 promoter and loss of NCoR. Appropriate therapeutic dosage of butyrate is essential to avoid potential adverse effects on patients' health, especially in the case of estrogen receptor-positive breast tumors. Sub-therapeutic doses of butyrate may induce undesirable cell processes, such as migration due to low-dose butyrate-mediated ERα activation. These findings shed light on the complex effects of butyrate in breast cancer and provide insights for research in the development of antitumoral drugs.

N-terminal region is responsible for mHv1 channel activity in MDSCs.

Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance-voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.

Expression of Hv1 proton channels in myeloid-derived suppressor cells (MDSC) and its potential role in T cell regulation.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.

Enzyme activity and expression of catalases in response to oxidative stress in Sporothrix schenckii.

Sporothrix schenckii is a dimorphic fungus, pathogenic to humans and animals, which is usually infective in the yeast form. Reactive oxygen species (ROS) play an important role in the host's defense, damaging the pathogen's DNA, proteins, and lipids. To prevent oxidative damage, the ROS are detoxified by pathogen-derived antioxidant enzymes such as catalases (CATs). In this work, we analyzed the activity and expression level of three S. schenckii genes, designated as CAT1, CAT2, and CAT3, that putatively encoded for three isoforms of monofunctional CAT with a predicted molecular weight of 57.6, 56.2, and 81.4 kDa, respectively. Our results demonstrate that oxidative stress induced by exogenous H2O2 leads to an altered lipid peroxidation, modifying CAT activity and the expression levels of the CAT genes, being CAT1 and CAT3 the genes with the highest expression in response to the oxidizing agent. These results show that CAT isoforms in S. schenckii can be regulated in response to oxidative stress and might help to control ROS homeostasis in the fungus-host interaction.

Assistance for Folding of Disease-Causing Plasma Membrane Proteins.

An extensive catalog of plasma membrane (PM) protein mutations related to phenotypic diseases is associated with incorrect protein folding and/or localization. These impairments, in addition to dysfunction, frequently promote protein aggregation, which can be detrimental to cells. Here, we review PM protein processing, from protein synthesis in the endoplasmic reticulum to delivery to the PM, stressing the main repercussions of processing failures and their physiological consequences in pathologies, and we summarize the recent proposed therapeutic strategies to rescue misassembled proteins through different types of chaperones and/or small molecule drugs that safeguard protein quality control and regulate proteostasis.

Identification of proteins in Sporothrixschenckiisensu stricto in response to oxidative stress induced by hydrogen peroxide / Identificación de proteínas en Sporothrix schenckii sensu stricto en respuesta al estrés oxidativo inducido por peróxido de hidrógeno

Background: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. In order to colonize the host, the pathogen must neutralize the reactive oxygen species produced by the phagocytic cells during the respiratory burst. Little is known about these mechanisms in S. schenckii. Aims: To identify the proteins differentially expressed after the exposure of S. schenckiisensu stricto to different concentrations of H2O2. Methods: Yeast cells of S. schenckiisensu stricto were exposed to increasing concentrations of H2O2. Proteins differentially expressed in response to oxidative stress were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-MS/MS. RT-PCR assays were performed to evaluate the transcription of genes of the identified proteins. Results: Concentrations of H2O2 as high as 800 mM allowed cell growth, and 200 mM and 400mM were selected for comparative analysis by 2D-PAGE. This analysis revealed at least five differentially expressed proteins, which were identified as heat shock 70 kDa protein (Hsp70), chaperonin GroEL, elongation factor 1-β (EF1-β), a hypothetical protein, and mitochondrial peroxiredoxin (Prx1). RT-PCR revealed that the transcription of the genes coding for some of these proteins are differentially regulated. Conclusions: Based on these results, it is proposed that these proteins may be involved in the resistance of S. schenckii to oxidative stress, and play an important role in the fungus survival in the host

Antecedentes: La esporotricosis es una infección fúngica causada por el complejo Sporothrix schenckii. Para colonizar al huésped, los patógenos deben neutralizar las especies reactivas de oxígeno producidas por las células fagocíticas durante el estallido respiratorio. Poco se conoce sobre este mecanismo en S. schenckii. Objetivos: Identificar proteínas diferencialmente expresadas durante la exposición de S. schenckiisensu stricto a diferentes concentraciones de H2O2. Métodos: Levaduras de S. schenckiisensu stricto fueron expuestas a concentraciones crecientes de H2O2. Las proteínas diferencialmente expresadas en respuesta el estrés oxidativo fueron analizadas mediante electroforesis en geles de poliacrilamida en doble dimensión (2D-PAGE) e identificadas por MALDI-MS/MS. Se realizaron ensayos de RT-PCR para evaluar la transcripción de genes de las proteínas identificadas. Resultados: Concentraciones altas de H2O2 (800 mM) permitieron el crecimiento celular, y se seleccionaron las concentraciones de 200 y 400 mM para el análisis comparativo mediante 2D-PAGE. Este análisis reveló al menos cinco proteínas diferencialmente expresadas, identificadas como proteína de choque térmico de 70 kDa (Hsp70), chaperonina GroEL, factor de alargamiento 1-β (EF1-β), una proteína hipotética y peroxirredoxina mitocondrial (Prx1). La RT-PCR reveló que la transcripción de los genes que codifican para algunas de estas proteínas se regula diferencialmente. Conclusiones: Con estos resultados pensamos que estas proteínas podrían estar involucradas en la resistencia de S. schenckiisensu stricto al estrés oxidativo y jugar un papel importante en la supervivencia del hongo en el huésped

Identification of proteins in Sporothrixschenckiisensu stricto in response to oxidative stress induced by hydrogen peroxide.

BACKGROUND: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. In order to colonize the host, the pathogen must neutralize the reactive oxygen species produced by the phagocytic cells during the respiratory burst. Little is known about these mechanisms in S. schenckii. AIMS: To identify the proteins differentially expressed after the exposure of S. schenckiisensu stricto to different concentrations of H2O2. METHODS: Yeast cells of S. schenckiisensu stricto were exposed to increasing concentrations of H2O2. Proteins differentially expressed in response to oxidative stress were analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and identified by MALDI-MS/MS. RT-PCR assays were performed to evaluate the transcription of genes of the identified proteins. RESULTS: Concentrations of H2O2 as high as 800mM allowed cell growth, and 200mM and 400mM were selected for comparative analysis by 2D-PAGE. This analysis revealed at least five differentially expressed proteins, which were identified as heat shock 70kDa protein (Hsp70), chaperonin GroEL, elongation factor 1-ß (EF1-ß), a hypothetical protein, and mitochondrial peroxiredoxin (Prx1). RT-PCR revealed that the transcription of the genes coding for some of these proteins are differentially regulated. CONCLUSIONS: Based on these results, it is proposed that these proteins may be involved in the resistance of S. schenckii to oxidative stress, and play an important role in the fungus survival in the host.

Deglycosylation of Shaker KV channels affects voltage sensing and the open-closed transition.

Most membrane proteins are subject to posttranslational glycosylation, which influences protein function, folding, solubility, stability, and trafficking. This modification has been proposed to protect proteins from proteolysis and modify protein-protein interactions. Voltage-activated ion channels are heavily glycosylated, which can result in up to 30% of the mature molecular mass being contributed by glycans. Normally, the functional consequences of glycosylation are assessed by comparing the function of fully glycosylated proteins with those in which glycosylation sites have been mutated or by expressing proteins in model cells lacking glycosylation enzymes. Here, we study the functional consequences of deglycosylation by PNGase F within the same population of voltage-activated potassium (KV) channels. We find that removal of sugar moieties has a small, but direct, influence on the voltage-sensing properties and final opening-closing transition of Shaker KV channels. Yet, we observe that the interactions of various ligands with different domains of the protein are not affected by deglycosylation. These results imply that the sugar mass attached to the voltage sensor neither represents a cargo for the dynamics of this domain nor imposes obstacles to the access of interacting molecules.

Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules.

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.

Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine's sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids' side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems.

Editing of human K(V)1.1 channel mRNAs disrupts binding of the N-terminus tip at the intracellular cavity.

In the nervous system, A→I RNA editing has an important role in regulating neuronal excitability. Ligand-gated membrane receptors, synaptic proteins, as well as ion channels, are targets for recoding by RNA editing. Although scores of editing sites have been identified in the mammalian brain, little is known about the functional alterations that they cause, and even less about the mechanistic underpinnings of how they change protein function. We have previously shown that an RNA editing event (I,400 V) alters the inner permeation pathway of human K(V)1.1, modifying the kinetics of fast inactivation. Here we show that the channel's inactivation gate enters deep into the ion permeation pathway and the very tip establishes a direct hydrophobic interaction with the edited position. By converting I to V, the intimacy of the interaction is reduced, allowing the inactivation gate to unbind with much faster kinetics.

The metal-coordinated Casiopeína IIIEa induces the petite-like phenotype in Saccharomyces cerevisiae.

The Casiopeínas® are mixed chelate copper (II) complexes and promising antineoplastics agents against cancer cells and tumors in vitro and in vivo. However, the action mode of these compounds is poorly characterized. In this work the effect of the antineoplastic Casiopeína IIIEa on the metabolism and ultrastructure of the yeast Saccharomyces cerevisiae was investigated. Exposure of cells growing in rich or in low-iron medium to 5 µM of the compound decreased duplication time and reduced oxygen consumption. Those cells formed smaller colonies when growing in a non-fermentable carbon source and low-iron medium, and under the light microscope, multiple folds were observed along the plasma membrane accompanied with a reduction in the diameter of the yeast. These observations were confirmed under the electron microscope, which also revealed a slight reduction of the mitochondrial size. A correlation was found with smaller colonies exhibiting lower rates of oxygen consumption, and yeast labelled with fluorescent MitoTracker(TM) consistently exhibited reduced mitochondrial activity. It appears that Casiopeína IIIEa gives rise to smaller yeast and petite-like colonies by reducing the mitochondrial respiratory activity without significantly affecting the mitochondrial structure.

The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl- across the plasma membrane.

Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl- channel that remained permanently open and was blocked by nitro-2-(3-phenyl-propylamino) benzoic acid and niflumic acid. NPPB induced petite-like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl- through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined.