Browsing by Author "Varas-Godoy, Manuel"

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    Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells
    (2023) Niechi, Ignacio; Erices, Jose I.; Carrillo-Beltran, Diego; Uribe-Ojeda, Atenea; Torres, Angelo; Rocha, Jose Dellis; Uribe, Daniel; Toro, Maria A.; Villalobos-Nova, Karla; Gaete-Ramirez, Belen; Mingo, Gabriel; Owen, Gareth I. I.; Varas-Godoy, Manuel; Jara, Lilian; Aguayo, Francisco; Burzio, Veronica A.; Quezada-Monras, Claudia; Tapia, Julio C. C.
    Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1c(K6R) mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1c(K6R) behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1c(K6R)-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1c(K6R) cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.
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    Frizzled-1 receptor regulates adult hippocampal neurogenesis
    (2016) Inestrosa Cantín, Nibaldo; Mardones, Muriel D.; Andaur, Gabriela A.; Varas-Godoy, Manuel; Henriquez, Jenny F.; Salech, Felipe; Behrens, María I.; Couve, Andrés; Varela-Nallar, Lorena
    Abstract Background In the adult hippocampus new neurons are continuously generated from neural stem cells (NSCs) present at the subgranular zone of the dentate gyrus. This process is controlled by Wnt signaling, which plays a complex role in regulating multiple steps of neurogenesis including maintenance, proliferation and differentiation of progenitor cells and the development of newborn neurons. Differential effects of Wnt signaling during progression of neurogenesis could be mediated by cell-type specific expression of Wnt receptors. Here we studied the potential role of Frizzled-1 (FZD1) receptor in adult hippocampal neurogenesis. Results In the adult dentate gyrus, we determined that FZD1 is highly expressed in NSCs, neural progenitors and immature neurons. Accordingly, FZD1 is expressed in cultured adult hippocampal progenitors isolated from mouse brain. To evaluate the role of this receptor in vivo we targeted FZD1 in newborn cells using retroviral-mediated RNA interference. FZD1 knockdown resulted in a marked decrease in the differentiation of newborn cells into neurons and increased the generation of astrocytes, suggesting a regulatory role for the receptor in cell fate commitment. In addition, FZD1 knockdown induced an extended migration of adult-born neurons within the granule cell layer. However, no differences were observed in total dendritic length and dendritic arbor complexity between control and FZD1-deficient newborn neurons. Conclusions Our results show that FZD1 regulates specific stages of adult hippocampal neurogenesis, being required for neuronal differentiation and positioning of newborn neurons into the granule cell layer, but not for morphological development of adult-born granule neurons.Abstract Background In the adult hippocampus new neurons are continuously generated from neural stem cells (NSCs) present at the subgranular zone of the dentate gyrus. This process is controlled by Wnt signaling, which plays a complex role in regulating multiple steps of neurogenesis including maintenance, proliferation and differentiation of progenitor cells and the development of newborn neurons. Differential effects of Wnt signaling during progression of neurogenesis could be mediated by cell-type specific expression of Wnt receptors. Here we studied the potential role of Frizzled-1 (FZD1) receptor in adult hippocampal neurogenesis. Results In the adult dentate gyrus, we determined that FZD1 is highly expressed in NSCs, neural progenitors and immature neurons. Accordingly, FZD1 is expressed in cultured adult hippocampal progenitors isolated from mouse brain. To evaluate the role of this receptor in vivo we targeted FZD1 in newborn cells using retroviral-mediated RNA interference. FZD1 knockdown resulted in a marked decrease in the differentiation of newborn cells into neurons and increased the generation of astrocytes, suggesting a regulatory role for the receptor in cell fate commitment. In addition, FZD1 knockdown induced an extended migration of adult-born neurons within the granule cell layer. However, no differences were observed in total dendritic length and dendritic arbor complexity between control and FZD1-deficient newborn neurons. Conclusions Our results show that FZD1 regulates specific stages of adult hippocampal neurogenesis, being required for neuronal differentiation and positioning of newborn neurons into the granule cell layer, but not for morphological development of adult-born granule neurons.
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    H3K9 Methyltransferases Suv39h1 and Suv39h2 Control the Differentiation of Neural Progenitor Cells in the Adult Hippocampus
    (2022) Guerra, Miguel V.; Caceres, Matias I.; Herrera-Soto, Andrea; Arredondo, Sebastian B.; Varas-Godoy, Manuel; van Zundert, Brigitte; Varela-Nallar, Lorena
    In the dentate gyrus of the adult hippocampus new neurons are generated from neural precursor cells through different stages including proliferation and differentiation of neural progenitor cells and maturation of newborn neurons. These stages are controlled by the expression of specific transcription factors and epigenetic mechanisms, which together orchestrate the progression of the neurogenic process. However, little is known about the involvement of histone posttranslational modifications, a crucial epigenetic mechanism in embryonic neurogenesis that regulates fate commitment and neuronal differentiation. During embryonic development, the repressive modification trimethylation of histone H3 on lysine 9 (H3K9me3) contributes to the cellular identity of different cell-types. However, the role of this modification and its H3K9 methyltransferases has not been elucidated in adult hippocampal neurogenesis. We determined that during the stages of neurogenesis in the adult mouse dentate gyrus and in cultured adult hippocampal progenitors (AHPs), there was a dynamic change in the expression and distribution of H3K9me3, being enriched at early stages of the neurogenic process. A similar pattern was observed in the hippocampus for the dimethylation of histone H3 on lysine 9 (H3K9me2), another repressive modification. Among H3K9 methyltransferases, the enzymes Suv39h1 and Suv39h2 exhibited high levels of expression at early stages of neurogenesis and their expression decreased upon differentiation. Pharmacological inhibition of these enzymes by chaetocin in AHPs reduced H3K9me3 and concomitantly decreased neuronal differentiation while increasing proliferation. Moreover, Suv39h1 and Suv39h2 knockdown in newborn cells of the adult mouse dentate gyrus by retrovirus-mediated RNA interference impaired neuronal differentiation of progenitor cells. Our results indicate that H3K9me3 and H3K9 methyltransferases Suv39h1 and Suv39h2 are critically involved in the regulation of adult hippocampal neurogenesis by controlling the differentiation of neural progenitor cells.
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    Membrane vesicles in Acidithiobacillia class extreme acidophiles: influence on collective behaviors of 'Fervidacidithiobacillus caldus'
    (2024) Rossoni, Stefano; Beard, Simon; Segura-Bidermann, Maria Ignacia; Duarte-Ramirez, Juan; Osorio, Francisco Kirhman; Varas-Godoy, Manuel; Martinez-Bellange, Patricio; Vera, Mario; Quatrini, Raquel; Castro, Matias
    Membrane vesicles (MVs) are envelope-derived extracellular sacs that perform a broad diversity of physiological functions in bacteria. While considerably studied in pathogenic microorganisms, the roles, relevance, and biotechnological potential of MVs from environmental bacteria are less well established. Acidithiobacillaceae family bacteria are active players in the sulfur and iron biogeochemical cycles in extremely acidic environments and drivers of the leaching of mineral ores contributing to acid rock/mine drainage (ARD/AMD) and industrial bioleaching. One key aspect of such a role is the ability of these bacteria to tightly interact with the mineral surfaces and extract electrons and nutrients to support their chemolithotrophic metabolism. Despite recent advances in the characterization of acidithiobacilli biofilms and extracellular matrix (ECM) components, our understanding of its architectural and mechanistic aspects remains scant. Using different microscopy techniques and nano-tracking analysis we show that vesiculation is a common phenomenon in distant members of the Acidithiobacillaceae family, and further explore the role of MVs in multicellular colonization behaviors using 'Fervidacidithiobacillus caldus' as a bacterial model. Production of MVs in 'F. caldus' occurred in both planktonic cultures and biofilms formed on sulfur surfaces, where MVs appeared individually or in chains resembling tube-shaped membranous structures (TSMSs) important for microbial communication. Liquid chromatography-mass spectrometry data and bioinformatic analysis of the MV-associated proteome revealed that 'F. caldus' MVs were enriched in proteins involved in cell-cell and cell-surface processes and largely typified the MVs as outer MVs (OMVs). Finally, microbiological assays showed that amendment of 'F. caldus' MVs to cells and/or biofilms affects collective colonizing behaviors relevant to the ecophysiology and applications of these acidophiles, providing grounds for their exploitation in biomining.
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    Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival
    (2022) Vargas, Gabriela; Cortes, Omar; Arias-Munoz, Eloisa; Hernandez, Sergio; Cerda-Troncoso, Cristobal; Hernandez, Laura; Gonzalez, Alexis E.; Tatham, Michael H.; Bustamante, Hianara A.; Retamal, Claudio; Cancino, Jorge; Varas-Godoy, Manuel; Hay, Ronald T.; Rojas-Fernandez, Alejandro; Cavieres, Viviana A.; Burgos, Patricia V.
    Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.
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    Selective Surface and Intraluminal Localization of Wnt Ligands on Small Extracellular Vesicles Released by HT-22 Hippocampal Neurons
    (2021) Torres, Viviana I.; Barrera, Daniela P.; Varas-Godoy, Manuel; Arancibia, Duxan; Inestrosa, Nibaldo C.
    The Wnt signaling pathway induces various responses underlying the development and maturation of the nervous system. Wnt ligands are highly hydrophobic proteins that limit their diffusion through an aqueous extracellular medium to a target cell. Nevertheless, their attachment to small extracellular vesicles-like exosomes is one of the described mechanisms that allow their transport under this condition. Some Wnt ligands in these vehicles are expected to be dependent on post-translational modifications such as acylation. The mechanisms determining Wnt loading in exosomes and delivery to the target cells are largely unknown. Here, we took advantage of a cell model that secret a highly enriched population of small extracellular vesicles (sEVs), hippocampal HT-22 neurons. First, to establish the cell model, we characterized the morphological and biochemical properties of an enriched fraction of sEVs obtained from hippocampal HT-22 neurons that express NCAM-L1, a specific exosomal neuronal marker. Transmission electron microscopy showed a highly enriched fraction of exosome-like vesicles. Next, the exosomal presence of Wnt3a, Wnt5a, and Wnt7a was confirmed by western blot analysis and electron microscopy combined with immunogold. Also, we studied whether palmitoylation is a necessary post-translational modification for the transport Wnt in these vesicles. We found that proteinase-K treatment of exosomes selectively decreased their Wnt5a and Wnt7a content, suggesting that their expression is delimited to the exterior membrane surface. In contrast, Wnt3a remained attached, suggesting that it is localized within the exosome lumen. On the other hand, Wnt-C59, a specific inhibitor of porcupine O-acyltransferase (PORCN), decreased the association of Wnt with exosomes, suggesting that Wnt ligand acylation is necessary for them to be secreted by exosomes. These findings may help to understand the action of the Wnt ligands in the target cell, which could be defined during the packaging of the ligands in the secretory cell sEVs.
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    TNF-α-activated eNOS signaling increases leukocyte adhesion through the S- nitrosylation pathway
    (2021) Aguilar, Gaynor; Cordova, Francisco; Koning, Tania; Sarmiento, Jose; Boric, Mauricio P.; Birukov, Konstantin; Cancino, Jorge; Varas-Godoy, Manuel; Soza, Andrea; Alves, Natascha G.; Mujica, Patricio E.; Duran, Walter N.; Ehrenfeld, Pamela; Sanchez, Fabiola A.
    Nitric oxide ( NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-alpha (TNF-alpha) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase C sigma (PKC sigma) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-alpha, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-alpha-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-alpha-induced NO also produced S-nitrosylation and phosphorylation of PKCf, association of PKCf with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCf blocked leukocyte adhesion induced by TNF-alpha. Mass spectrometry analysis of purified PKCf identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCf may be an important regulatory step in early leukocyte adhesion in inflammation.