Papers by Andrea Huerta Soza
Cancers, 2021
Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways... more Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by intera...
The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, wh... more The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findin...
Journal of Periodontal Research, 2020
BACKGROUND AND OBJECTIVE During cyclosporine-induced gingival overgrowth, the homeostatic balance... more BACKGROUND AND OBJECTIVE During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-β1-stimulated human gingival fibroblasts. Gal-8 interacted with α5β1-integrin and type II TGF-β receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through β1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.
ABSTRACTUbiquitination regulates several biological processes. Here, we search for ubiquitin-rela... more ABSTRACTUbiquitination regulates several biological processes. Here, we search for ubiquitin-related genes implicated in protein membrane trafficking performing a High-Content siRNA Screening including 1,187 genes of the human “ubiquitinome” using Amyloid Precursor Protein (APP) as a reporter. We identified the deubiquitinating enzyme PSMD14, a subunit of the 19S regulatory particle of the proteasome, specific for K63-Ub chains in cells, as a novel key regulator of Golgi-to-endoplasmic reticulum (ER) retrograde transport. Silencing or pharmacological inhibition of PSMD14 caused a robust and rapid inhibition of Golgi-to-ER retrograde transport which leads to a potent blockage of macroautophagy by a mechanism associated with the retention of Atg9A and Rab1A at the Golgi apparatus. Because pharmacological inhibition of the proteolytic core of the 20S proteasome did not recapitulate these effects, we concluded that PSMD14, and their K-63-Ub chains, act as a crucial regulator factor for ...
Molecular Neurobiology, 2019
Galectin-8 (Gal-8) is a glycan-binding protein that modulates a variety of cellular processes int... more Galectin-8 (Gal-8) is a glycan-binding protein that modulates a variety of cellular processes interacting with cell surface glycoproteins. Neutralizing anti-Gal-8 antibodies that block Gal-8 functions have been described in autoimmune and inflammatory disorders, likely playing pathogenic roles. In the brain, Gal-8 is highly expressed in the choroid plexus and accordingly has been detected in human cerebrospinal fluid. It protects against central nervous system autoimmune damage through its immunesuppressive potential. Whether Gal-8 plays a direct role upon neurons remains unknown. Here, we show that Gal-8 protects hippocampal neurons in primary culture against damaging conditions such as nutrient deprivation, glutamate-induced excitotoxicity, hydrogen peroxide (H 2 O 2)-induced oxidative stress, and β-amyloid oligomers (Aβo). This protective action is manifested even after 2 h of exposure to the harmful condition. Pull-down assays demonstrate binding of Gal-8 to selected β1integrins, including α3 and α5β1. Furthermore, Gal-8 activates β1-integrins, ERK1/2, and PI3K/AKT signaling pathways that mediate neuroprotection. Hippocampal neurons in primary culture produce and secrete Gal-8, and their survival decreases upon incubation with human function-blocking Gal-8 autoantibodies obtained from lupus patients. Despite the low levels of Gal-8 expression detected by real-time PCR in hippocampus, compared with other brain regions, the complete lack of Gal-8 in Gal-8 KO mice determines higher levels of apoptosis upon H 2 O 2 stereotaxic injection in this region. Therefore, endogenous Gal-8 likely contributes to generate a neuroprotective environment in the brain, which might be eventually counteracted by human function-blocking autoantibodies.
Frontiers in Immunology, 2019
Carcinogenesis, 2019
The permeability of endothelial cells is regulated by the stability of the adherens junctions, wh... more The permeability of endothelial cells is regulated by the stability of the adherens junctions, which is highly sensitive to kinase-mediated phosphorylation and endothelial nitric oxide synthase (eNOS)-mediated S-nitrosylation of its protein components. Solid tumors can produce a variety of factors that stimulate these signaling pathways leading to endothelial cell hyperpermeability. This generates stromal conditions that facilitate tumoral growth and dissemination. Galectin-8 (Gal-8) is overexpressed in several carcinomas and has a variety of cellular effects that can contribute to tumor pathogenicity, including angiogenesis. Here we explored whether Gal-8 has also a role in endothelial permeability. We show that recombinant Gal-8 activates eNOS, induces S-nitrosylation of p120-catenin (p120) and dissociation of adherens junction, leading to hyperpermeability of the human endothelial cell line EAhy926. This pathway involves focal-adhesion kinase (FAK) activation downstream of eNOS as a requirement for eNOS-mediated p120 S-nitrosylation. This suggests a reciprocal, yet little understood, regulation of phosphorylation and S-nitrosylation events acting upon adherens junction permeability. In addition, glutathione S-transferase (GST)-Gal-8 pull-down experiments and function-blocking β1-integrin antibodies point to β1-integrins as cell surface components involved in Gal-8-induced hyperpermeability. Endogenous Gal-8 secreted from the breast cancer cell line MCF-7 has similar hyperpermeability and signaling effects. Furthermore, the mouse cremaster model system showed that Gal-8 also activates eNOS, induces S-nitrosylation of adherens junction components and is an effective hyperpermeability agent in vivo. These results add endothelial permeability regulation by S-nitrosylation as a new function of Gal-8 that can potentially contribute to the pathogenicity of tumors overexpressing this lectin.
Frontiers in cellular neuroscience, 2018
Alzheimer's disease (AD) is the most common cause of age-related dementia leading to severe i... more Alzheimer's disease (AD) is the most common cause of age-related dementia leading to severe irreversible cognitive decline and massive neurodegeneration. While therapeutic approaches for managing symptoms are available, AD currently has no cure. AD associates with a progressive decline of the two major catabolic pathways of eukaryotic cells-the autophagy-lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS)-that contributes to the accumulation of harmful molecules implicated in synaptic plasticity and long-term memory impairment. One protein recently highlighted as the earliest initiator of these disturbances is the amyloid precursor protein (APP) intracellular C-terminal membrane fragment β (CTFβ), a key toxic agent with deleterious effects on neuronal function that has become an important pathogenic factor for AD and a potential biomarker for AD patients. This review focuses on the involvement of regulatory molecules and specific post-translational modifications (P...
Molecular biology of the cell, Jan 3, 2018
Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal... more Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein Galectin-8 (Gal-8) is over-expressed by certain carcinomas and activates selective β1-integrins involved in EMT. Here we show that Gal-8 over-expression or exogenous addition promotes proliferation, migration and invasion in non-tumoral MDCK cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the EGFR, likely triggered by α5β1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin and Snail expression, as well as increased β-catenin activity. Changes related with migration/invasion included higher expression of α5β1 integrin and extracellular matrix-degrading MMP13 and uPA/uPAR protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome over-activity characteristi...
PloS one, 2017
Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune syste... more Galectin-8 (Gal-8) is a member of a glycan-binding protein family that regulates the immune system, among other functions, and is a target of antibodies in autoimmune disorders. However, its role in multiple sclerosis (MS), an autoimmune inflammatory disease of the central nervous system (CNS), remains unknown. We study the consequences of Gal-8 silencing on lymphocyte subpopulations and the development of experimental autoimmune encephalitis (EAE), to then assess the presence and clinical meaning of anti-Gal-8 antibodies in MS patients. Lgals8/Lac-Z knock-in mice lacking Gal-8 expression have higher polarization toward Th17 cells accompanied with decreased CCR6+ and higher CXCR3+ regulatory T cells (Tregs) frequency. These conditions result in exacerbated MOG35-55 peptide-induced EAE. Gal-8 eliminates activated Th17 but not Th1 cells by apoptosis and ameliorates EAE in C57BL/6 wild-type mice. β-gal histochemistry reflecting the activity of the Gal-8 promoter revealed Gal-8 expressi...
Biological research, Jan 27, 2016
Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma... more Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model. We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in ...
Revista médica de Chile, 2006
Antibodies against galectin-8 in patients with systemic lupus erythematosus Background: The famil... more Antibodies against galectin-8 in patients with systemic lupus erythematosus Background: The family of lectins known as galectins (galectins 1-14) are involved in the regulation of the immune system and in oncogenesis. During a search for antigens recognized by antibodies produced by a patient with systemic lupus erythematosus (SLE) we found reactivity against galectin-8, for which autoantibodies have not been previously described. Aim: To determine the frequency of autoantibodies against galectin-8 in lupus patients compared with healthy controls. Patients and Methods: Galectin-8 was purified from a bacterial expression system and used in immunoblot assays as antigen to screen the sera of 55 SLE patients and matched controls. Disease activity was evaluated using the Mexican Modification of the Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI). Results: Reactivity against galectin-8 was detected in 30% of SLE patients, compared to 7% of controls (p=0.003). We could not detect any particular SLE manifestation associated to the presence of these autoantibodies. Conclusions: This is the first description of autoantibodies against galectin-8. Its higher frequency in patients with SLE suggests a pathogenic role. Further studies are needed to determine their clinical relevance (Rev Méd Chile 2006; 134: 159-66).
Molecular Biology of the Cell, 2007
The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both bios... more The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit μ1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3–5 min. By contrast, the antibody did not block trafficking o...
The Journal of Immunology, 2000
The proteasome is a large protease complex that generates most of the peptide ligands of MHC clas... more The proteasome is a large protease complex that generates most of the peptide ligands of MHC class I molecules either in their final form or in the form of N-terminally extended precursors. Upon the stimulation of cells with IFN-γ, three constitutively expressed subunits of the 20S proteasome are replaced by the inducible subunits LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1 (multicatalytic endopeptidase complex-like-1) to form so-called immunoproteasomes. We show in this study that overexpression of these three subunits in triple transfectants led to a marked enhancement in the H-2Ld-restricted presentation of the immunodominant nonameric epitope NP118, which is derived from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. Overexpression of the α and β subunits of the IFN-γ-inducible proteasome regulator PA28, in contrast, did not have a comparable effect. In vitro, immunoproteasomes as compared with constitutive proteasomes generated higher amounts of 11-...
Journal of Biological Chemistry, 2004
Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in th... more Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosinebased motifs and an unconventional basolateral motif.
Journal of Biological Chemistry, 2009
Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. H... more Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by functionblocking autoantibodies in autoimmunity.
FEBS Letters, 1997
We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mR... more We have cloned the mouse PA28 proteasome activator cDNAs. Northern blot demonstrates high PA28 mRNA levels in liver, kidney and lung. mRNA levels are low in thymus, spleen and brain. In contrast, PA28 protein levels vary little between these tissues. Immunocytological analysis and cell fractionation experiments demonstrate that both subunits are almost equally distributed between the cytoplasm and the nucleus. Interestingly, PA28oc spares nucleoli, while PA28P is strongly enhanced in the nucleolus. This indicates for the first time that the PA28a and PA28P subunits may serve nuclear functions which may be different from and independent of each other.
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Papers by Andrea Huerta Soza