Browsing by Author "Gonzalez, Alfonso"

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    Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes
    (AMER SOC CELL BIOLOGY, 2007) Cancino, Jorge; Torrealba, Carolina; Soza, Andrea; Yuseff, Maria Isabel; Gravotta, Diego; Henklein, Peter; Rodriguez Boulan, Enrique; Gonzalez, Alfonso
    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 mu 1B. 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 of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in similar to 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.
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    D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy
    (2021) Barra, Jonathan; Cerda-Infante, Javier; Sandoval, Lisette; Gajardo-Meneses, Patricia; Henriquez, Jenny F.; Labarca, Mariana; Metz, Claudia; Venegas, Jaime; Retamal, Claudio; Oyanadel, Claudia; Cancino, Jorge; Soza, Andrea; Cuello, Mauricio A.; Carlos Roa, Juan; Montecinos, Viviana P.; Gonzalez, Alfonso
    Simple Summary Cancer progression is frequently driven by altered functions of EGFR belonging to the tyrosine-kinase family of growth factor receptors and by the transcription factor p53, which is called the "genome guardian". We report that D-Propranolol, previously used for other purposes in human patients, has antitumor effects involving a redistribution of cell surface EGFR to intracellular compartments and degradation of gain-of-function mutants of p53 (GOF-mutp53). These effects can be seen in cancer cell lines expressing EGFR and GOF-mutp53 and are reproduced in vivo, reducing tumor growth and prolonging survival of xenografted mice. D-Propranolol is proposed as a prototype drug for a new strategy against highly aggressive EGFR- and mutp53-expressing tumors. 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-alpha. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.
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    Galectin-8 counteracts folic acid-induced acute kidney injury and prevents its transition to fibrosis
    (2024) Perez-Moreno, Elisa; Toledo, Tomas; Campusano, Pascale; Zuniga, Sebastian; Azocar, Lorena; Feuerhake, Teo; Mendez, Gonzalo P.; Labarca, Mariana; Perez-Molina, Francisca; de la Pena, Adely; Herrera-Cid, Cristian; Ehrenfeld, Pamela; Godoy, Alejandro S.; Gonzalez, Alfonso; Soza, Andrea
    Acute kidney injury (AKI), characterized by a sudden decline in kidney function involving tubular damage and epithelial cell death, can lead to progressive tissue fibrosis and chronic kidney disease due to interstitial fibroblast activation and tissue repair failures that lack direct treatments. After an AKI episode, surviving renal tubular cells undergo cycles of dedifferentiation, proliferation and redifferentiation while fibroblast activity increases and then declines to avoid an exaggerated extracellular matrix deposition. Appropriate tissue recovery versus pathogenic fibrotic progression depends on fine-tuning all these processes. Identifying endogenous factors able to affect any of them may offer new therapeutic opportunities to improve AKI outcomes. Galectin-8 (Gal-8) is an endogenous carbohydrate-binding protein that is secreted through an unconventional mechanism, binds to glycosylated proteins at the cell surface and modifies various cellular activities, including cell proliferation and survival against stress conditions. Here, using a mouse model of AKI induced by folic acid, we show that pretreatment with Gal-8 protects against cell death, promotes epithelial cell redifferentiation and improves renal function. In addition, Gal-8 decreases fibroblast activation, resulting in less expression of fibrotic genes. Gal-8 added after AKI induction is also effective in maintaining renal function against damage, improving epithelial cell survival. The ability to protect kidneys from injury during both pre- and post-treatments, coupled with its anti-fibrotic effect, highlights Gal-8 as an endogenous factor to be considered in therapeutic strategies aimed at improving renal function and mitigating chronic pathogenic progression.
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    Galectin-8 Favors the Presentation of Surface-Tethered Antigens by Stabilizing the B Cell Immune Synapse
    (2018) Obino, Dorian; Fetler, Luc; Soza Gajardo, Andrea; Malbec, Odile; Saez, Jose; Labarca, Mariana; Oyanadel, Claudia; Del Valle Batalla, Felipe; Goles, Nicolas; Chikina, Aleksandra; Lankar, Danielle; Yuseff Sepúlveda, María Isabel; Segovia Miranda, Fabián Josué; Garcia, Camille; Léger, Thibaut; Gonzalez, Alfonso; Espéli, Marion; Lennon-Duménil, Ana-Maria
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    Galectin-8 Induces Apoptosis in Jurkat T Cells by Phosphatidic Acid-mediated ERK1/2 Activation Supported by Protein Kinase A Down-regulation
    (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009) Norambuena, Andres; Metz, Claudia; Vicuna, Lucas; Silva, Antonia; Pardo, Evelyn; Oyanadel, Claudia; Massardo, Loreto; Gonzalez, Alfonso; Soza, Andrea
    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 function-blocking autoantibodies in autoimmunity.
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    Galectin-8 induces endothelial hyperpermeability through the eNOS pathway involving S-nitrosylation-mediated adherens junction disassembly
    (2019) Zamorano, Patricia; Koning, Tania; Oyanadel, Claudia; Mardones, Gonzalo A.; Ehrenfeld, Pamela; Boric, Mauricio P.; Gonzalez, Alfonso; Soza, Andrea; Sanchez, Fabiola A.
    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 beta 1-integrin antibodies point to beta 1-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.
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    Galectin-8 induces partial epithelial-mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin-Darby canine kidney cells
    (2018) Oyanadel, Claudia; Holmes, Christopher; Pardo, Evelyn; Retamal, Claudio; Shaughnessy, Ronan; Smith, Patricio; Cortes, Priscilla; Bravo-Zehnder, Marcela; Metz, Claudia; Feuerhake, Teo; Romero, Diego, V; Carlos Roa, Juan; Montecinos, Viviana; Soza, Andrea; Gonzalez, Alfonso
    Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal- transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective beta 1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by alpha 5 beta 1 integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8(H)) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased beta-catenin activity. Changes related to migration/invasion included higher expression of alpha 5 beta 1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and beta 1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8(H) cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.
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    Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling
    (PUBLIC LIBRARY SCIENCE, 2012) Diskin, Shiri; Chen, Wei Sheng; Cao, Zhiyi; Gyawali, Smita; Gong, Haiyan; Soza, Andrea; Gonzalez, Alfonso; Panjwani, Noorjahan
    Purpose: The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.
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    Identification of a Novel Mono-Leucine Basolateral Sorting Motif Within the Cytoplasmic Domain of Amphiregulin
    (WILEY, 2011) Gephart, Jonathan D.; Singh, Bhuminder; Higginbotham, James N.; Franklin, Jeffrey L.; Gonzalez, Alfonso; Foelsch, Heike; Coffey, Robert J.
    Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.
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    Interpretable Machine Learning Model for Characterizing Magnetic Susceptibility based Biomarkers in First Episode Psychosis
    (2025) Franco, Pamela ; Montalba Zalaquett, Cristian Andres; Caulier-Cisterna, Raul; Milovic Fabregat, Carlos Andrés; Gonzalez, Alfonso ; Ramirez Mahaluf, Juan Pablo; Undurraga, Juan ; Salas, Rodrigo ; Crossley, Nicolás; Tejos Núñez, Cristián Andrés; Uribe, Sergio
    Altered neurochemicals in deep-brain nuclei, especially dopamine dysfunction, arelinked to psychosis. Quantitative Susceptibility Mapping (QSM) measures brainmagnetic susceptibility changes, including iron concentration, which affects dopaminepathways. This study used machine learning (ML) to analyze MRI data and build aclassifier distinguishing healthy individuals from First-Episode Psychosis (FEP)patients while predicting their response to antipsychotic treatment. A random forestmodel was trained, with the SHAP framework assessing feature importance andinterpretability. Hierarchical clustering identified relationships among features. Themodel achieved performance, with 76.48 ± 10.73% accuracy for classifying FEPpatients (based on R2* values in the nucleus accumbens and amygdala, and QSM inthe thalamus) and 76.43 ± 12.57% accuracy for predicting treatment response (basedon R2* values in the hippocampus, caudate, and putamen, and QSM in the amygdala).MRI-based biomarkers and ML could help tailor personalized treatments for FEPpatients, especially those not responding to standard therapies.
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    Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells
    (2007) Buvinic, Sonja; Bravo-Zehnder, Marcela; Boyer, Jose Luis; Huidobro-Toro, Juan Pablo; Gonzalez, Alfonso
    Epidermal growth factor receptor ( EGFR) function is transregulated by a variety of stimuli, including agonists of certain G-protein-coupled receptors (GPCRs). One of the most ubiquitous GPCRs is the P2Y(1) receptor (P2RY1, hereafter referred to as P2Y(1)R) for extracellular nucleotides, mainly ADP. Here, we show in tumoral HeLa cells and normal FRT epithelial cells that P2Y(1)R broadcasts mitogenic signals by transactivating the EGFR. The pathway involves PKC, Src and cell surface metalloproteases. Stimulation of P2Y(1)R for as little as 1560 minutes triggers mitogenesis, mirroring the half-life of extracellular ADP. Apyrase degradation of extracellular nucleotides and drug inhibition of P2Y(1)R, both reduced basal cell proliferation of HeLa and FRT cells, but not MDCK cells, which do not express P2Y(1)R. Thus, cell-released nucleotides constitute strong mitogenic stimuli, which act via P2Y(1)R. Strikingly, MDCK cells ectopically expressing P2Y(1)R display a highly proliferative phenotype that depends on EGFR activity associated with an increased level of EGFR, thus disclosing a novel aspect of GPCR-mediated regulation of EGFR function. These results highlight a role of P2Y(1)R in EGFR-dependent epithelial cell proliferation. P2Y(1)R could potentially mediate both trophic stimuli of basally released nucleotides and first-line mitogenic stimulation upon tissue damage. It could also contribute to carcinogenesis and serve as target for antitumor therapies.
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    P2Y(1) Receptor Activation Elicits Its Partition out of Membrane Rafts and Its Rapid Internalization from Human Blood Vessels: Implications for Receptor Signaling
    (AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2008) Norambuena, Andres; Poblete, M. Ines; Donoso, M. Veronica; Espinoza, C. Sofia; Gonzalez, Alfonso; Huidobro Toro, J. Pablo
    The nucleotide P2Y(1) receptor ( P2Y(1) R) is expressed in both the endothelial and vascular smooth muscle cells; however, its plasma membrane microregionalization and internalization in human tissues remain unknown. We report on the role of membrane rafts in P2Y(1) R signaling by using sodium carbonate or OptiPrep sucrose density gradients, Western blot analysis, reduction of tissue cholesterol content, and vasomotor assays of endothelium-denuded human chorionic arteries. In tissue extracts prepared either in sodium carbonate or OptiPrep, approximately 20 to 30% of the total P2Y(1) R mass consistently partitioned into raft fractions and correlated with vasomotor activity. Vessel treatment with methyl beta-cyclodextrin reduced the raft partitioning of the P2Y(1) R and obliterated the P2Y(1) R-mediated contractions but not the vasomotor responses elicited by either serotonin or KCl. Perfusion of chorionic artery segments with 100 nM 2-methylthio ADP or 10 nM [[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl] 2,3dihydroxybicyclo[3.1.0] hex-1-yl]methyl] diphosphoric acid mono ester trisodium salt (MRS 2365), a selective P2Y(1) R agonist, not only displaced within 4 min the P2Y(1) R localization out of membrane rafts but also induced its subsequent internalization. 2'-Deoxy-N-6-methyladenosine 3',5'-bisphosphate tetrasodium salt (MRS 2179), a specific P2Y(1) R antagonist, did not cause a similar displacement but blocked the agonist-induced exit from rafts. Neither adenosine nor uridine triphosphate displaced the P2Y(1) R from the membrane raft, further evidencing the pharmacodynamics of the receptor-ligand interaction. Vascular reactivity assays showed fading of the ligand-induced vasoconstrictions, a finding that correlated with the P2Y(1) R exit from raft domains and internalization. These results demonstrate in intact human vascular smooth muscle the association of the P2Y(1) R to membrane rafts, highlighting the role of this microdomain in P2Y(1) R signaling.
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    Pex3p-Dependent Peroxisomal Biogenesis Initiates in the Endoplasmic Reticulum of Human Fibroblasts
    (WILEY-BLACKWELL, 2009) Toro, Andres A.; Araya, Claudia A.; Cordova, Gonzalo J.; Arredondo, Cristian A.; Cardenas, Hugo G.; Moreno, Regina E.; Venegas, Alejandro; Koenig, Cecilia S.; Cancino, Jorge; Gonzalez, Alfonso; Santos, Manuel J.
    The mechanisms of peroxisomal biogenesis remain incompletely understood, specially regarding the role of the endoplasmic reticulum (ER) in human cells, where genetic disorders of peroxisome biogenesis lead to Zellweger syndrome (ZS). The Pex3p peroxisomal membrane protein (PMP) required for early steps of peroxisome biogenesis has been detected in the ER in yeast but not in mammalian cells. Here, we show that Pex3p-GFP expressed in a new ZS cell line (MR), which lacks peroxisomes due to a mutation in the PEX3 gene, localizes first in the ER and subsequently in newly formed peroxisomes. Pex3p bearing an artificial N-glycosylation site shows an electrophoretic shift indicative of ER targeting while en route to preformed peroxisomes in normal fibroblast. A signal peptide that forces its entry into the ER does not eliminate its capability to drive peroxisome biogenesis in ZS cells. Thus, Pex3p is able to drive peroxisome biogenesis from the ER and its ER pathway is not privative of ZS cells. Cross-expression experiments of Pex3p in GM623 cells lacking Pex16p or Pex16p in MR cells lacking Pex3p, showed evidence that Pex3p requires Pex16p for ER location but: is dispensable for the ER location of Pex16p. These results indicate that Pex3p follows the ER-to-peroxisomal route in mammalian cells and provides new clues to understand its function. J. Cell. Biochem. 107: 10831096, 2009. (C) 2009 Wiley-Liss, Inc.
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    Phosphatidic Acid Induces Ligand-independent Epidermal Growth Factor Receptor Endocytic Traffic through PDE4 Activation
    (AMER SOC CELL BIOLOGY, 2010) Norambuena, Andres; Metz, Claudia; Jung, Juan E.; Silva, Antonia; Otero, Carolina; Cancino, Jorge; Retamal, Claudio; Valenzuela, Juan C.; Soza, Andrea; Gonzalez, Alfonso
    Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.
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    Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis
    (2021) Metz, Claudia; Oyanadel, Claudia; Jung, Juan; Retamal, Claudio; Cancino, Jorge; Barra, Jonathan; Venegas, Jaime; Du, Guangwei; Soza, Andrea; Gonzalez, Alfonso
    Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.
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    Quantitative Susceptibility Mapping MRI in Deep-Brain Nuclei in First-Episode Psychosis
    (2023) García Saborit, Marisleydis; Jara Vallejos, Alejandro Antonio; Muñoz Camelo, Néstor Andrés; Milovic, Carlos; Tepper, Angeles; Alliende Correa, Luz María; Mena, Carlos; Iruretagoyena Bruce, Bárbara Arantzazu; Ramírez Mahaluf, Juan Pablo; Diaz, Camila; Nachar, Rubén; Castaneda, Carmen Paz; Gonzalez, Alfonso; Undurraga, Juan; Crossley, Nicolás; Tejos Núñez, Cristián Andrés
    Background Psychosis is related to neurochemical changes in deep-brain nuclei, particularly suggesting dopamine dysfunctions. We used an magnetic resonance imaging-based technique called quantitative susceptibility mapping (QSM) to study these regions in psychosis. QSM quantifies magnetic susceptibility in the brain, which is associated with iron concentrations. Since iron is a cofactor in dopamine pathways and co-localizes with inhibitory neurons, differences in QSM could reflect changes in these processes. Methods We scanned 83 patients with first-episode psychosis and 64 healthy subjects. We reassessed 22 patients and 21 control subjects after 3 months. Mean susceptibility was measured in 6 deep-brain nuclei. Using linear mixed models, we analyzed the effect of case-control differences, region, age, gender, volume, framewise displacement (FD), treatment duration, dose, laterality, session, and psychotic symptoms on QSM. Results Patients showed a significant susceptibility reduction in the putamen and globus pallidus externa (GPe). Patients also showed a significant R2* reduction in GPe. Age, gender, FD, session, group, and region are significant predictor variables for QSM. Dose, treatment duration, and volume were not predictor variables of QSM. Conclusions Reduction in QSM and R2* suggests a decreased iron concentration in the GPe of patients. Susceptibility reduction in putamen cannot be associated with iron changes. Since changes observed in putamen and GPe were not associated with symptoms, dose, and treatment duration, we hypothesize that susceptibility may be a trait marker rather than a state marker, but this must be verified with long-term studies.
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    Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE
    (2022) Sandoval, Lisette; Labarca, Mariana; Retamal, Claudio; Sanchez, Paula; Larrain, Juan; Gonzalez, Alfonso
    Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin-Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.
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    The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells
    (2013) Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; Gonzalez, Alfonso; Bonifacino, Juan S.
    An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the mu 1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous mu 1A and the epithelial-specific mu 1B. Previous studies led to the notion that mu 1A and mu 1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that mu 1A and mu 1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, mu 1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a mu 1B-dependent manner. We conclude that expression of distinct mu 1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface.
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    The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release
    (2014) Escudero, Claudia A.; Lazo, Oscal M.; Galleguillos, Carolina; Parraguez, Jose I.; Lopez-Verrilli, Maria A.; Cabeza, Carolina; Leon, Luisa; Saeed, Uzma; Retamal, Claudio; Gonzalez, Alfonso; Marzolo, Maria-Paz; Carter, Bruce D.; Court, Felipe A.; Bronfman, Francisca C.
    The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.