RESUMEN

PDZ (PSD-95 [postsynaptic density protein 95]/Dlg [Discs large]/ZO-1 [zonula occludens-1]) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks and are mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens that express proteins containing PDZ-binding motifs (PDZbms). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of 7 host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of 3 of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and interleukin-16), in human monocyte-derived macrophages and in dendritic cells upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells, and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, supports the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. An inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed.

Asunto(s)

Proteínas de la Envoltura de Coronavirus , Células Dendríticas , Macrófagos , Dominios PDZ , SARS-CoV-2 , Humanos , Células Dendríticas/metabolismo , Células Dendríticas/virología , SARS-CoV-2/metabolismo , Proteínas de la Envoltura de Coronavirus/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , COVID-19/metabolismo , COVID-19/virología , Unión Proteica , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular

RESUMEN

Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF's catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.

Asunto(s)

Melanoma , Neoplasias Cutáneas , Humanos , Melanoma/genética , Células Endoteliales/metabolismo , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo

RESUMEN

The COVID-19 pandemic caused by the SARS-CoV-2 virus is still a global health concern. Several spike (S) protein-based vaccines have been developed that efficiently protect the human population against severe forms of COVID-19. However, some SARS-CoV-2 variants of concern (VOCs) have emerged that evade the protective effect of vaccine-induced antibodies. Therefore, efficient and specific antiviral treatments to control COVID-19 are indispensable. To date, two drugs have been approved for mild COVID-19 treatment; nevertheless, more drugs, preferably broad-spectrum and ready-to-use therapeutic agents for new pandemics, are needed. Here, I discuss the PDZ-dependent protein-protein interactions of the viral E protein with host proteins as attractive alternatives for the development of antivirals against coronavirus.

Asunto(s)

COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Pandemias/prevención & control , Tratamiento Farmacológico de COVID-19 , Antivirales/farmacología , Antivirales/uso terapéutico , Anticuerpos Antivirales , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo

RESUMEN

BACKGROUND: Oncogenic Human Papillomaviruses (HPVs) base their transforming potential on the action of both E6 and E7 viral oncoproteins, which perform cooperative or antagonistic actions and thus interfere with a variety of relevant cellular targets. Among them, the expression of some PDZ-containing polarity proteins, as DLG1 and hScrib, is altered during the HPV life cycle and the consequent malignant transformation. Together with the well-established interference of E6 with PDZ proteins, we have recently shown that E7 viral oncoprotein is also responsible for the changes in abundance and localization of DLG1 observed in HPV-associated lesions. Given that the mechanisms involved remained only partially understood, we here thoroughly analyse the contribution of a crucial E7 post-translational modification: its CKII-dependent phosphorylation. Moreover, we extended our studies to hScrib, in order to investigate possible conserved regulatory events among diverse PDZ targets of HPV. METHODS: We have acutely analysed the expression of DLG1 and hScrib in restrictive conditions for E7 phosphorylation by CKII in epithelial culture cells by western blot and confocal fluorescence microscopy. We made use of genome-edited HPV-positive cells, specific inhibitors of CKII activity and transient expression of the viral oncoproteins, including a mutant version of E7. RESULTS: We here demonstrate that the functional phosphorylation of E7 oncoprotein by the CKII cellular kinase, a key regulatory event for its activities, is also crucial to counteract the E6-mediated degradation of the PDZ-polarity protein DLG1 and to promote its subcellular redistribution. Moreover, we show that the CKII-dependent phosphorylation of E7 is able to control the expression of another PDZ target of HPV: hScrib. Remarkably, we found this is a shared feature among different oncogenic HPV types, suggesting a common path towards viral pathogenesis. CONCLUSIONS: The present study sheds light into the mechanisms behind the misexpression of PDZ-polarity proteins during HPV infections. Our findings stress the relevance of the CKII-mediated regulation of E7 activities, providing novel insights into the joint action of HPV oncoproteins and further indicating a conserved and most likely crucial mechanism during the viral life cycle and the associated transformation.

Asunto(s)

Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Transformación Celular Neoplásica , Humanos , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Procesamiento Proteico-Postraduccional

RESUMEN

The PDZ (PSD95, Dlg and ZO-1) genes encode proteins that primarily function as scaffolds of diverse signaling pathways. To date, 153 PDZ genes have been identified in the human genome, most of which have multiple protein isoforms widely studied in epithelial and neural cells. However, their expression and function in immune cells have been poorly studied. Herein, we aimed to assess the transcriptional profiles of 83 PDZ genes in human macrophages (Mɸ) and dendritic cells (DCs) and changes in their relative expression during cell PRR stimulation. Significantly distinct PDZ gene transcriptional profiles were identified under different stimulation conditions. Furthermore, a distinct PDZ gene transcriptional signature was found in Mɸ and DCs under the same phagocytic stimuli. Notably, more than 40 PDZ genes had significant changes in expression, with potentially relevant functions in antigen-presenting cells (APCs). Given that several PDZ proteins are targeted by viral products, our results support that many of these proteins might be viral targets in APCs as part of evasion mechanisms. Our results suggest a distinct requirement for PDZ scaffolds in Mɸ and DCs signaling pathways activation. More assessments on the functions of PDZ proteins in APCs and their role in immune evasion mechanisms are needed.

Asunto(s)

Evasión Inmune , Macrófagos , Células Dendríticas , Humanos , Macrófagos/metabolismo , Transducción de Señal

RESUMEN

LMO7 is a multifunctional PDZ-LIM protein that can interact with different molecular partners and is found in several intracellular locations. The aim of this work was to shed light on LMO7 evolution, alternative transcripts, protein structure and gene regulation through multiple in silico analyses. We also explored the intracellular distribution of the LMO7 protein in chicken and zebrafish embryonic skeletal muscle cells by means of confocal fluorescence microscopy. Our results revealed a single LMO7 gene in mammals, sauropsids, Xenopus and in the holostean fish spotted gar while two lmo7 genes (lmo7a and lmo7b) were identified in teleost fishes. In addition, several different transcripts were predicted for LMO7 in human and in major vertebrate model organisms (mouse, chicken, Xenopus and zebrafish). Bioinformatics tools revealed several structural features of the LMO7 protein including intrinsically disordered regions. We found the LMO7 protein in multiple intracellular compartments in chicken and zebrafish skeletal muscle cells, such as membrane adhesion sites and the perinuclear region. Curiously, the LMO7 protein was detected within the nuclei of muscle cells in chicken but not in zebrafish. Our data showed that a conserved regulatory element may be related to muscle-specific LMO7 expression. Our findings uncover new and important information about LMO7 and open new challenges to understanding how the diverse regulation, structure and distribution of this protein are integrated into highly complex vertebrate cellular milieux, such as skeletal muscle cells.

Asunto(s)

Evolución Molecular , Proteínas con Dominio LIM/metabolismo , Modelos Moleculares , Fibras Musculares Esqueléticas/metabolismo , Factores de Transcripción/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Pollos , Simulación por Computador , Humanos , Proteínas con Dominio LIM/genética , Ratones , Modelos Animales , Conformación Proteica , Factores de Transcripción/genética , Pez Cebra

RESUMEN

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein-protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

Asunto(s)

Dominios PDZ/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Animales , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/etiología , Enfermedades del Sistema Inmune/metabolismo , Modelos Moleculares , Terapia Molecular Dirigida , Péptidos/uso terapéutico , Unión Proteica/efectos de los fármacos , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.

Asunto(s)

Proteínas de la Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Codón sin Sentido , Hipotiroidismo Congénito/genética , Hipotiroidismo Congénito/metabolismo , Secuencia Conservada , Perros , Endosomas/metabolismo , Células HEK293 , Humanos , Lisosomas/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Dominios PDZ/genética , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Simportadores/química , Simportadores/genética , Glándula Tiroides/metabolismo , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética

RESUMEN

Gßγ marks the inner side of the plasma membrane where chemotactic GPCRs activate Rac to lead the assembly of actin filaments that push the cell to move forward. Upon dissociation from heterotrimeric Gi, Gßγ recruits and activates P-Rex1, a Rac guanine nucleotide exchange factor (RacGEF). This cytosolic chemotactic effector is kept inactive by intramolecular interactions. The mechanism by which Gßγ stimulates P-Rex1 has been debated. We hypothesized that Gßγ activates P-Rex1 by a two-step mechanism based on independent interaction interfaces to recruit and unroll this RacGEF. Using pulldown assays, we found that Gßγ binds P-Rex1-DH/PH as well as PDZ-PDZ domains. These domains and the DEP-DEP tandem interact among them and dissociate upon binding with Gßγ, arguing for a stimulatory allosteric effect. In addition, P-Rex1 catalytic activity is inhibited by its C-terminal domain. To discern P-Rex1 recruitment from activation, we studied Q-Rhox, a synthetic RhoGEF having the PDZ-RhoGEF catalytic DH/PH module, insensitive to Gßγ, swapped into P-Rex1. Gßγ recruited Q-Rhox to the plasma membrane, indicating that Gßγ/PDZ-PDZ interaction interface plays a role on P-Rex1 recruitment. In conclusion, we reconcile previous findings and propose a mechanistic model of P-Rex1 activation; accordingly, Gßγ recruits P-Rex1 via the Gßγ/PDZ-PDZ interface followed by a second contact involving the Gßγ/DH/PH interface to unleash P-Rex1 RacGEF activity at the plasma membrane.

Asunto(s)

Membrana Celular/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Citoesqueleto de Actina/metabolismo , Células HEK293 , Humanos , Dominios PDZ , Unión Proteica , Transducción de Señal

RESUMEN

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13 Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

Asunto(s)

Movimiento Celular , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Dominios Homólogos a Pleckstrina/genética , Seudópodos/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Proteína de Unión al GTP cdc42/metabolismo , Animales , Línea Celular , Humanos , Ratones , Fosforilación

RESUMEN

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.

Asunto(s)

Presentación de Antígeno , Células Dendríticas/inmunología , Homólogo 1 de la Proteína Discs Large/fisiología , Proteínas de la Membrana/fisiología , Proteínas Supresoras de Tumor/fisiología , Inmunidad Adaptativa , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígeno B7-2/biosíntesis , Antígeno B7-2/genética , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Homólogo 1 de la Proteína Discs Large/antagonistas & inhibidores , Homólogo 1 de la Proteína Discs Large/genética , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad Innata , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/genética , Interleucina-12/metabolismo , Interleucina-6/biosíntesis , Interleucina-6/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Densidad Postsináptica/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Antígeno CD83

RESUMEN

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD-95, DLG, and ZO-1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual ß3 α1 ß4 ß5 α2 ß6 ß1 ß2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable ß2 -strand and a more flexible and unstable α2 -helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.

Asunto(s)

Proteínas de la Matriz de Golgi/química , Proteínas de la Matriz de Golgi/metabolismo , Dominios PDZ , Conformación Proteica , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Homología de Secuencia

RESUMEN

PDLIM's protein family is involved in the rearrangement of the actin cytoskeleton. In the present study, we describe the localization of PDLIM1 in chicken photoreceptors. This study provides evidence that this protein is present at the cone pedicles, as well as in other synapses of the chicken retina. Here, we demonstrate the expression pattern of PDLIM1 through immunofluorescence staining, immunoblots, subcellular fractionation, and immunoprecipitation experiments. Also, we consider the possibility that PDLIM1 may be involved in the synaptic vesicle endocytosis and/or the presynaptic trafficking of synaptic vesicles back to the nonready releasable pool. This endocytotic/exocytotic coupling requires a tight link between exocytic vesicle fusion at defined release sites and endocytic retrieval of synaptic vesicle membranes. In turn, photoreceptor ribbon synaptic structure depends on the cytoskeleton arrangement, both at the active zone-related with exocytosis-as well as at the endocytic zone-periactive zone. To our knowledge, the PDLIM1 protein has not been observed in the pre synapses of the retina. Thus, the present study describes the expression and subcellular localization of PDLIM1 for the first time, as well as its modulation by visual environment in the chicken retina.

Asunto(s)

Proteínas Aviares/metabolismo , Proteínas con Dominio LIM/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Sinapsis/metabolismo , Animales , Pollos

RESUMEN

PDZ proteins are highly conserved through evolution; the principal function of this large family of proteins is to assemble protein complexes that are involved in many cellular processes, such as cell-cell junctions, cell polarity, recycling, or trafficking. Many PDZ proteins that have been identified as targets of viral pathogens by promoting viral replication and spread are also involved in epithelial cell polarity. Here, we briefly review the PDZ polarity proteins in cells of the immune system to subsequently focus on our hypothesis that the viral PDZ-dependent targeting of PDZ polarity proteins in these cells may alter the cellular fitness of the host to favor that of the virus; we further hypothesize that this modification of the cellular fitness landscape occurs as a common and widespread mechanism for immune evasion by viruses and possibly other pathogens.-Gutiérrez-González, L. H., Santos-Mendoza, T. Viral targeting of PDZ polarity proteins in the immune system as a potential evasion mechanism.

Asunto(s)

Polaridad Celular/inmunología , Interacciones Microbiota-Huesped/inmunología , Dominios PDZ/inmunología , Animales , Virus de la Encefalitis Transmitidos por Garrapatas/inmunología , Virus de la Encefalitis Transmitidos por Garrapatas/patogenicidad , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Humanos , Evasión Inmune , Virus de la Influenza A/inmunología , Virus de la Influenza A/patogenicidad , Modelos Inmunológicos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Virus Vaccinia/inmunología , Virus Vaccinia/patogenicidad

RESUMEN

Human T cell leukemia virus (HTLV)-1 Tax (Tax) protein is very important in viral replication and cell transformation. Tax localizes in the nucleus and cytoplasm in association with organelles. Some activities of Tax depend on interactions with PDZ (PSD-95/Discs Large/Z0-1) domain-containing proteins such as Discs large protein 1 (DLG1) which is involved in cell polarity and proliferation. The DLG1 interaction results in a cytoplasmic co-localization pattern resembling vesicular aggregates, the nature of which is still unknown. To further explore the role of PDZ proteins in HTLV-1 cell transformation, we deeply investigated the Tax-DLG1 association. By fluorescence resonance energy transfer (FRET), we detected, for the first time, the direct binding of Tax to DLG1 within the cell. We showed that the interaction specifically affects the cellular distribution of not only DLG1, but also Tax. After studying different cell structures, we demonstrated that the aggregates distribute into the Golgi apparatus in spatial association with the microtubule-organizing center (MTOC). This study contributes to understand the biological significance of Tax-PDZ interactions.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Polaridad Celular , Productos del Gen tax/metabolismo , Virus Linfotrópico T Tipo 1 Humano/fisiología , Proteínas de la Membrana/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Núcleo Celular/metabolismo , Transformación Celular Viral , Citoplasma/metabolismo , Homólogo 1 de la Proteína Discs Large , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Productos del Gen tax/genética , Aparato de Golgi/metabolismo , Células HEK293 , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Microscopía , Agregado de Proteínas , Transporte de Proteínas , Replicación Viral

RESUMEN

Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting.

Asunto(s)

Neoplasias de la Mama/enzimología , Núcleo Celular/enzimología , Codón sin Sentido , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Polimerasa II/metabolismo , Transporte Activo de Núcleo Celular/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Núcleo Celular/genética , Citoplasma/enzimología , Citoplasma/genética , Femenino , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Dominios PDZ , ARN Polimerasa II/genética

RESUMEN

BACKGROUND: High-risk human papillomaviruses (HPVs) are strongly associated with the development of some malignancies. The E6 and E7 viral oncoproteins are the primary proteins responsible for cell homeostasis alteration and immortalization. Furthermore, the E6 protein from high-risk HPVs can interact with the PDZ (PSD-90/Dlg/ZO-1) domains of cellular proteins, triggering cell transformation. One protein that is associated with pathological conditions and has a PDZ domain is the protease HTRA1 (high temperature requirement 1). This protein is poorly expressed in some cancers, suggesting a tumor suppressor role. The aim of this study was to evaluate the effect of HTRA1 overexpression in HPV16-positive (CasKi) and HPV-negative (C33) cervical cell lines. METHODS: The cells were transfected with a vector containing the HTRA1 ORF or an empty vector. HTRA1 overexpression was confirmed by qRT-PCR. The cells were subjected to cell proliferation, colony formation, apoptosis and cell cycle assays. RESULTS: C33 cells expressing HTRA1 grew significantly fewer colonies and showed less proliferation than cells without HTRA1 expression. In contrast, in the CasKi cells overexpressing HTRA1, there was an increase in the cell growth rate and in the colonies density compared to cells expressing low levels of HTRA1. An apoptosis assay showed that HTRA1 does not interfere with the apoptosis rate in these cells. A cell cycle immunofluorescence assay revealed more CasKi cells overexpressing HTRA1 in the S phase and more C33 HTRA1-transfected cells in the G0/G1 phase, suggesting that HTRA1 plays different roles in the cell cycle progression of these cells. CONCLUSIONS: HTRA1 overexpression prevents cell proliferation in the HPV-negative cell line and increases cell proliferation in the HPV-positive cell line. Although the E6/HTRA1 interaction has already been described in the literature, more studies are required to confirm whether the present functional findings are a result of this interaction.

Asunto(s)

Proliferación Celular , Transformación Celular Neoplásica/patología , Papillomaviridae/patogenicidad , Infecciones por Papillomavirus/patología , Serina Endopeptidasas/metabolismo , Neoplasias del Cuello Uterino/patología , Apoptosis , Ciclo Celular , Transformación Celular Neoplásica/metabolismo , Femenino , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología

RESUMEN

The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3ß, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.

RESUMEN

Tight junctions (TJs) are highly specialized membrane domains involved in many important cellular processes such as the regulation of the passage of ions and macromolecules across the paracellular space and the establishment of cell polarity in epithelial cells. Over the past few years there has been increasing evidence that different components of the TJs can be hijacked by viruses in order to complete their infectious cycle. Viruses from at least nine different families of DNA and RNA viruses have been reported to use TJ proteins in their benefit. For example, TJ proteins such as JAM-A or some members of the claudin family of proteins are used by members of the Reoviridae family and hepatitis C virus as receptors or co-receptors during their entry into their host cells. Reovirus, in addition, takes advantage of the TJ protein Junction Adhesion Molecule-A (JAM-A) to achieve its hematogenous dissemination. Some other viruses are capable of regulating the expression or the localization of TJ proteins to induce cell transformation or to improve the efficiency of their exit process. This review encompasses the importance of TJs for viral entry, replication, dissemination, and egress, and makes a clear statement of the importance of studying these proteins to gain a better understanding of the replication strategies used by viruses that infect epithelial and/or endothelial cells.

Asunto(s)

Virus ADN/fisiología , Virus ARN/fisiología , Uniones Estrechas/virología , Internalización del Virus , Liberación del Virus , Replicación Viral , Animales , Humanos , Receptores Virales/metabolismo , Proteínas de Uniones Estrechas/metabolismo

RESUMEN

The aim of the present study was to investigate novel molecular markers that could improve the diagnosis of ovarian cancer patients or be of predictive value. The sequence of the sodium-hydrogen antiporter 3 regulator 1 (SLC9A3R1) gene that codes for the PDZ2 motif of the Na+/H+ exchanger regulatory factor 1 (NHERF1) protein was analyzed. Changes in migration and cell transformation, and alterations of growth factor signaling pathways have been described in cells lacking endogenous NHERF1 or expressing an isoform lacking the function of the PDZ2 domain. Exons 2 and 3, together with flanking intronic sequences of the SLC9A3R1 gene, were amplified and bi-directionally sequenced in 31 primary tumor samples from epithelial ovarian cancer patients. In total, 3 different previously undescribed mutations were detected in 8 out of 31 serous adenocarcinoma tumor samples (25.8%). Bioinformatics analysis predicted a significant effect in the splicing process as a result of the mutations that could disrupt the NHERF1 PDZ2 domain. Point mutations in consensus splicing recognition are a major cause of the splicing defects that are found in several diseases, including cancer. It has previously been shown that a lack of exon 2 and disruption of the PDZ2 domain contribute to cell transformation and leads to modifications in the physiological regulation of the conformational state of NHERF1. Further studies in bigger groups of ovarian cancer patients will determine the importance of this mutation in disease progression and patient survival.