RESUMO
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.
Assuntos
Domínios PDZ/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Animais , Gerenciamento Clínico , Suscetibilidade a Doenças , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/etiologia , Doenças do Sistema Imunitário/metabolismo , Modelos Moleculares , Terapia de Alvo Molecular , Peptídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Células HEK293 , Humanos , Domínios PDZ , Ligação Proteica , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas da Matriz do Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/metabolismo , Domínios PDZ , Conformação Proteica , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Homologia de SequênciaRESUMO
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.
Assuntos
Polaridade Celular/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Domínios PDZ/imunologia , Animais , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Evasão da Resposta Imune , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Modelos Imunológicos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Vaccinia virus/imunologia , Vaccinia virus/patogenicidadeRESUMO
In this work, we identified the expression, regulation, and viral targeting of Scribble and Dlg1 in antigen-presenting cells. Scribble and Dlg1 belong to the family of PDZ (postsynaptic density (PSD95), disc large (Dlg), and zonula occludens (ZO-1)) proteins involved in cell polarity. The relevance of PDZ proteins in cellular functions is reinforced by the fact that many viruses interfere with host PDZ-dependent interactions affecting cellular mechanisms thus favoring viral replication. The functions of Scribble and Dlg have been widely studied in polarized cells such as epithelial and neuron cells. However, within the cells of the immune system, their functions have been described only in T and B lymphocytes. Here we demonstrated that Scribble and Dlg1 are differentially expressed during antigen-presenting cell differentiation and dendritic cell maturation. While both Scribble and Dlg1 seem to participate in distinct dendritic cell functions, both are targeted by the viral protein NS1 of influenza A in a PDZ-dependent manner in dendritic cells. Our findings suggest that these proteins might be involved in the mechanisms of innate immunity and/or antigen processing and presentation that can be hijacked by viral pathogens.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/imunologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Proteína 1 Homóloga a Discs-Large , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Membrana/genética , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/virologia , Domínios PDZ , Proteínas Supressoras de Tumor/genética , Proteínas não Estruturais Virais/genéticaRESUMO
OBJECTIVE: The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif. MATERIALS AND METHODS: The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence. RESULTS: The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151. CONCLUSION: The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.
Assuntos
Queratinas/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Células-Tronco/metabolismo , Transcrição Gênica , Motivos de Aminoácidos , Animais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Queratinócitos/citologia , Queratinas/genética , Camundongos Transgênicos , Domínios PDZ , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pele/metabolismo , Relação Estrutura-Atividade , Transcriptoma , Cicatrização , beta Catenina/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/enzimologia , Núcleo Celular/enzimologia , Códon sem Sentido , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Transporte Ativo do Núcleo Celular/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Citoplasma/enzimologia , Citoplasma/genética , Feminino , GTP Fosfo-Hidrolases/genética , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Domínios PDZ , RNA Polimerase II/genéticaRESUMO
Among all proteins localized in the Golgi apparatus, a two-PDZ (PSD95/DlgA/Zo-1) domain protein plays an important role in the assembly of the cisternae. This Golgi Reassembly and Stacking Protein (GRASP) has puzzled researchers due to its large array of functions and relevance in Golgi functionality. We report here a biochemical and biophysical study of the GRASP55/65 homologue in Cryptococcus neoformans (CnGRASP). Bioinformatic analysis, static fluorescence and circular dichroism spectroscopies, calorimetry, small angle X-ray scattering, solution nuclear magnetic resonance, size exclusion chromatography and proteolysis assays were used to unravel structural features of the full-length CnGRASP. We detected the coexistence of regular secondary structures and large amounts of disordered regions. The overall structure is less compact than a regular globular protein and the high structural flexibility makes its hydrophobic core more accessible to solvent. Our results indicate an unusual behavior of CnGRASP in solution, closely resembling a class of intrinsically disordered proteins called molten globule proteins. To the best of our knowledge, this is the first structural characterization of a full-length GRASP and observation of a molten globule-like behavior in the GRASP family. The possible implications of this and how it could explain the multiple facets of this intriguing class of proteins are discussed.
Assuntos
Proteínas de Transporte/química , Proteínas de Membrana/química , Conformação Proteica , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Domínios PDZ , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Desdobramento de Proteína , Soluções , Relação Estrutura-AtividadeRESUMO
Allosteric communication in proteins is a fundamental and yet unresolved problem of structural biochemistry. Previous findings, from computational biology ( Ota, N.; Agard, D. A. J. Mol. Biol. 2005 , 351 , 345 - 354 ), have proposed that heat diffuses in a protein through cognate protein allosteric pathways. This work studied heat diffusion in the well-known PDZ-2 protein, and confirmed that this protein has two cognate allosteric pathways and that heat flows preferentially through these. Also, a new property was also observed for protein structures: heat diffuses asymmetrically through the structures. The underling structure of this asymmetrical heat flow was a normal length hydrogen bond (â¼2.85 Å) that acted as a thermal rectifier. In contrast, thermal rectification was compromised in short hydrogen bonds (â¼2.60 Å), giving rise to symmetrical thermal diffusion. Asymmetrical heat diffusion was due, on a higher scale, to the local, structural organization of residues that, in turn, was also mediated by hydrogen bonds. This asymmetrical/symmetrical energy flow may be relevant for allosteric signal communication directionality in proteins and for the control of heat flow in materials science.