RESUMEN
Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia wStri, isolated from Laodelphax striatellus, was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that wStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by wStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into wStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in wStri-infected cells. This study's findings increase the potential for application of wStri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection.IMPORTANCE Dengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont, Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use of Wolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.
Asunto(s)
Aedes/virología , Antibiosis , Mosquitos Vectores/virología , Replicación Viral , Wolbachia/fisiología , Virus Zika/fisiología , Animales , Virus Chikungunya/genética , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/fisiología , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Virus del Dengue/fisiología , Masculino , Internalización del Virus , Wolbachia/genética , Virus Zika/genética , Virus Zika/crecimiento & desarrolloRESUMEN
Liver-resident CD8+ T cells are highly motile cells that patrol the vasculature and provide protection against liver pathogens. A key question is: how can these liver CD8+ T cells be simultaneously present in the circulation and tissue-resident? Because liver-resident T cells do not express CD103 - a key integrin for T cell residence in epithelial tissues - we investigated other candidate adhesion molecules. Using intra-vital imaging we found that CD8+ T cell patrolling in the hepatic sinusoids is dependent upon LFA-1-ICAM-1 interactions. Interestingly, liver-resident CD8+ T cells up-regulate LFA-1 compared to effector-memory cells, presumably to facilitate this behavior. Finally, we found that LFA-1 deficient CD8+ T cells failed to form substantial liver-resident memory populations following Plasmodium or LCMV immunization. Collectively, our results demonstrate that it is adhesion through LFA-1 that allows liver-resident memory CD8+ T cells to patrol and remain in the hepatic sinusoids.
RESUMEN
Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.
Asunto(s)
Antivirales/metabolismo , Gránulos Citoplasmáticos/metabolismo , Virus Vaccinia/patogenicidad , Animales , Antivirales/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Chlorocebus aethiops , Cricetinae , ADN Helicasas , Células HeLa , Humanos , Ratones , Mutación , Orthopoxvirus/genética , Orthopoxvirus/patogenicidad , Fosforilación , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/genética , Antígeno Intracelular 1 de las Células T , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Virus Vaccinia/genética , Células Vero , Proteínas Virales/genética , Replicación Viral , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
BACKGROUND: Drugs that block platelet-platelet and platelet-fibrin interactions via the alpha(IIb)beta(3) (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. OBJECTIVES: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the alpha(IIb)beta(3) receptor in an activated state. It compared the effects on platelet function and alpha(IIb)beta(3) conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine-glycine-aspartate (RGD) disintegrin. METHODS: The integrin antagonist concentrations required to saturate platelets and to block platelet-platelet and platelet-fibrin interactions were determined by flow cytometry, aggregometry, and clot-based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of alpha(IIb)beta(3). Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. RESULTS: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing alpha(IIb)beta(3) conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating alpha(IIb)beta(3):echistatin complex. CONCLUSIONS: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the alpha(IIb)beta(3) receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists.
Asunto(s)
Plaquetas/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/efectos de los fármacos , Administración Oral , Alanina/farmacología , Amidinas/farmacología , Unión Competitiva , Plaquetas/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Eptifibatida , Femenino , Fibrinógeno/metabolismo , Humanos , Técnicas In Vitro , Inyecciones Intravenosas , Péptidos y Proteínas de Señalización Intercelular , Isoxazoles/farmacología , Cinética , Masculino , Modelos Moleculares , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/metabolismo , Pruebas de Función Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Conformación Proteica/efectos de los fármacos , Pirrolidinas/farmacología , Valores de Referencia , Relación Estructura-Actividad , Tirofibán , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.
Asunto(s)
Proteínas Portadoras , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Plantas/metabolismo , Proteínas/química , Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Antígenos de Diferenciación , Apoptosis , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Biblioteca de Genes , Humanos , Cinética , Modelos Genéticos , Fosfoproteínas Fosfatasas , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteína Fosfatasa 1 , Proteínas Recombinantes/metabolismo , Sciuridae , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
We investigated the role of postsynaptic protein phosphatase 1 (PP1) in regulating synaptic strength by loading CA1 pyramidal cells either with peptides that disrupt PP1 binding to synaptic targeting proteins or with active PP1. The peptides blocked synaptically evoked LTD but had no effect on basal synaptic currents mediated by either AMPA or NMDA receptors. They did, however, cause an increase in synaptic strength following the induction of LTD. Similarly, PP1 had no effect on basal synaptic strength but enhanced LTD. In cultured neurons, synaptic activation of NMDA receptors increased the proportion of PP1 localized to synapses. These results suggest that PP1 does not significantly regulate basal synaptic strength. Appropriate NMDA receptor activation, however, allows PP1 to gain access to synaptic substrates and be recruited to synapses where its activity is necessary for sustaining LTD.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Sinapsis/enzimología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/citología , Hipocampo/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Inhibición Neural/fisiología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Proteína Fosfatasa 1 , Células Piramidales/fisiología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.
Asunto(s)
Hipocampo/metabolismo , Activación del Canal Iónico/fisiología , Potenciación a Largo Plazo/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Ritmo Teta , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Potenciales de Acción/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Plasticidad Neuronal/fisiología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 1 , Proteínas/genética , Proteínas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transmisión Sináptica/efectos de los fármacosRESUMEN
Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIalpha (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Memoria/fisiología , Plasticidad Neuronal/fisiología , Membranas Sinápticas/metabolismo , Compuestos de Anilina , Animales , Animales Recién Nacidos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/efectos de los fármacos , Células Cultivadas , Estimulación Eléctrica , Colorantes Fluorescentes , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/ultraestructura , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Fosforilación , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/ultraestructura , XantenosRESUMEN
Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Cartilla de ADN , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Unión Proteica , Proteína Fosfatasa 1 , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.
Asunto(s)
Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/metabolismo , Bovinos , Membrana Celular/metabolismo , Cromatografía de Afinidad , Humanos , Datos de Secuencia Molecular , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/aislamiento & purificación , Fosforilación , Unión Proteica , Proteína Fosfatasa 1 , Conejos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Médula Espinal/metabolismoRESUMEN
Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.
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Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Piranos , Compuestos de Espiro , Alquenos/farmacología , Secuencia de Aminoácidos , Antifúngicos/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Microcistinas , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/genética , Polienos , Unión Proteica , Proteína Fosfatasa 1 , Estructura Secundaria de Proteína , Proteínas/farmacología , Pironas , Homología de Secuencia de AminoácidoAsunto(s)
Sustitutos Sanguíneos/metabolismo , Proteínas Portadoras/metabolismo , Hemoglobinas/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Proteínas Portadoras/aislamiento & purificación , Bovinos , Reactivos de Enlaces Cruzados , Humanos , Masculino , Ácido Poliglutámico , Polímeros , Ratas , Superóxido Dismutasa/aislamiento & purificaciónRESUMEN
Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta12-beta13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta12-beta13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta12-beta13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta12-beta13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta12-beta13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.
Asunto(s)
Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Proteínas/metabolismo , Sitios de Unión , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Inhibidores Enzimáticos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Toxinas Marinas , Microcistinas , Modelos Moleculares , Mutagénesis , Ácido Ocadaico/farmacología , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 1Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas/aislamiento & purificación , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Músculo Esquelético/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Biosíntesis de Proteínas , Proteína Fosfatasa 1 , Proteínas/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Portadoras , AMP Cíclico/metabolismo , Hipocampo/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Potenciación a Largo Plazo , Fosfoproteínas Fosfatasas/metabolismo , Sinapsis/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estimulación Eléctrica , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Masculino , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteína Fosfatasa 1 , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Tionucleótidos/farmacologíaRESUMEN
The phosphorylase phosphatase activity of protein phosphatase 1 (PP1) catalytic subunit from freshly purified rabbit skeletal muscle was inhibited by MnCl2. Prolonged storage or inhibition by nonspecific phosphatase inhibitors ATP, sodium pyrophosphate, and NaF converted the muscle PP1 to a form that required Mn2+ for enzyme activity. Recombinant PP1 catalytic subunit expressed in Escherichia coli was also a Mn2+-dependent enzyme. While native PP1 was inhibited by the phosphoprotein inhibitor I (I-1), with an IC50 of 1 nM, 40-50-fold higher concentrations of I-1 were required to inhibit the Mn2+-dependent PP1 enzymes. Conversion to the Mn2+-dependent state was accompanied by a 20-fold increase in PP1's ability to dephosphorylate and inactivate I-1. Inhibition by thiophosphorylated I-1 established that dephosphorylation does not play a significant role in I-1's reduced potency as an inhibitor of Mn2+-dependent PP1. The Mn2+-dependent PP1 enzymes were poorly inhibited by N-terminal phosphopeptides of I-1, indicating their impaired interaction with the I-1 functional domain. Mutation of a residue conserved in I-1 and DARPP-32, a structurally related PP1 inhibitor, preferentially attenuated I-1's activity as an inhibitor of Mn2+-dependent PP1. These data showed that, in addition to changes in its catalytic properties, Mn2+-dependent PP1 was modified in its interaction with I-1 at a site that was distinct from its catalytic domain. Our studies suggest that conversion to a Mn2+-dependent state alters multiple structural elements in PP1 catalytic subunit that together define its regulation by I-1.