RESUMO
The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein-centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein-coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., "self-activating" behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.
Assuntos
Amoeba , Naegleria fowleri , Proteínas RGS , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/metabolismo , Naegleria fowleri/metabolismo , Proteínas RGS/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologiaRESUMO
G protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors, regulate a wide range of physiological processes, and are the major targets of pharmaceutical drugs. Canonical signaling from GPCRs is relayed to intracellular effector proteins by trimeric G proteins, composed of α, ß, and γ subunits (Gαßγ). Here, we report that G protein ß subunits (Gß) bind to DDB1 and that Gß2 targets GRK2 for ubiquitylation by the DDB1-CUL4A-ROC1 ubiquitin ligase. Activation of GPCR results in PKA-mediated phosphorylation of DDB1 at Ser645 and its dissociation from Gß2, leading to increase of GRK2 protein. Deletion of Cul4a results in cardiac hypertrophy in male mice that can be partially rescued by the deletion of one Grk2 allele. These results reveal a non-canonical function of the Gß protein as a ubiquitin ligase component and a mechanism of feedback regulation of GPCR signaling.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Estabilidade Proteica , Proteólise , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
The T1R and T2R families of G protein-coupled receptors (GPCRs) initiate tastant perception by signaling via guanine nucleotide exchange and hydrolysis performed by associated heterotrimeric G proteins (Gαßγ). Heterotrimeric G protein signal termination is sped up by Gα-directed GTPase-accelerating proteins (GAPs) known as the Regulators of G protein Signaling (RGS proteins). Of this family, RGS21 is highly expressed in lingual epithelial cells and we have shown it acting in vitro to decrease the potency of bitterants on cultured cells. However, constitutive RGS21 loss in mice reduces organismal response to GPCR-mediated tastants-opposite to expectations arising from observed in vitro activity of RGS21 as a GAP and inhibitor of T2R signaling. Here, we show reduced quinine aversion and reduced sucrose preference by mice lacking RGS21 does not result from post-ingestive effects, as taste-salient brief-access tests confirm the reduced bitterant aversion and reduced sweetener preference seen using two-bottle choice testing. Eliminating Rgs21 expression after chemosensory system development, via tamoxifen-induced Cre recombination in eight week-old mice, led to a reduction in quinine aversive behavior that advanced over time, suggesting that RGS21 functions as a negative regulator to sustain stable bitter tastant reception. Consistent with this notion, we observed downregulation of multiple T2R proteins in the lingual tissue of Rgs21-deficient mice. Reduced tastant-mediated responses exhibited by mice lacking Rgs21 expression either since birth or in adulthood has highlighted the potential requirement for a GPCR GAP to maintain the full character of tastant signaling, likely at the level of mitigating receptor downregulation.
Assuntos
Proteínas RGS , Animais , Proteínas de Ligação ao GTP , Camundongos , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , PaladarRESUMO
The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.
Assuntos
Autofagia/fisiologia , Interferon Tipo I/biossíntese , Proteínas Mitocondriais/fisiologia , Fator Tu de Elongação de Peptídeos/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Aminoácidos , Animais , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/fisiologia , Citocinas/biossíntese , Citocinas/genética , Proteína DEAD-box 58 , RNA Helicases DEAD-box/fisiologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Interferon Tipo I/genética , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Complexos Multiproteicos/fisiologia , Fator Tu de Elongação de Peptídeos/química , Mapeamento de Interação de Proteínas , Proteínas/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Organismos Livres de Patógenos Específicos , Vesiculovirus/fisiologiaRESUMO
A serious adverse effect of prescription opioid analgesics is addiction, both to these analgesics and to illicit drugs like heroin that also activate the µ-opioid receptor (MOR). Opioid use disorder (OUD) and opioid overdose deaths represent a current American health crisis, and the prescription of opioid analgesics has contributed significantly to this crisis. While prescription opioids are highly effective analgesics, there currently exists no facile way to use them for extended periods without the risk of addiction. If addiction caused by MOR-targeting analgesics could be blocked by blending in a new "antiaddiction" ingredient that does not diminish analgesia and does not introduce its own therapeutically limiting side effects, then continued clinical use of prescription opioids for treating pain could be maintained (or even enhanced) instead of curtailed. In this narrative review, we contextualize this hypothesis, first with a brief overview of the current American opioid addiction crisis. The neurobiology of 2 key receptors in OUD development, MOR and the κ-opioid receptor (KOR), is then discussed to highlight the neuroanatomical features and circuitry in which signal transduction from these receptors lie in opposition-creating opportunities for pharmacological intervention in curtailing the addictive potential of MOR agonism. Prior findings with mixed MOR/KOR agonists are considered before exploring new potential avenues such as biased KOR agonists. New preclinical data are highlighted, demonstrating that the G protein-biased KOR agonist nalfurafine reduces the rewarding properties of MOR-targeting analgesics and enhances MOR-targeting analgesic-induced antinociception. Finally, we discuss the recent discovery that a regulator of G protein signaling (namely, RGS12) is a key component of signaling bias at KOR, presenting another drug discovery target toward identifying a single agent or adjuvant to be added to traditional opioid analgesics that could reduce or eliminate the addictive potential of the latter drug.
Assuntos
Desenho de Fármacos , Antagonistas de Entorpecentes/farmacologia , Nociceptividade/efeitos dos fármacos , Dor Nociceptiva/tratamento farmacológico , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Animais , Humanos , Estrutura Molecular , Antagonistas de Entorpecentes/efeitos adversos , Antagonistas de Entorpecentes/química , Dor Nociceptiva/metabolismo , Dor Nociceptiva/fisiopatologia , Dor Nociceptiva/psicologia , Transtornos Relacionados ao Uso de Opioides/etiologia , Proteínas RGS/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria). Recent data suggest that some functionally selective, or biased, KOR-targeting agonists might retain the therapeutic effects of KOR activation without inducing undesirable side effects. Nalfurafine, used safely in Japan since 2009 for uremic pruritus, is one such functionally selective KOR-targeting agonist. Here, we quantify the bias of nalfurafine and several other KOR agonists relative to an unbiased reference standard (U50,488) and show that nalfurafine and EOM-salvinorin-B demonstrate marked G protein-signaling bias. While nalfurafine (0.015 mg/kg) and EOM-salvinorin-B (1 mg/kg) produced spinal antinociception equivalent to 5 mg/kg U50,488, only nalfurafine significantly enhanced the supraspinal analgesic effect of 5 mg/kg morphine. In addition, 0.015 mg/kg nalfurafine did not produce significant conditioned place aversion, yet retained the ability to reduce morphine-induced conditioned place preference in C57BL/6J mice. Nalfurafine and EOM-salvinorin-B each produced robust inhibition of both spontaneous and morphine-stimulated locomotor behavior, suggesting a persistence of sedative effects when coadministered with morphine. Taken together, these findings suggest that nalfurafine produces analgesic augmentation, while also reducing opioid-induced reward with less risk of dysphoria. Thus, adjuvant administration of G protein-biased KOR agonists like nalfurafine may be beneficial in enhancing the therapeutic potential of MOR-targeting analgesics, such as morphine.
Assuntos
Analgesia/métodos , Sistemas de Liberação de Medicamentos/métodos , Morfinanos/administração & dosagem , Morfina/administração & dosagem , Medição da Dor/efeitos dos fármacos , Receptores Opioides mu/metabolismo , Compostos de Espiro/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Medição da Dor/métodos , Distribuição Aleatória , Receptores Opioides kappa/administração & dosagem , Receptores Opioides mu/agonistasRESUMO
BACKGROUND: Chronic rhinosinusitis (CRS) is a multifaceted disease with a significant genetic component. The importance of taste receptor signaling has recently been highlighted in CRS; single nucleotide polymorphisms (SNPs) of bitter tastant-responsive G-protein-coupled receptors have been linked with CRS and with altered innate immune responses to multiple bacterially derived signals. OBJECTIVE: To determine in CRS the frequency of six SNPs in genes with known bitter tastant signaling function. METHODS: Genomic DNA was isolated from 74 CRS volunteers in West Virginia, and allele frequency was determined and compared with demographically matched data from the 1,000 Genomes database. RESULTS: For two SNPs in a gene recently associated with bitterant signaling regulation, RGS21, there were no associations with CRS (although the frequency of the minor allele of RGS21, rs7528947, was seen to increase with increasing Lund-Mackay CT staging score). Two TAS2R bitter taste receptor gene variants (TAS2R19 rs10772420 and TAS2R38 rs713598), identified in prior CRS genetics studies, were found to have similar associations in this study. CONCLUSION: Unique to our study is the establishment of an association between CRS in this patient population and GNB3 SNP rs5443, a variation in an established G protein component downstream of bitterant receptor signal transduction.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/genética , Polimorfismo de Nucleotídeo Único , Receptores Acoplados a Proteínas G/genética , Rinite/diagnóstico , Rinite/genética , Sinusite/diagnóstico , Sinusite/genética , Adulto , Idoso , Alelos , Doença Crônica , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Rinite/imunologia , Fatores de Risco , Sinusite/imunologiaRESUMO
The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21-a GPCR signaling negative regulator-should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.
Assuntos
Preferências Alimentares , Proteínas RGS/metabolismo , Paladar , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas RGS/deficiência , Proteínas RGS/genéticaRESUMO
Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.
Assuntos
Ativação Plaquetária/fisiologia , Proteínas RGS/fisiologia , Animais , Plaquetas/efeitos dos fármacos , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/fisiologia , Epoprostenol/farmacologia , Transplante de Tecido Fetal , Fígado/embriologia , Transplante de Fígado , Camundongos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Quimera por Radiação , Receptores de Trombina/agonistas , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Trombina/farmacologiaRESUMO
Rho family GTPases modulate actin cytoskeleton dynamics by signaling through multiple effectors, including the p21-activated kinases (PAKs). The intestinal parasite Entamoeba histolytica expresses â¼20 Rho family GTPases and seven isoforms of PAK, two of which have been implicated in pathogenesis-related processes such as amoebic motility and invasion and host cell phagocytosis. Here, we describe two previously unstudied PAK isoforms, EhPAK4 and EhPAK5, as highly specific effectors of EhRacC. A structural model based on 2.35 Å X-ray crystallographic data of a complex between EhRacC(Q65L)·GTP and the EhPAK4 p21 binding domain (PBD) reveals a fairly well-conserved Rho/effector interface despite deviation of the PBD α-helix. A structural comparison with EhRho1 in complex with EhFormin1 suggests likely determinants of Rho family GTPase signaling specificity in E. histolytica. These findings suggest a high degree of Rho family GTPase diversity and specificity in the single-cell parasite E. histolytica. Because PAKs regulate pathogenesis-related processes in E. histolytica, they may be valid pharmacologic targets for anti-amoebiasis drugs.
Assuntos
Entamoeba histolytica/enzimologia , Entamebíase/parasitologia , Quinases Ativadas por p21/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Entamoeba histolytica/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Quinases Ativadas por p21/química , Proteínas rho de Ligação ao GTP/químicaRESUMO
The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than ß-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas RGS/metabolismo , Receptor PAR-1/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Mutação , Proteínas RGS/genética , Receptor PAR-1/genética , Transdução de SinaisRESUMO
Inflammasomes are multi-protein complexes that regulate maturation of the interleukin 1ß-related cytokines IL-1ß and IL-18 through activation of the cysteine proteinase caspase-1. NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is a key component of inflammasomes that assemble in response to a wide variety of endogenous and pathogen-derived danger signals. Activation of the NLRP3-inflammasome and subsequent secretion of IL-1ß is highly regulated by at least three processes: transcriptional activation of both NLRP3 and pro-IL-1ß genes, non-transcriptional priming of NLRP3, and final activation of NLRP3. NLRP3 is predominantly expressed in cells of the hematopoietic lineage. Using a yeast two-hybrid screen, we identified the hematopoietic-restricted protein, G protein signaling modulator-3 (GPSM3), as a NLRP3-interacting protein and a negative regulator of IL-1ß production triggered by NLRP3-dependent inflammasome activators. In monocytes, GPSM3 associates with the C-terminal leucine-rich repeat domain of NLRP3. Bone marrow-derived macrophages lacking GPSM3 expression exhibit an increase in NLRP3-dependent IL-1ß, but not TNF-α, secretion. Furthermore, GPSM3-null mice have enhanced serum and peritoneal IL-1ß production following Alum-induced peritonitis. Our findings suggest that GPSM3 acts as a direct negative regulator of NLRP3 function.
Assuntos
Proteínas de Transporte/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Inflamassomos/metabolismo , Adjuvantes Imunológicos/efeitos adversos , Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/efeitos adversos , Compostos de Alúmen/farmacologia , Animais , Proteínas de Transporte/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/induzido quimicamente , Peritonite/metabolismo , Peritonite/patologia , Estrutura Terciária de ProteínaRESUMO
Ubiquitination is important for numerous cellular processes in most eukaryotic organisms, including cellular proliferation, development, and protein turnover by the proteasome. The intestinal parasite Entamoeba histolytica harbors an extensive ubiquitin-proteasome system. Proteasome inhibitors are known to impair parasite proliferation and encystation, suggesting the ubiquitin-proteasome pathway as a viable therapeutic target. However, no functional studies of the E. histolytica ubiquitination enzymes have yet emerged. Here, we have cloned and characterized multiple E. histolytica ubiquitination components, spanning ubiquitin and its activating (E1), conjugating (E2), and ligating (E3) enzymes. Crystal structures of EhUbiquitin reveal a clustering of unique residues on the α1 helix surface, including an eighth surface lysine not found in other organisms, which may allow for a unique polyubiquitin linkage in E. histolytica. EhUbiquitin is activated by and forms a thioester bond with EhUba1 (E1) in vitro, in an ATP- and magnesium-dependent fashion. EhUba1 exhibits a greater maximal initial velocity of pyrophosphate:ATP exchange than its human homolog, suggesting different kinetics of ubiquitin activation in E. histolytica. EhUba1 engages the E2 enzyme EhUbc5 through its ubiquitin-fold domain to transfer the EhUbiquitin thioester. However, EhUbc5 has a >10-fold preference for EhUba1â¼Ub compared with unconjugated EhUba1. A crystal structure of EhUbc5 allowed prediction of a noncovalent "backside" interaction with EhUbiquitin and E3 enzymes. EhUbc5 selectively engages EhRING1 (E3) to the exclusion of two HECT family E3 ligases, and mutagenesis indicates a conserved mode of E2/RING-E3 interaction in E. histolytica.
Assuntos
Entamoeba histolytica/metabolismo , Regulação Enzimológica da Expressão Gênica , Ubiquitina/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cinética , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/química , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/químicaRESUMO
In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting ß2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and COPD.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/administração & dosagem , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/administração & dosagem , Proteínas RGS/genética , Mucosa Respiratória/metabolismo , Combinação de Medicamentos , Células Epiteliais/efeitos dos fármacos , Humanos , Proteínas RGS/biossíntese , Proteínas RGS/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacos , Fatores de TempoRESUMO
Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGßγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism.
Assuntos
Entamoeba histolytica/imunologia , Entamebíase/imunologia , Subunidades alfa de Proteínas de Ligação ao GTP/imunologia , Proteínas de Protozoários/imunologia , Fatores de Virulência/imunologia , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Entamebíase/enzimologia , Entamebíase/genética , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/imunologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Células Jurkat , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Transcrição Gênica/imunologia , Fatores de Virulência/biossíntese , Fatores de Virulência/químicaRESUMO
Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαßγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gα(i1)(G42R) binding to GDP·AlF(4)(-) or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gßγ and GoLoco motifs in any nucleotide state. The corresponding Gα(q)(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Magnaporthe/patogenicidade , Micoses/genética , Mutação Puntual , Dobramento de Proteína , Substituição de Aminoácidos/fisiologia , Domínio Catalítico/genética , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Hordeum/microbiologia , Magnaporthe/genética , Magnaporthe/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Mutação Puntual/fisiologia , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genéticaRESUMO
In asthma and chronic obstructive pulmonary disease, activation of G(q)-protein-coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting ß(2)-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates G(q) signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Broncoconstrição/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas RGS/genética , Albuterol/análogos & derivados , Albuterol/farmacologia , Animais , Western Blotting , Broncoconstrição/genética , Broncoconstrição/fisiologia , Budesonida/farmacologia , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Dexametasona/farmacologia , Sinergismo Farmacológico , Etanolaminas/farmacologia , Fumarato de Formoterol , Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Proteínas RGS/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xinafoato de SalmeterolRESUMO
Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.
Assuntos
Descoberta de Drogas/métodos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/química , Proteínas RGS/metabolismo , Transdução de Sinais , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Guanosina Trifosfato/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Terapia de Alvo Molecular , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas RGS/classificação , Proteínas RGS/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Cognitive decline in Parkinson's disease (PD) is a critical premotor sign that may occur in approximately 40% of PD patients up to 10 years prior to clinical recognition and diagnosis. Delineating the mechanisms and specific behavioral signs of cognitive decline associated with PD prior to motor impairment is a critical unmet need. Rodent PD models that have an impairment in a cognitive phenotype for a time period sufficiently long enough prior to motor decline can be useful to establish viable candidate mechanisms. Arguably, the methods used to evaluate cognitive decline in rodent models should emulate methods used in the assessment of humans to optimize translation. Premotor cognitive decline in human PD can potentially be examined in the genetically altered PINK1-/- rat model, which exhibits a protracted onset of motor decline in most studies. To increase translation to cognitive assessment in human PD, we used a modified non-water multiple T-maze, which assesses attention, cognitive flexibility, and working memory similarly to the Trail Making Test (TMT) in humans. Similar to the deficiencies revealed in TMT test outcomes in human PD, 4-month-old PINK1-/- rats made more errors and took longer to complete the maze, despite a hyperkinetic phenotype, compared to wild-type rats. Thus, we have identified a potential methodological tool with cross-species translation to evaluate executive functioning in an established PD rat model.
RESUMO
Cognitive decline in Parkinson's disease (PD) emerges up to 10 years before clinical recognition. Neurobiological mechanisms underlying premotor cognitive impairment in PD can potentially be examined in the PINK1 -/- rat, which exhibits a protracted motor onset. To enhance translation to human PD cognitive assessments, we tested a modified multiple T-maze, which measures cognitive flexibility similarly to the Trail-Making Test in humans. Like human PD outcomes, PINK1 -/- rats made more errors and took longer to complete the maze than wild types. Thus, we have identified a potential tool for assessing cross-species translation of cognitive functioning in an established PD animal model.