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1.
Mol Cell ; 73(6): 1138-1149.e6, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901564

RESUMO

The nuclear factor (NF)-κB pathway plays a central role in inflammatory and immune responses, with aberrant activation of NF-κB signaling being implicated in various human disorders. Here, we show that mammalian ste20-like kinase 1 (MST1) is a previously unrecognized component of the tumor necrosis factor α (TNFα) receptor 1 signaling complex (TNF-RSC) and attenuates TNFα-induced NF-κB signaling. Genetic ablation of MST1 in mouse embryonic fibroblasts and bone marrow-derived macrophages potentiated the TNFα-induced increase in IκB kinase (IKK) activity, as well as the expression of NF-κB target genes. TNFα induced the recruitment of MST1 to TNF-RSC and its interaction with HOIP, the catalytic component of the E3 ligase linear ubiquitin assembly complex (LUBAC). Furthermore, MST1 activated in response to TNFα stimulation mediates the phosphorylation of HOIP and thereby inhibited LUBAC-dependent linear ubiquitination of NEMO/IKKγ. Together, our findings suggest that MST1 negatively regulates TNFα-induced NF-κB signaling by targeting LUBAC.


Assuntos
Fibroblastos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Fibroblastos/enzimologia , Células HEK293 , Humanos , Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multienzimáticos , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
EMBO Rep ; 21(2): e48290, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31885126

RESUMO

The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.


Assuntos
Aurora Quinase A , Tubulina (Proteína) , Animais , Aurora Quinase A/genética , Cílios , Humanos , Microtúbulos , Proteínas , Tubulina (Proteína)/genética , Peixe-Zebra/genética
3.
Cell Mol Life Sci ; 79(1): 15, 2021 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-34967918

RESUMO

Excessive activation of the ionotropic N-methyl-D-aspartate (NMDA) receptor has been shown to cause abnormally high levels of Ca2+ influx, thereby leading to excitotoxic neuronal death. In this study, exposure of mouse primary cortical neurons to NMDA resulted in the cleavage and activation of mammalian sterile 20-like kinase-1 (MST1), both of which were mediated by calpain 1. In vitro cleavage assay data indicated that calpain 1 cleaves out the autoinhibitory domain of MST1 to generate an active form of the kinase. Furthermore, calpain 1 mediated the cleavage and activation of wild-type MST1, but not of MST1 (G339A). Intriguingly, NMDA/calpain-induced MST1 activation promoted the nuclear translocation of the kinase and the phosphorylation of histone H2B in mouse cortical neurons, leading to excitotoxicity. Thus, we propose a previously unrecognized mechanism of MST1 activation associated with NMDA-induced excitotoxic neuronal death.


Assuntos
Córtex Cerebral/patologia , N-Metilaspartato/toxicidade , Neurônios/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Camundongos Endogâmicos C57BL , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotoxinas/toxicidade , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos
4.
Cell Mol Life Sci ; 78(5): 2315-2328, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32975614

RESUMO

Pattern-recognition receptors including Toll-like receptors (TLRs) recognize invading pathogens and trigger an immune response in mammals. Here we show that mammalian ste20-like kinase 1/serine/threonine kinase 4 (MST1/STK4) functions as a negative regulator of lipopolysaccharide (LPS)-induced activation of the TLR4-NF-κB signaling pathway associated with inflammation. Myeloid-specific genetic ablation of MST1/STK4 increased the susceptibility of mice to LPS-induced septic shock. Ablation of MST1/STK4 also enhanced NF-κB activation triggered by LPS in bone marrow-derived macrophages (BMDMs), leading to increased production of proinflammatory cytokines by these cells. Furthermore, MST1/STK4 inhibited TRAF6 autoubiquitination as well as TRAF6-mediated downstream signaling induced by LPS. In addition, we found that TRAF6 mediates the LPS-induced activation of MST1/STK4 by catalyzing its ubiquitination, resulting in negative feedback regulation by MST1/STK4 of the LPS-induced pathway leading to cytokine production in macrophages. Together, our findings suggest that MST1/STK4 functions as a negative modulator of the LPS-induced NF-κB signaling pathway during macrophage activation.


Assuntos
Macrófagos/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Sepse/sangue , Sepse/genética , Sepse/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Fator 6 Associado a Receptor de TNF/genética , Receptor 4 Toll-Like/genética , Ubiquitinação/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 115(43): 11078-11083, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30297409

RESUMO

In the descending analgesia pathway, opioids are known to disinhibit the projections from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), leading to suppression of pain signals at the spinal cord level. The locus coeruleus (LC) has been proposed to engage in the descending pathway through noradrenergic inputs to the spinal cord. Nevertheless, how the LC is integrated in the descending analgesia circuit has remained unknown. Here, we show that the opioidergic analgesia pathway is bifurcated in structure and function at the PAG. A knockout as well as a PAG-specific knockdown of phospholipase C ß4 (PLCß4), a signaling molecule for G protein-coupled receptors, enhanced swim stress-induced and morphine-induced analgesia in mice. Immunostaining after simultaneous retrograde labeling from the RVM and the LC revealed two mutually exclusive neuronal populations at the PAG, each projecting either to the LC or the RVM, with PLCß4 expression only in the PAG-LC projecting cells that provide a direct synaptic input to LC-spinal cord (SC) projection neurons. The PAG-LC projection neurons in wild-type mice turned quiescent in response to opiates, but remained active in the PLCß4 mutant, suggesting a possibility that an increased adrenergic function induced by the persistent PAG-LC activity underlies the enhanced opioid analgesia in the mutant. Indeed, the enhanced analgesia in the mutant was reversed by blocking α2-noradrenergic receptors. These findings indicate that opioids suppress descending analgesia through the PAG-LC pathway, while enhancing it through the PAG-RVM pathway, i.e., two distinct pathways with opposing effects on opioid analgesia. These results point to a therapeutic target in pain control.


Assuntos
Analgesia/métodos , Mesencéfalo/fisiopatologia , Manejo da Dor/métodos , Analgésicos Opioides/farmacologia , Animais , Masculino , Mesencéfalo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Dor/fisiopatologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/fisiologia , Yin-Yang
6.
PLoS Genet ; 13(5): e1006819, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28558013

RESUMO

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid ß-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.


Assuntos
Autofagia , Doença de Depósito de Glicogênio Tipo I/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Doença de Depósito de Glicogênio Tipo I/genética , Hepatócitos/metabolismo , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Sirtuína 1/genética
7.
Hum Mol Genet ; 26(18): 3615-3629, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28666328

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron loss in the spinal cord and brain. Mutations in the superoxide dismutase 1 (SOD1) gene have been linked to familial ALS. To elucidate the role of SOD1 mutations in ALS, we investigated 14-3-3, a crucial regulator of cell death that was identified in patients with familial ALS. In a transgenic mouse model (SOD1-G93A) of ALS, 14-3-3 co-localized with mutant SOD1 aggregates and was more insoluble in the spinal cords of mutant SOD1 transgenic mice than in those of wild-type mice. Immunofluorescence and co-immunoprecipitation experiments showed that the 14-3-3ɛ and θ isoforms interact with mutant SOD1 aggregates in the juxtanuclear quality control compartment of N2a neuroblastoma cells. Fluorescence loss in photobleaching experiments revealed that movement of the isoforms of 14-3-3 was markedly reduced in SOD1 aggregates. Bax translocation into and cytochrome c release from the mitochondria were promoted by the sequestration of 14-3-3 into mutant SOD1 aggregates, increasing cell death. Mutant SOD1 aggregates were dissolved by the Hsp104 chaperone, which increased the interaction of 14-3-3 with Bax, reducing cell death. Our study demonstrates that mutant SOD1 inhibits 14-3-3-mediated cell survival. This information may contribute to the identification of a novel therapeutic target for ALS.


Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Esclerose Lateral Amiotrófica/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Proteínas 14-3-3/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Morte Celular/fisiologia , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação , Medula Espinal/metabolismo , Medula Espinal/patologia
8.
Exp Cell Res ; 368(1): 50-58, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29665354

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.


Assuntos
Macrófagos/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA/efeitos dos fármacos , Proteínas Repressoras/efeitos dos fármacos
9.
Biochim Biophys Acta Mol Cell Res ; 1864(5): 760-770, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28214532

RESUMO

Survival motor neuron (SMN) is a 38-kDa protein, whose deficiency in humans develops spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease with muscular atrophy due to motor neuron death in the spinal cord. We now report that SMN prevents the activation of TRAF6 and IκB kinase (IKK) and thereby negatively regulates the NF-κB signaling processes. SMN physically interacted with TRAF6 and with each component of the IKK complex, IKK-α, IKK-ß, and IKK-γ in BV2 microglia cells. Moreover, SMN1 inhibited the E3 ubiquitin ligase activity of TRAF6 as well as the kinase activity of IKK. Furthermore, depletion of endogenous SMN by RNA interference enhanced the IL-1ß-induced activation of IKK and production of inflammatory mediators such as TNF-α and nitric oxide in BV2 cells. Consistently, the potentiation of IL-1ß-induced IKK activity was also found in SMA patient fibroblasts, compared with that of normal ones. Our results thus suggest that SMN functions as a natural inhibitor for IL-1ß-induced NF-κB signaling by targeting TRAF6 and the IKK complex.


Assuntos
NF-kappa B/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Camundongos , NF-kappa B/antagonistas & inibidores , Transdução de Sinais/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores
10.
Biochem Biophys Res Commun ; 490(3): 1093-1099, 2017 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-28668392

RESUMO

LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel ß-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Sequência de Aminoácidos , Autofagia , Proteínas de Bactérias/química , Humanos , Legionella pneumophila/química , Proteínas Associadas aos Microtúbulos/química , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência
11.
Proc Natl Acad Sci U S A ; 111(32): 11828-33, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25071191

RESUMO

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca(2+) channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca(2+) channel, CaV3.3, was deleted (CaV3.3(-/-)). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in CaV3.3(-/-) mice and in mice in which the CaV3.3 gene was silenced predominantly in the TRN. CaV3.3(-/-) mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both CaV3.3 and CaV3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Epilepsia Tipo Ausência/fisiopatologia , Núcleos Talâmicos/fisiopatologia , 4-Butirolactona/toxicidade , Potenciais de Ação , Animais , Canais de Cálcio Tipo T/deficiência , Canais de Cálcio Tipo T/genética , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Epilepsia Tipo Ausência/induzido quimicamente , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Patch-Clamp
12.
Biochim Biophys Acta ; 1852(11): 2517-24, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26264610

RESUMO

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the selective degeneration of upper and lower motor neurons associated with the abnormal aggregation of ubiquitinated proteins. The molecular mechanisms underlying the pathogenesis of ALS remain unclear, however. Autophagy is a major pathway for the elimination of protein aggregates and damaged organelles and therefore contributes to cellular homeostasis. This catabolic process begins with the formation of the double membrane-bound autophagosome that engulfs portions of the cytoplasm and subsequently fuses with a lysosome to form an autolysosome, in which lysosomal enzymes digest autophagic substrates. Defects at various stages of autophagy have been associated with pathological mutations of several ALS-linked genes including SOD1, p62, TDP-43, and optineurin, suggesting that such defects may play a causative role in the pathogenesis of this condition. In this review, we summarize the dysregulation of autophagy associated with ALS as well as potential therapeutic strategies based on modulation of the autophagic process.

13.
J Cell Sci ; 127(Pt 8): 1640-6, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24522193

RESUMO

Son of sevenless 1 (SOS1) is a Ras-specific guanine-nucleotide-exchange factor (GEF) that mediates intracellular signaling processes induced by receptor tyrosine kinases. In this study, we show that CIIA (also known as VPS28) physically associates with SOS1 and thereby inhibits the GEF activity of SOS1 on Ras, which prevents the epidermal growth factor (EGF)-induced activation of the Ras-Erk1/2 pathway. Furthermore, CIIA inhibited cyclin D1 expression, as well as DNA, synthesis in response to EGF. Intriguingly, CIIA failed to inhibit the Ras-specific GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L and E846K). Taken together, our results suggest that CIIA functions as a negative modulator of the SOS1-Ras signaling events initiated by peptide growth factors including EGF.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Sistema de Sinalização das MAP Quinases , Proteína SOS1/fisiologia , Proteínas ras/metabolismo , Animais , Ciclina D1/genética , Ciclina D1/metabolismo , Replicação do DNA , Cães , Fator de Crescimento Epidérmico/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células HEK293 , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Síndrome de Noonan/genética
14.
Biochem Biophys Res Commun ; 478(2): 784-90, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27501752

RESUMO

TNF receptor-associated factor 6 (TRAF6) plays a critical role in NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, both of which mediate macrophage activation in response to pathogen-associated molecular patterns such as bacterial endotoxin, lipopolysaccharides (LPS). In this study, we investigated whether HLA-B associated transcript-3 (BAT3) regulates LPS-induced macrophage activation. BAT3 physically interacted with TRAF6 in macrophages, and this interaction was enhanced in the cells after LPS treatment. Furthermore, BAT3 inhibited the homo-oligomerization of TRAF6 as well as the interaction between TRAF6 and its downstream kinase transforming growth factor beta-activated kinase 1 (TAK1), thereby suppressing TRAF6-mediated signaling events. Intriguingly, TRAF6 mediated ubiquitination of BAT3 and this ubiquitination was crucial for its inhibitory effect on TRAF6-mediated signaling. Depletion of BAT3 by RNA interference resulted in enhancement of LPS-induced activation of the NF-κB signaling with increasing expression levels of pro-inflammatory cytokines. These findings suggest that BAT3 functions as the negative regulator of LPS-induced macrophage activation.


Assuntos
Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Chaperonas Moleculares/genética , NF-kappa B/genética , Proteínas Nucleares/genética , Fator 6 Associado a Receptor de TNF/genética , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/imunologia , NF-kappa B/imunologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/imunologia , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/imunologia , Ubiquitinação
15.
Proc Natl Acad Sci U S A ; 110(29): 12066-71, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23818595

RESUMO

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by loss of motor neurons. Dominant mutations in the gene for superoxide dismutase 1 (SOD1) give rise to familial ALS by an unknown mechanism. Here we show that genetic deficiency of mammalian sterile 20-like kinase 1 (MST1) delays disease onset and extends survival in mice expressing the ALS-associated G93A mutant of human SOD1. SOD1(G93A) induces dissociation of MST1 from a redox protein thioredoxin-1 and promotes MST1 activation in spinal cord neurons in a reactive oxygen species-dependent manner. Moreover, MST1 was found to mediate SOD1(G93A)-induced activation of p38 mitogen-activated protein kinase and caspases as well as impairment of autophagy in spinal cord motoneurons of SOD1(G93A) mice. Our findings implicate MST1 as a key determinant of neurodegeneration in ALS.


Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Ativação Enzimática/fisiologia , Neurônios Motores/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Superóxido Dismutase/genética , Adulto , Esclerose Lateral Amiotrófica/metabolismo , Análise de Variância , Animais , Autofagia/genética , Autofagia/fisiologia , Ativação Enzimática/genética , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Mutação de Sentido Incorreto/genética , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Medula Espinal/citologia , Superóxido Dismutase-1 , Tiorredoxinas/metabolismo
16.
Neurobiol Dis ; 80: 63-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26002422

RESUMO

Oxidative stress contributes to degeneration of motor neurons in patients with amyotrophic lateral sclerosis (ALS) as well as transgenic mice overexpressing ALS-associated human superoxide dismutase 1 (SOD1) mutants. However, the molecular mechanism by which the ALS-linked SOD1 mutants including SOD1(G93A) induce oxidative stress remains unclear. Here, we show that iron was accumulated in ventral motor neurons from SOD1(G93A)-transgenic mice even at 4 weeks of age, subsequently inducing oxidative stress. Iron chelation with deferoxamine mesylate delayed disease onset and extended lifespan of SOD1(G93A) mice. Furthermore, SOD1(G93A)-induced iron accumulation mediated the increase in the enzymatic activity of TNF-α converting enzyme (TACE), leading to secretion of TNF-α at least in part through iron-dependent oxidative stress. Our findings suggest iron as a key determinant of early motor neuron degeneration as well as proinflammatory responses at symptomatic stage in SOD1(G93A) mice.


Assuntos
Proteínas ADAM/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Ferro/toxicidade , Neurônios Motores/metabolismo , Superóxido Dismutase/genética , Fator de Necrose Tumoral alfa/sangue , Proteína ADAM17 , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Modelos Animais de Doenças , Humanos , Ferro/metabolismo , Camundongos , Camundongos Transgênicos , Atividade Motora , Neurônios Motores/patologia , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia
17.
EMBO J ; 30(12): 2465-76, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21572393

RESUMO

Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. Although the persistent secretion of inflammatory mediators by the activated cells contributes to the pathogenesis of various neurological disorders, most activated microglia eventually undergo apoptosis through the process of activation-induced cell death (AICD). The molecular mechanism of AICD, however, has remained unclear. Here, we show that Daxx and mammalian Ste20-like kinase-1 (MST1) mediate apoptosis elicited by interferon-γ (IFN-γ) in microglia. IFN-γ upregulated the expression of Daxx, which in turn mediated the homodimerization, activation, and nuclear translocation of MST1 and apoptosis in microglial cells. Depletion of Daxx or MST1 by RNA interference also attenuated IFN-γ-induced cell death in primary rat microglia. Furthermore, the extent of IFN-γ-induced death of microglia in the brain of MST1-null mice was significantly reduced compared with that apparent in wild-type mice. Our results thus highlight new functions of Daxx and MST1 that they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-γ.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Microglia/citologia , Microglia/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Animais , Apoptose/genética , Células COS , Proteínas de Transporte/genética , Morte Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Chlorocebus aethiops , Proteínas Correpressoras , Fibroblastos/citologia , Fibroblastos/fisiologia , Células HEK293 , Células HeLa , Fator de Crescimento de Hepatócito/deficiência , Fator de Crescimento de Hepatócito/genética , Humanos , Mediadores da Inflamação , Interferon gama/administração & dosagem , Interferon gama/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética
18.
Mol Cell Biochem ; 402(1-2): 33-40, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25547065

RESUMO

RC3/neurogranin is a postsynaptic protein and plays pivotal roles in spatial learning and emotional anxiety as well as synaptic plasticity. The expression level of RC3 is dynamically changed during developmental stages, but the function of RC3 in brain development is not well understood yet. Neurotrophins interact with tropomyosin-related kinase receptors to activate Ras-extracellular signal-regulated kinase (ERK) pathway and can also induce neuronal differentiation. In this study, we demonstrate that RC3 inhibits Ras-ERK pathway by interaction with Ras and controls neurite outgrowth induced by neurotrophins. In PC12 cells, RC3 inhibits nerve growth factor (NGF)-induced activation of Ras and thereby ERK1/2 signaling cascade as well as neurite outgrowth induced by NGF. We found Ras is the target of the inhibitory function of RC3, because RC3 interacts with Ras and suppresses the elevated affinity of Ras to Ras-binding domain of Raf-1. Meanwhile, already activated Raf-1 by Ras activity is not affected by RC3. Furthermore, depletion of RC3 by RNA interference drastically enhances the stimulation of ERK1/2 and neurite outgrowth induced by brain-derived neurotrophic factor in hippocampal neurons. These findings suggest that RC3 is a novel natural inhibitor of Ras-ERK1/2 signaling axis, leading to negatively regulate neuronal differentiation induced by neurotrophins.


Assuntos
Sistema de Sinalização das MAP Quinases , Neurogranina/fisiologia , Proteínas ras/metabolismo , Animais , Fator de Crescimento Neural/fisiologia , Neuritos/fisiologia , Células PC12 , Ligação Proteica , Ratos
19.
Anesth Analg ; 120(3): 671-677, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25695583

RESUMO

BACKGROUND: The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation. METHODS: Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe4, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection. RESULTS: Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol). CONCLUSIONS: These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.


Assuntos
Analgésicos Opioides/farmacologia , Analgésicos/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Formaldeído , Nociceptividade/efeitos dos fármacos , Dor Nociceptiva/prevenção & controle , Proteínas RGS/antagonistas & inibidores , Medula Espinal/efeitos dos fármacos , Tiazolidinedionas/administração & dosagem , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Injeções Espinhais , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antagonistas de Entorpecentes/farmacologia , Dor Nociceptiva/genética , Dor Nociceptiva/metabolismo , Dor Nociceptiva/fisiopatologia , Dor Nociceptiva/psicologia , Medição da Dor , Proteínas RGS/deficiência , Proteínas RGS/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Fatores de Tempo
20.
Arch Toxicol ; 89(6): 867-82, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25690731

RESUMO

The mitogen-activated protein kinases (MAPKs) in mammals include c-Jun NH2-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). These enzymes are serine-threonine protein kinases that regulate various cellular activities including proliferation, differentiation, apoptosis or survival, inflammation, and innate immunity. The compromised MAPK signaling pathways contribute to the pathology of diverse human diseases including cancer and neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The JNK and p38 MAPK signaling pathways are activated by various types of cellular stress such as oxidative, genotoxic, and osmotic stress as well as by proinflammatory cytokines such as tumor necrosis factor-α and interleukin 1ß. The Ras-Raf-MEK-ERK signaling pathway plays a key role in cancer development through the stimulation of cell proliferation and metastasis. The p38 MAPK pathway contributes to neuroinflammation mediated by glial cells including microglia and astrocytes, and it has also been associated with anticancer drug resistance in colon and liver cancer. We here summarize recent research on the roles of MAPK signaling pathways in human diseases, with a focus on cancer and neurodegenerative conditions.


Assuntos
Doença de Alzheimer/enzimologia , Esclerose Lateral Amiotrófica/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/enzimologia , Doença de Parkinson/enzimologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/patologia , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/patologia , Proliferação de Células , Humanos , Imunidade Inata , Sistema de Sinalização das MAP Quinases , Neoplasias/imunologia , Neoplasias/patologia , Estresse Oxidativo , Doença de Parkinson/imunologia , Doença de Parkinson/patologia
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