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BACKGROUND: Danger-associated molecular patterns (DAMPs) may be implicated in the pathophysiological pathways associated with an unfavorable outcome after acute brain injury (ABI). METHODS: We collected samples of ventricular cerebrospinal fluid (vCSF) for 5 days in 50 consecutive patients at risk of intracranial hypertension after traumatic and nontraumatic ABI. Differences in vCSF protein expression over time were evaluated using linear models and selected for functional network analysis using the PANTHER and STRING databases. The primary exposure of interest was the type of brain injury (traumatic vs. nontraumatic), and the primary outcome was the vCSF expression of DAMPs. Secondary exposures of interest included the occurrence of intracranial pressure ≥20 or ≥ 30 mm Hg during the 5 days post-ABI, intensive care unit (ICU) mortality, and neurological outcome (assessed using the Glasgow Outcome Score) at 3 months post-ICU discharge. Secondary outcomes included associations of these exposures with the vCSF expression of DAMPs. RESULTS: A network of 6 DAMPs (DAMP_trauma; protein-protein interaction [PPI] P=0.04) was differentially expressed in patients with ABI of traumatic origin compared with those with nontraumatic ABI. ABI patients with intracranial pressure ≥30 mm Hg differentially expressed a set of 38 DAMPS (DAMP_ICP30; PPI P< 0.001). Proteins in DAMP_ICP30 are involved in cellular proteolysis, complement pathway activation, and post-translational modifications. There were no relationships between DAMP expression and ICU mortality or unfavorable versus favorable outcomes. CONCLUSIONS: Specific patterns of vCSF DAMP expression differentiated between traumatic and nontraumatic types of ABI and were associated with increased episodes of severe intracranial hypertension.
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Friedreich ataxia is an autosomal recessive multisystem disorder with prominent neurological manifestations and cardiac involvement. The disease is caused by large GAA expansions in the first intron of the FXN gene, encoding the mitochondrial protein frataxin, resulting in downregulation of gene expression and reduced synthesis of frataxin. The selective loss of proprioceptive neurons is a hallmark of Friedreich ataxia, but the cause of the specific vulnerability of these cells is still unknown. We herein perform an in vitro characterization of human induced pluripotent stem cell-derived sensory neuronal cultures highly enriched for primary proprioceptive neurons. We employ neurons differentiated from healthy donors, Friedreich ataxia patients and Friedreich ataxia sibling isogenic control lines. The analysis of the transcriptomic and proteomic profile suggests an impairment of cytoskeleton organization at the growth cone, neurite extension and, at later stages of maturation, synaptic plasticity. Alterations in the spiking profile of tonic neurons are also observed at the electrophysiological analysis of mature neurons. Despite the reversal of the repressive epigenetic state at the FXN locus and the restoration of FXN expression, isogenic control neurons retain many features of Friedreich ataxia neurons. Our study suggests the existence of abnormalities affecting proprioceptors in Friedreich ataxia, particularly their ability to extend towards their targets and transmit proper synaptic signals. It also highlights the need for further investigations to better understand the mechanistic link between FXN silencing and proprioceptive degeneration in Friedreich ataxia.
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Clinical trials in rare diseases as Friedreich ataxia (FRDA) offer special challenges, particularly when multiple treatments become ready for clinical testing. Regulatory health authorities have developed specific pathways for "orphan" drugs allowing the use of a validated biomarker for initial approval. This study aimed to identify changes in cerebrospinal fluid (CSF) proteins occurring in FRDA patients that may be potential biomarkers in therapeutic trials. CSF was obtained from 5 FRDA patients (4 females, 1 male) from the Brussels site of the European Friedreich Ataxia Consortium for Translational Studies (EFACTS). Two patients were ambulatory, three used a wheelchair. Residual CSF samples from 19 patients who had had a lumbar puncture as part of a diagnostic workup were used as controls. All CSF samples had normal cells, total protein and glucose levels. Proteins were identified by label-free data-dependent acquisition mass spectrometry (MS) coupled to micro-high performance liquid chromatography. We found 172 differentially expressed proteins (DEPs) (92 up, 80 down) between FRDA patients and controls at P < 0.05, 34 DEPs (28 up, 6 down) at P < 0.0001. Remarkably, there was no overlap between FRDA patients and controls for seven upregulated and six downregulated DEPs. Represented pathways included extracellular matrix organization, signaling, the complement cascade, adhesion molecules, synaptic proteins, neurexins and neuroligins. This study supports the hypothesis that the quantitative analysis CSF proteins may provide robust biomarkers for clinical trials as well as shed light on pathogenic mechanisms. Interestingly, DEPs in FA patients CSF point to neurodegeneration and neuroinflammation processes that may respond to treatment.
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BACKGROUND: Quantitative analysis of ventricular cerebrospinal fluid (vCSF) proteins following acute brain injury (ABI) may help identify pathophysiological pathways and potential biomarkers that can predict unfavorable outcome. METHODS: In this prospective proteomic analysis study, consecutive patients with severe ABI expected to require intraventricular catheterization for intracranial pressure (ICP) monitoring for at least 5 days and patients without ABI admitted for elective clipping of an unruptured cerebral aneurysm were included. vCSF samples were collected within the first 24 h after ABI and ventriculostomy insertion and then every 24 h for 5 days. In patients without ABI, a single vCSF sample was collected at the time of elective clipping. Data-independent acquisition and sequential window acquisition of all theoretical spectra (SWATH) mass spectrometry were used to compare differences in protein expression in patients with ABI and patients without ABI and in patients with traumatic and nontraumatic ABI. Differences in protein expression according to different ICP values, intensive care unit outcome, subarachnoid hemorrhage (SAH) versus traumatic brain injury (TBI), and good versus poor 3-month functional status (assessed by using the Glasgow Outcome Scale) were also evaluated. vCSF proteins with significant differences between groups were compared by using linear models and selected for gene ontology analysis using R Language and the Panther database. RESULTS: We included 50 patients with ABI (SAH n = 23, TBI n = 15, intracranial hemorrhage n = 6, ischemic stroke n = 3, others n = 3) and 12 patients without ABI. There were significant differences in the expression of 255 proteins between patients with and without ABI (p < 0.01). There were intraday and interday differences in expression of seven proteins related to increased inflammation, apoptosis, oxidative stress, and cellular response to hypoxia and injury. Among these, glial fibrillary acidic protein expression was higher in patients with ABI with severe intracranial hypertension (ICH) (ICP ≥ 30 mm Hg) or death compared to those without (log 2 fold change: + 2.4; p < 0.001), suggesting extensive primary astroglial injury or death. There were differences in the expression of 96 proteins between patients with traumatic and nontraumatic ABI (p < 0.05); intraday and interday differences were observed for six proteins related to structural damage, complement activation, and cholesterol metabolism. Thirty-nine vCSF proteins were associated with an increased risk of severe ICH (ICP ≥ 30 mm Hg) in patients with traumatic compared with nontraumatic ABI (p < 0.05). No significant differences were found in protein expression between patients with SAH versus TBI or between those with good versus poor 3-month Glasgow Outcome Scale score. CONCLUSIONS: Dysregulated vCSF protein expression after ABI may be associated with an increased risk of severe ICH and death.
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Lesões Encefálicas Traumáticas , Lesões Encefálicas , Hipertensão Intracraniana , Hemorragia Subaracnóidea , Biomarcadores , Colesterol , Proteína Glial Fibrilar Ácida , Humanos , Hipertensão Intracraniana/etiologia , Pressão Intracraniana/fisiologia , Estudos Prospectivos , Proteômica , Hemorragia Subaracnóidea/complicaçõesRESUMO
OBJECTIVES: Anti-carbamylated protein antibodies (anti-CarPAs) are present in RA sera and have been associated with erosive disease. The exact targets of anti-CarPAs in vivo are currently not well known; we used a proteomic approach on serum and SF of RA patients to assess the human carbamylome and to identify carbamylated autoantigens as potential biomarkers in early RA. METHODS: Mass spectrometry was performed on SF and serum from RA patients. Carbamylated proteins present in both sample types were selected as candidate autoantigens for the establishment of ELISAs. A cohort of early RA patients was tested for positivity for specific anti-CarPAs. RESULTS: Eleven novel carbamylated proteins were identified, and five were selected as potential autoantigens for detection of anti-CarPAs. Among them, antibodies against carbamylated hemopexin (anti-CaHPX) and alpha-2-macroglobulin (anti-CaA2M) showed comparable diagnostic value to the established carbamylated foetal calf serum-based ELISA. A cohort of 189 early RA patients was studied. The combination of these new biomarkers with anti-citrullinated protein antibodies and RF identified 89% of early RA patients in our cohort. There was little correlation between the tested biomarkers, and each one of the tested antigens could identify a different subset of seronegative RA patients. Anti-CaA2M positivity showed clinical potential, being associated with higher disease disability. CONCLUSION: We highlight the detection of novel carbamylated autoantigens in vivo using a combined proteomics approach in the SF and serum of RA patients. Anti-CaHPX and anti-CaA2M are promising clinical biomarkers, especially in seronegative RA.
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Artrite Reumatoide , Autoantígenos , Hemopexina , alfa 2-Macroglobulinas Associadas à Gravidez , Artrite Reumatoide/diagnóstico , Autoanticorpos , Biomarcadores , Humanos , Peptídeos Cíclicos , Proteínas , ProteômicaRESUMO
BACKGROUND: Acute brain injuries are associated with high mortality rates and poor long-term functional outcomes. Measurement of cerebrospinal fluid (CSF) biomarkers in patients with acute brain injuries may help elucidate some of the pathophysiological pathways involved in the prognosis of these patients. METHODS: We performed a systematic search and descriptive review using the MEDLINE database and the PubMed interface from inception up to June 29, 2021, to retrieve observational studies in which the relationship between CSF concentrations of protein biomarkers and neurological outcomes was reported in patients with acute brain injury [traumatic brain injury, subarachnoid hemorrhage, acute ischemic stroke, status epilepticus or post-cardiac arrest]. We classified the studies according to whether or not biomarker concentrations were associated with neurological outcomes. The methodological quality of the studies was evaluated using the Newcastle-Ottawa quality assessment scale. RESULTS: Of the 39 studies that met our criteria, 30 reported that the biomarker concentration was associated with neurological outcome and 9 reported no association. In TBI, increased extracellular concentrations of biomarkers related to neuronal cytoskeletal disruption, apoptosis and inflammation were associated with the severity of acute brain injury, early mortality and worse long-term functional outcome. Reduced concentrations of protein biomarkers related to impaired redox function were associated with increased risk of neurological deficit. In non-traumatic acute brain injury, concentrations of CSF protein biomarkers related to dysregulated inflammation and apoptosis were associated with a greater risk of vasospasm and a larger volume of brain ischemia. There was a high risk of bias across the studies. CONCLUSION: In patients with acute brain injury, altered CSF concentrations of protein biomarkers related to cytoskeletal damage, inflammation, apoptosis and oxidative stress may be predictive of worse neurological outcomes.
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Biomarcadores/análise , Lesões Encefálicas/complicações , Líquido Cefalorraquidiano , Proteínas/análise , Adulto , Biomarcadores/líquido cefalorraquidiano , Lesões Encefálicas/fisiopatologia , Humanos , PrognósticoRESUMO
Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.
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Condrócitos/metabolismo , MAP Quinase Quinase Quinases/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Osteocalcina/genética , Osteocondrodisplasias/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Aminoacetonitrila/análogos & derivados , Aminoacetonitrila/farmacologia , Animais , Calcificação Fisiológica/genética , Diferenciação Celular , Condrócitos/patologia , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteocalcina/antagonistas & inibidores , Osteocalcina/metabolismo , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patologia , Osteogênese/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/deficiência , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tiofenos/farmacologiaRESUMO
Shigella invasion and dissemination in intestinal epithelial cells relies on a type 3 secretion system (T3SS), which mediates translocation of virulence proteins into host cells. T3SSs are composed of three major parts: an extracellular needle, a basal body, and a cytoplasmic complex. Three categories of proteins are hierarchically secreted: (a) the needle components, (b) the translocator proteins which form a pore (translocon) inside the host cell membrane and (c) the effectors interfering with the host cell signaling pathways. In the absence of host cell contact, the T3SS is maintained in an "off" state by the presence of a tip complex. Secretion is activated by host cell contact which allows the release of a gatekeeper protein called MxiC. In this work, we have investigated the role of Spa33, a component of the cytoplasmic complex, in the regulation of secretion. The spa33 gene encodes a 33-kDa protein and a smaller fragment of 12 kDa (Spa33C ) which are both essential components of the cytoplasmic complex. We have shown that the spa33 gene gives rise to 5 fragments of various sizes. Among them, three are necessary for T3SS. Interestingly, we have shown that Spa33 is implicated in the regulation of secretion. Indeed, the mutation of a single residue in Spa33 induces an effector mutant phenotype, in which MxiC is sequestered. Moreover, we have shown a direct interaction between Spa33 and MxiC.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Shigella/fisiologia , Sistemas de Secreção Tipo III/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Códon de Iniciação , Mutação , Ligação Proteica , Virulência/genéticaRESUMO
The regulation of the striatum by the GPCR signaling through neuromodulators is essential for its physiology and physiopathology, so it is necessary to know all the compounds of these pathways. In this study, we identified a new important partner of the dopaminergic pathway: GPRIN3 (a member of the GPRIN family). GPRIN3 is highly expressed in the striatum but with undefined function. Cell sorting of medium spiny neurons (MSNs) in indirect MSNs and direct MSNs indicated the presence of the GPRIN3 gene in both populations with a preferential expression in indirect MSNs. This led us to generate GPRIN3 KO mice by CRISPR/Cas9 and test male animals to access possible alterations in morphological, electrophysiological, and behavioral parameters following its absence. 3D reconstruction analysis of MSNs revealed increased neuronal arborization in GPRIN3 KO and modified passive and active electrophysiological properties. These cellular alterations were coupled with increased motivation and cocaine-induced hyperlocomotion. Additionally, using a specific indirect MSN knockdown, we showed a preferential role for GPRIN3 in indirect MSNs related to the D2R signaling. Together, these results show that GPRIN3 is a mediator of D2R function in the striatum playing a major role in striatal physiology.SIGNIFICANCE STATEMENT The striatum is the main input of the basal ganglia processing information from different brain regions through the combined actions of direct pathway neurons and indirect pathway neurons. Both neuronal populations are defined by the expression of dopamine D1R or D2R GPCRs, respectively. How these neurons signal to the respective G-protein is still debatable. Here we identified GPRIN3 as a putative selective controller of D2R function in the striatum playing a critical role in striatal-associated behaviors and cellular functions. This study represents the identification of a new target to tackle striatal dysfunction associated with the D2R, such as schizophrenia, Parkinson's disease, and drug addiction.
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Corpo Estriado/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaRESUMO
Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.
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Fatores Quimiotáticos/metabolismo , Células Dendríticas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Vírus da Pneumonia Murina/imunologia , Pneumonia Viral/imunologia , Infecções por Pneumovirus/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Quimiocinas , Fatores Quimiotáticos/biossíntese , Células Dendríticas/metabolismo , Mediadores da Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Interferon Tipo I/biossíntese , Interferon Tipo I/deficiência , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vírus da Pneumonia Murina/metabolismo , Vírus da Pneumonia Murina/patogenicidade , Pneumonia Viral/metabolismo , Infecções por Pneumovirus/metabolismo , Receptores de Quimiocinas , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/genética , Carga ViralRESUMO
The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.
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Proteínas de Transporte/metabolismo , Catepsina D/fisiologia , Fatores Quimiotáticos/agonistas , Hemeproteínas/metabolismo , Peptídeos/agonistas , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Receptores de Formil Peptídeo/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Proteínas de Transporte/biossíntese , Catepsina D/deficiência , Células Cultivadas , Fatores Quimiotáticos/biossíntese , Fatores Quimiotáticos/metabolismo , Cricetinae , Cricetulus , Proteínas Ligantes de Grupo Heme , Hemeproteínas/biossíntese , Humanos , Ligantes , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos/metabolismo , Ligação Proteica/imunologia , Precursores de Proteínas/biossíntese , Receptores de Formil Peptídeo/biossínteseRESUMO
Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin's metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O(2)(*-)-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser(473) and Thr(308) as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3alpha phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr(156/139), close to their active site Asp(166/149), in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.
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Angiotensina II/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitratos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina/metabolismo , Animais , Células Cultivadas , Transportador de Glucose Tipo 4/antagonistas & inibidores , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Transporte Proteico , RatosRESUMO
The SH2 domain containing inositol 5-phosphatase SHIP2 contains several interacting domains that are important for scaffolding properties. We and others have previously reported that SHIP2 interacts with the E3 ubiquitin ligase c-Cbl. Here, we identified human SHIP2 monoubiquitination on lysine 315. SHIP2 could also be polyubiquitinated but was not degraded by the 26 S proteasome. Furthermore, we identified a ubiquitin-interacting motif at the C-terminal end of SHIP2 that confers ubiquitin binding capacity. However, this ubiquitin-interacting motif is dispensable for its monoubiquitination. We showed that neither c-Cbl nor Nedd4-1 play the role of ubiquitin ligase for SHIP2. Strikingly, monoubiquitination of the DeltaSH2-SHIP2 mutant (lacking the N-terminal SH2 domain) is strongly increased, suggesting an intrinsic inhibitory effect of the SHIP2 SH2 domain on its monoubiquitination. Moreover, SHIP2 monoubiquitination was increased upon 30 min of epidermal growth factor stimulation. This correlates with the loss of interaction between the SHIP2 SH2 domain and c-Cbl. In this model, c-Cbl could mask the monoubiquitination site and thereby prevent SHIP2 monoubiquitination. The present study thus reveals an unexpected and novel role of SHIP2 SH2 domain in the regulation of its newly identified monoubiquitination.
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Fator de Crescimento Epidérmico/metabolismo , Modelos Biológicos , Monoéster Fosfórico Hidrolases/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Células COS , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Dual oxidases were initially identified as NADPH oxidases producing H(2)O(2) necessary for thyroid hormone biosynthesis. The crucial role of Duox2 has been demonstrated in patients suffering from partial iodide organification defect caused by bi-allelic mutations in the DUOX2 gene. However, the Duox1 function in thyroid remains elusive. We optimized a functional assay by co-expressing Duox1 or Duox2 with their respective maturation factors, DuoxA1 and DuoxA2, to compare their intrinsic enzymatic activities under stimulation of the major signaling pathways active in the thyroid in relation to their membrane expression. We showed that basal activity of both Duox isoenzymes depends on calcium and functional EF-hand motifs. However, the two oxidases are differentially regulated by activation of intracellular signaling cascades. Duox1 but not Duox2 activity is stimulated by forskolin (EC(50) = 0.1 microm) via protein kinase A-mediated Duox1 phosphorylation on serine 955. In contrast, phorbol esters induce Duox2 phosphorylation via protein kinase C activation associated with high H(2)O(2) generation (phorbol 12-myristate 13-acetate EC(50) = 0.8 nm). These results were confirmed in human thyroid cells, suggesting that Duox1 is also involved in thyroid hormonogenesis. Our data provide, for the first time, detailed insights into the mechanisms controlling the activation of Duox1-2 proteins and reveal additional phosphorylation-mediated regulation.
Assuntos
Membrana Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , NADPH Oxidases/metabolismo , Proteína Quinase C/metabolismo , Glândula Tireoide/enzimologia , Animais , Células COS , Carcinógenos/farmacologia , Membrana Celular/genética , Chlorocebus aethiops , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Oxidases Duais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Iodetos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , NADPH Oxidases/genética , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Doenças da Glândula Tireoide/enzimologia , Doenças da Glândula Tireoide/genética , Hormônios Tireóideos/biossínteseRESUMO
Chemerin is a potent chemotactic factor that was identified recently as the ligand of ChemR23, a G protein-coupled receptor expressed by mononuclear phagocytes, dendritic cells (DCs), and NK cells. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active on ChemR23. However, prochemerin can be converted rapidly into a full ChemR23 agonist by proteolytic removal of a carboxy-terminal peptide. This maturation step is mediated by the neutrophil-derived serine proteases elastase and cathepsin G. In the present work, we have investigated proteolytic events that negatively control chemerin activity. We demonstrate here that neutrophil-derived proteinase 3 (PR3) and mast cell (MC) chymase are involved in the generation of specific chemerin variants, which are inactive, as they do not induce calcium release or DC chemotaxis. Mass spectrometry analysis showed that PR3 specifically converts prochemerin into a chemerin form, lacking the last eight carboxy-terminal amino acids, and is inactive on ChemR23. Whereas PR3 had no effect on bioactive chemerin, MC chymase was shown to abolish chemerin activity by the removal of additional amino acids from its C-terminus. This effect was shown to be specific to bioactive chemerin (chemerin-157 and to a lesser extent, chemerin-156), as MC chymase does not use prochemerin as a substrate. These mechanisms, leading to the production of inactive variants of chemerin, starting from the precursor or the active variants, highlight the complex interplay of proteases regulating the bioactivity of this novel mediator during early innate immune responses.
Assuntos
Quimiocinas/metabolismo , Quimases/fisiologia , Células Dendríticas/metabolismo , Mastócitos/enzimologia , Mieloblastina/fisiologia , Neutrófilos/enzimologia , Equorina/metabolismo , Animais , Apoproteínas/metabolismo , Células da Medula Óssea/metabolismo , Células CHO , Cálcio/metabolismo , Células Cultivadas , Quimiotaxia , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/metabolismo , Neutrófilos/citologia , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We were looking by a proteomic approach for new phospho-proteins involved during the early steps of the TNF + cycloheximide (CHX)-induced apoptosis-preceding mitochondrial membrane permeabilization-of endothelial cells (BAEC). In the present study, we observed on the autoradiography from 2D gel of (32)P-labeled samples a string of proteins undergoing a complete dephosphorylation after 1 h of stimulation with TNF + CHX-while mitochondrial membrane permeabilization was observed after 3 h-identified the different spots by mass spectrometry as one and only protein, HDGF, and confirmed the identity by western blot. The intensity of the 2D phosphorylation pattern of HDGF was correlated with the amount of apoptosis induced by TNF + CHX and TNF or CHX alone and this event was inhibited by the Caspase specific inhibitor zVADfmk. Moreover the TNF + CHX-treatment did not affect the nuclear localization of GFP-HDGF. Taken together, our data suggest an involvement of HDGF during the initiation phase of the apoptotic process downstream from an initiator Caspase and a regulation of this protein by phosphorylation in the nucleus.
Assuntos
Apoptose , Caspases/fisiologia , Células Endoteliais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Animais , Aorta/citologia , Apoptose/efeitos dos fármacos , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Fosforilação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Chemerin is a chemotactic agonist recently identified as the ligand of ChemR23, a serpentine receptor expressed by mononuclear phagocytes and dendritic cells (DCs). This study shows that blood CD56(low)CD16(+) natural killer (NK) cells selectively express functional ChemR23 and that this receptor is coexpressed with CXCR1, the CXCL8 receptor, and the KIR receptors. In vitro culturing of NK cells with IL-2 or IL-15 induced a delayed and time-dependent down-regulation of ChemR23 that was associated with the inhibition of NK cell migration to chemerin. Biopsies obtained from patients with oral lichen planus presented an infiltration of CD94(+)CD3(-)CD56(+) NK cells that coexpressed ChemR23. The same biopsies were infiltrated by myeloid, DC-SIGN(+) and plasmacytoid, CD123(+)BDCA2(+), ChemR23(+) dendritic cells that were occasionally associated with NK cells. In the same histologic sections, chemerin was expressed by inflamed dermal endothelium. These findings propose a role for the ChemR23/chemerin axis in the recruitment of blood NK cells and strongly implicate chemerin as a key factor for the colocalization of NK cells and DC subsets in pathologic peripheral tissues.
Assuntos
Movimento Celular/imunologia , Quimiocinas/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Líquen Plano Bucal/imunologia , Receptores de Quimiocinas/imunologia , Quimiocinas/biossíntese , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Derme/imunologia , Derme/metabolismo , Derme/patologia , Endotélio/imunologia , Endotélio/metabolismo , Endotélio/patologia , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-15/biossíntese , Interleucina-15/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Líquen Plano Bucal/metabolismo , Líquen Plano Bucal/patologia , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Receptores de Quimiocinas/biossínteseRESUMO
Despite the ecological and evolutionary importance of echinoderms, very little is known about the immune mechanisms in this group especially regarding humoral immunity. In this paper, we screened for proteins putatively involved in immunity in the common European seastar Asterias rubens using a mass spectrometry-based proteomic approach. Two proteins showed striking sequence similarities with peptidoglycan recognition proteins (PGRPs). The two seastar proteins were identified as a single protein, termed PGRP-S1a, occurring in two forms in the coelomic plasma, one of 20kDa and another of 22kDa. We also cloned and sequenced a second member of the PGRP family, termed PGRP-S2a. It has a calculated molecular mass of 21.3kDa and is expressed in circulating phagocytes. Both the S1a-cDNA from coelomic epithelium RNA and the S2a-cDNA from phagocytes code for the amino acid residues necessary for peptidoglycan degradation. PGRP-S1a did not affect the phagocytic activity of seastar immune cells towards Micrococcus luteus but inhibited their production of reactive oxygen species (ROS). A recombinant, His-tagged, PGRP-S2a degrades peptidoglycan and increases the phagocytosis of M. luteus cells by seastar phagocytes.
Assuntos
Amidoidrolases/genética , Asterias/enzimologia , Asterias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Amidoidrolases/química , Amidoidrolases/metabolismo , Sequência de Aminoácidos , Animais , Asterias/genética , Sequência de Bases , Proteínas de Transporte/química , Clonagem Molecular , Imunidade Inata , Micrococcus luteus , Dados de Sequência Molecular , Peptidoglicano/metabolismo , Fagocitose , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Homologia de Sequência de AminoácidosRESUMO
F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.