RESUMO
NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.
Assuntos
Proteínas Reguladoras de Apoptose , Inflamassomos , Adenosina Trifosfatases/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Flagelina/metabolismo , Inflamassomos/metabolismo , Proteína Inibidora de Apoptose Neuronal/química , Proteína Inibidora de Apoptose Neuronal/metabolismo , Proteínas NLR/metabolismoRESUMO
Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Caspase 8/metabolismo , Necroptose , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Animais , Ceco/lesões , Ceco/patologia , Linhagem Celular , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Mutação/genética , Necroptose/efeitos dos fármacos , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Proteínas Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects â¼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.
Assuntos
Fosfopeptídeos , Proteômica , Proteômica/métodos , Espectrometria de Massas/métodos , Proteoma/análiseRESUMO
PURPOSE: This study aimed to investigate the diagnostic performance of [68Ga]Ga-FAPI PET/CT for primary and metastatic pancreatic carcinoma lesions and compare the results with those of [18F]-fluorodeoxyglucose ([18F]FDG) PET/CT. METHODS: Patients with suspected or diagnosed pancreatic malignancy, who underwent contemporaneous [18F]FDG and [68Ga]Ga-FAPI PET/CT between June 2020 and January 2021, were retrospectively analyzed. Routine contrast-enhanced CT (CE-CT) is performed in all patients as standardized care. Findings were confirmed by histopathology or radiographic follow-up. We compared radiotracer uptake, diagnostic performance, and TNM (tumor-node-metastasis) classifications. RESULTS: We evaluated 36 participants (25/36 men; median age, 60 years), including 26 patients with pancreatic malignancies and ten patients with pancreatic benign lesions. [68Ga]Ga-FAPI PET/CT showed higher radiotracer uptake and higher sensitivity than [18F]FDG PET/CT in evaluating primary tumors (SUVmax, 21.4 vs. 4.8; sensitivity, 100% vs. 73.1%), involved lymph nodes (SUVmax, 8.6 vs. 2.7; sensitivity, 81.8% vs. 59.1%), and metastases (SUVmax, 7.9 vs. 3.5; sensitivity, 91.5% vs. 44.0%); Compared with [18F]FDG, [68Ga]Ga-FAPI PET/CT upstaged six patients' TNM staging (6/23, 26.1%) and changed two patients' clinical management (2/23, 8.7%). Compared with CE-CT, [68Ga]Ga-FAPI PET/CT upgraded TNM staging in five patients (5/23, 21.7%) and changed the therapeutic regimen in only one patient (1/23, 4.3%). Intense [68Ga]Ga-FAPI uptake was observed throughout the pancreas in 12/26 pancreatic malignancies; dual-time point [68Ga]Ga-FAPI PET/CT may differentiate pancreatitis from malignancy. CONCLUSIONS: Compared with [18F]FDG PET/CT, [68Ga]Ga-FAPI PET/CT shows higher sensitivity in detecting primary pancreatic tumors, involved lymph nodes, and metastases and is superior in terms of TNM staging. Prospective trials with larger patient population are needed to evaluate whether [68Ga]Ga-FAPI PET/CT could elicit treatment modification in pancreatic cancer when compared with standard of care imaging.
Assuntos
Neoplasias Pancreáticas , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Feminino , Fibroblastos , Fluordesoxiglucose F18 , Radioisótopos de Gálio , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Estudos Prospectivos , Compostos Radiofarmacêuticos , Estudos Retrospectivos , Neoplasias PancreáticasRESUMO
PURPOSE: This prospective study aimed to evaluate the potential usefulness of [68Ga]Ga-DOTA-FAPI-04 positron emission tomography/computed tomography (PET/CT) in the oncological evaluation of patients presenting with inconclusive [18F]FDG PET/CT findings. METHODS: [68Ga]Ga-DOTA-FAPI-04 was performed in patients presenting with inconclusive [18F]FDG PET/CT findings. Tumour uptake was quantified by the maximum standard uptake value (SUV). Histopathology or follow-up imaging served as the standard for the final diagnosis. RESULTS: A total of 68 patients with inconclusive [18F]FDG PET/CT findings underwent additional [68Ga]Ga-DOTA-FAPI-04 PET/CT. Of them, 18 (26.5%) were for discrimination of mass lesions detected on conventional imaging, 6 (8.8%) for detection of the unknown primary site in biopsy-proven metastatic malignancy, 21 (30.9%) for the staging of cancer, and the other 23 (33.8%) for evaluation of suspected disease recurrence. Most of the primary and metastatic lesions demonstrated higher uptake of [68Ga]Ga-DOTA-FAPI-04 than did [18F]FDG, which resulted in favourable tumour-to-background contrast in various types of cancer. As a result, [68Ga]Ga-DOTA-FAPI-04 PET/CT identified suspicious mass lesions with an accuracy of 12/18 (66.7%), detected the primary site in 4/6 patients (66.7%) with unknown malignancy, upgraded tumour staging in 7/21 patients (33.3%), and detected disease recurrence in 20/23 patients (87.0%). CONCLUSIONS: In patients undergoing oncological evaluation with inconclusive [18F]FDG PET/CT findings, [68Ga]Ga-DOTA-FAPI-04 may have a complementary role in discriminating mass lesions on conventional imaging, locating the primary site of unknown malignancy, modifying tumour staging, and detecting suspected disease recurrence. Nevertheless, careful attention should be paid when reading the [68Ga]Ga-DOTA-FAPI-04 PET/CT images in tumours complicated with inflammation.
Assuntos
Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioisótopos de Gálio , Humanos , Recidiva Local de Neoplasia , Estudos Prospectivos , QuinolinasRESUMO
Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.
Assuntos
Lipopolissacarídeos/metabolismo , Espectrometria de Massas/métodos , Transdução de Sinais , Animais , Bases de Dados de Proteínas , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação , Ligação Proteica , Células RAW 264.7 , Fator 6 Associado a Receptor de TNF , Receptor 4 Toll-Like/metabolismoRESUMO
The purpose of this study was to determine the difference in discrimination between high- and low-grade supratentorial nonenhancing gliomas (HGGs and LGGs, respectively) when using apparent diffusion coefficient (ADC) values with high or standard b-value. Thirty-nine patients underwent conventional magnetic resonance imaging and diffusion-weighted imaging (DWI) with standard and high b-values (b = 1000 and 3000 s/mm2, respectively). Minimum, maximum, and mean ADC values (ADCMIN, ADCMAX, and ADCMEAN, respectively) were measured from ADC maps with both b-values. Receiver operating curve analysis was used to determine the cutoff ADC values for distinguishing between nonenhancing HGGs and LGGs. ADCMIN, ADCMAX, and ADCMEAN values for the nonenhancing HGGs were lower than those for LGGs. These differences were much larger when a high b-value was used (all P < 0.0001) than when a standard b-value was used (P = 0.0001, <0.0001, and <0.0001, respectively). Discriminant analysis indicated that the greatest likelihood for discriminating HGGs and LGGs when ADCMEAN was obtained with a high b-value, with cutoff value of 0.814 × 10-3 mm2/s. ADC values obtained with a high b-value can be useful for grading and surgical management of nonenhancing HGGs and LGGs. The lowest degree of overlap was obtained when ADCMEAN was determined with a b-value of 3000 s/mm2.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética , Glioma/diagnóstico por imagem , Gradação de Tumores/métodos , Adulto , Idoso , Área Sob a Curva , Encéfalo/patologia , Encéfalo/cirurgia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Imagem de Difusão por Ressonância Magnética/métodos , Análise Discriminante , Feminino , Glioma/patologia , Glioma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Cuidados Pré-OperatóriosRESUMO
Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection.
Assuntos
Dicistroviridae/fisiologia , Proteínas de Drosophila/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Virais/biossíntese , Replicação Viral/fisiologia , Animais , Capsídeo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutação , Proteínas Nucleares/genética , Proteínas Virais/genéticaRESUMO
Tetrastigma hemsleyanum is a rare and endangered herb, which is commercialized as the resource of anti-cancer drugs. Wild T. hemsleyanum plants are on the verge of extinction recently, there are increasing numbers of counterfeits on the market. In the present study, inter-simple sequence repeat (ISSR), Cleaved amplified polymorphic sequence (CAPS), and the internal transcribed spacer region II (ITS2) barcode were used for the first time for the authentication of T. hemsleyanum from its commonly counterfeits. ISSR analysis suggested that it was a useful method for distinguishing T. hemsleyanum from its adulterants of different genus. However, it was insufficient to distinguish T. hemsleyanum from those adulterants of the same genus. ITS2 of T. hemsleyanum and the commonly counterfeits were amplified and sequenced. The Neighbor-Joining tree constructed from the ITS2 sequences showed that T. hemsleyanum was clearly differentiated from all counterfeits samples. A mutation site in the ITS2 region of T. hemsleyanum had been found which could be recognized by the restriction endonuclease NcoI. T. hemsleyanum could be readily distinguished from counterfeits as the PCR products from T. hemsleyanum could be digested sufficiently by NcoI, while the PCR products from counterfeits could not be digested. The results indicated that CAPS and ITS2 barcode methods provided effective and accurate identification of T. hemsleyanum from all its adulterants, while ISSR could only distinguish T. hemsleyanum from its adulterants of different genus. The CAPS method developed in the present study will serve as a reliable tool for safe and effective use of T. hemsleyanum in the clinic application. It will also play an important role for the identification, management and conservation of this endangered species.
Assuntos
Espécies em Perigo de Extinção , Vitaceae/genética , Sequência de Bases , Código de Barras de DNA Taxonômico , DNA de Plantas/genética , DNA Espaçador Ribossômico/genética , Medicamentos de Ervas Chinesas , Genes de Plantas , Repetições de Microssatélites , Filogenia , Polimorfismo Genético , Vitaceae/classificaçãoRESUMO
The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.
Assuntos
Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Aconitato Hidratase/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Complexo I de Transporte de Elétrons/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND AND OBJECTIVE: Patients with chronic pancreatitis often require surgical treatment. The aim of this study was to evaluate the published evidence for Frey procedure in patients with chronic pancreatitis. METHODS: Literature search was undertaken to identify eligible studies until February 2015. Using meta-analytical techniques, Frey procedure was compared with pancreatoduodenectomy or Beger procedure, and the short- and long-term outcomes were analysed. RESULTS: Twenty-three studies comprising a total of 800 patients were reviewed. The postoperative morbidity and mortality were 23.2% and 0.4% respectively. The percentage of postoperative pain-relief patients was 89.4%. New onset of diabetes and exocrine insufficiency was present in 17.3% and 30.7% of patients, respectively. Compared with pancreatoduodenectomy, Frey procedure had favorable outcomes in terms of operation time, blood transfusion, overall morbidity, length of hospital and intensive care unit stay, pancreatic function and quality of life. Compared with Beger procedure, Frey procedure had shorter operation time and lower morbidity. CONCLUSIONS: Frey procedure is a safe and effective surgical procedure for chronic pancreatitis with dilated duct in the absence of neoplasia.
Assuntos
Pancreaticoduodenectomia/métodos , Pancreatite Crônica/cirurgia , Medicina Baseada em Evidências , Humanos , Pancreatectomia/métodos , Ductos Pancreáticos/patologia , Pancreaticoduodenectomia/efeitos adversos , Pancreaticojejunostomia/métodos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/terapia , Resultado do TratamentoRESUMO
Although more than half of genomic loci are believed to have antisense transcription, whether antisense transcription is involved in cytokine expression has not been studied. In this study, we show that some loci of innate immunity related genes do have antisense transcripts. We investigated the effect of several antisense RNAs, including anti-4-1BBL, anti-p100, and anti-IL-1ß, on their cognate sense gene's expression in macrophages. We found that overexpression of antisense IL-1ß transcript suppressed IL-1ß expression. Anti-IL-1ß is complementary to the sequence in the 5' upstream region of the IL-1ß promoter. Its mediated inhibition of IL-1ß production occurred at the transcriptional level. Anti-IL-1ß did not alter the methylation status of the IL-1ß promoter. However, chromatin immunoprecipitation assays revealed that the anti-IL-1ß transcript can change the chromatin structure of the IL-1ß promoter by decreasing H3K4 trimethylation on the promoter, which is at least part of the mechanism underlying the reduced binding of RNA polymerase II to the IL-1ß promoter upon anti-IL-1ß expression. Our data suggest that some antisense transcripts of innate immunity-related genes play a role by regulating cytokine expression.
Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucina-1beta/genética , RNA Antissenso/genética , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/genética , Imunidade Inata/imunologia , Interleucina-1beta/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , RNA Antissenso/imunologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.
Assuntos
Células Musculares/enzimologia , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Amiloide/genética , Amiloide/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Células Musculares/patologia , Proteínas Musculares/genética , Necrose/enzimologia , Necrose/genética , Necrose/patologia , Fosforilação/genética , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3(+/+)) and RIP3 knockout (RIP3(-/-)) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3(+/+) and RIP3(-/-) mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3(+/+) and RIP3(-/-) mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3(+/+) macrophages, 121 were detected exclusively from RIP3(+/+) MEFs, 286 phosphopeptides were induced more in RIP3(+/+) macrophages than in RIP3(-/-) macrophages and 26 phosphopeptides had higher induction in RIP3(+/+) MEFs than in RIP3(-/-) cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists.
Assuntos
Macrófagos Peritoneais/metabolismo , Necrose/metabolismo , Fosfopeptídeos/análise , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos , Animais , Ciclo Celular , Linhagem Celular , Cromatografia de Afinidade , Cromatografia Líquida , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Marcação por Isótopo , Lipopolissacarídeos , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fosforilação , Proteoma/análise , Proteômica/métodos , Análise de Sequência de Proteína , Transdução de Sinais , Coloração e Rotulagem , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Processamento Eletrônico de Dados/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Algoritmos , Linhagem Celular , Interpretação Estatística de Dados , Bases de Dados de Proteínas , Processamento Eletrônico de Dados/normas , Humanos , Espectrometria de Massas/normas , Fragmentos de Peptídeos/análise , Proteínas/análise , Proteômica/normas , SoftwareRESUMO
The signaling network of innate immunity in Drosophila is constructed by multiple evolutionarily conserved pathways, including the Toll- or Imd-regulated NF-κB and JNK pathways. The p38 MAPK pathway is evolutionarily conserved in stress responses, but its role in Drosophila host defense is not fully understood. Here we show that the p38 pathway also participates in Drosophila host defense. In comparison with wild-type flies, the sensitivity to microbial infection was slightly higher in the p38a mutant, significantly higher in the p38b mutant, but unchanged in the p38c mutant. The p38b;p38a double-mutant flies were hypersensitive to septic injury. The immunodeficiency of p38b;p38a mutant flies was also demonstrated by hindgut melanization and larvae stage lethality that were induced by microbes naturally presented in fly food. A canonical MAP3K-MKK cascade was found to mediate p38 activation in response to infection in flies. However, neither Toll nor Imd was required for microbe-induced p38 activation. We found that p38-activated heat-shock factor and suppressed JNK collectively contributed to host defense against infection. Together, our data demonstrate that the p38 pathway-mediated stress response contribute to Drosophila host defense against microbial infection.
Assuntos
Bactérias/imunologia , Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Fungos/imunologia , Imunidade/imunologia , Sistema de Sinalização das MAP Quinases , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Meios de Cultura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Ativação Enzimática , Alimentos , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Regulação da Expressão Gênica , Genes de Insetos/genética , Proteínas de Choque Térmico/metabolismo , Imunidade/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melaninas/metabolismo , Mutação/genética , Sepse/enzimologia , Sepse/prevenção & controleRESUMO
Inflammasomes are multimeric protein complexes that have crucial functions in innate immunity. Here, we present a protocol to reconstitute the PELO-driven assembly of NAIP5-NLRC4 inflammasome in vitro. We describe steps for expression and purification of recombinant PELO and flagellin, preparation of native cell lysate containing NAIP5-NLRC4, and in vitro assembly of NAIP5-NLRC4 inflammasome. We then detail analysis of NAIP5-NLRC4 inflammasome by blue native polyacrylamide gel electrophoresis and immunoblotting. This protocol can be adapted to monitor the oligomeric assembly of other inflammasome types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2023).1.
Assuntos
Proteínas Reguladoras de Apoptose , Inflamassomos , Inflamassomos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Inibidora de Apoptose Neuronal/genética , Proteína Inibidora de Apoptose Neuronal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Imunidade InataRESUMO
Obesity is regarded as an abnormal or excessive buildup of fat that may be bad for health and is influenced by a combination of intestinal flora, genetic background, physical activity level and environment. Symbiotic supplementation may be a realistic and easy therapy for the reversal of obesity and associated metabolic problems. In this study, we chose two Bifidobacterium species, three Lactobacilli species and four prebiotics to make a new symbiotic formulation. High or low doses of the symbiotic were administered to rats, and biochemical indicators were recorded to assess the biological effects in a high-fat-diet-induced rat model. The underlying mechanisms were explored by integrating 16S rRNA sequencing with an extensively targeted metabolome. High-dose symbiotic supplementation was effective in reducing obesity and concomitant metabolic syndrome. The high-dose symbiotic also significantly increased the abundance of Blautia, which was negatively correlated with taurocholic acid and the main differential metabolites involved in amino acid and bile acid metabolism. While the low-dose symbiotic had some therapeutic effects, they were not as strong as those at the high dose, demonstrating that the effects were dose-dependent. Overall, our novel symbiotic combination improved plasma glucose and lipid levels, shrunk adipocyte size, restored liver function, increased the abundance of Blautia and adjusted bile acid and amino acid metabolism.