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In the version of this article initially published, the institution name for affiliation 3 (Maryland Anderson Cancer Center) was incorrect. The correct institution is MD Anderson Cancer Center. The error has been corrected in the HTML and PDF versions of the article.
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This corrects the article DOI: 10.1038/ni.3748.
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An imbalance in the lineages of immunosuppressive regulatory T cells (Treg cells) and the inflammatory TH17 subset of helper T cells leads to the development of autoimmune and/or inflammatory disease. Here we found that TAZ, a coactivator of TEAD transcription factors of Hippo signaling, was expressed under TH17 cell-inducing conditions and was required for TH17 differentiation and TH17 cell-mediated inflammatory diseases. TAZ was a critical co-activator of the TH17-defining transcription factor RORγt. In addition, TAZ attenuated Treg cell development by decreasing acetylation of the Treg cell master regulator Foxp3 mediated by the histone acetyltransferase Tip60, which targeted Foxp3 for proteasomal degradation. In contrast, under Treg cell-skewing conditions, TEAD1 expression and sequestration of TAZ from the transcription factors RORγt and Foxp3 promoted Treg cell differentiation. Furthermore, deficiency in TAZ or overexpression of TEAD1 induced Treg cell differentiation, whereas expression of a transgene encoding TAZ or activation of TAZ directed TH17 cell differentiation. Our results demonstrate a pivotal role for TAZ in regulating the differentiation of Treg cells and TH17 cells.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Diferenciação Celular/imunologia , Colite/imunologia , Citocinas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Artrite Reumatoide/imunologia , Estudos de Casos e Controles , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Células HEK293 , Células HeLa , Histona Acetiltransferases/metabolismo , Humanos , Immunoblotting , Lisina Acetiltransferase 5 , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Síndrome de Sjogren/imunologia , Proteínas Smad/imunologia , Proteínas Smad/metabolismo , Fatores de Transcrição de Domínio TEA , Transativadores/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.
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Hipoglicemiantes , Metformina , ATPases Vacuolares Próton-Translocadoras , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina Trifosfatases/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Caenorhabditis elegans/metabolismo , Diabetes Mellitus/tratamento farmacológico , Glucose/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Lisossomos/metabolismo , Proteínas de Membrana , Metformina/agonistas , Metformina/metabolismo , Metformina/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.
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Fagócitos/imunologia , Fagócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Infecções Bacterianas/etiologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Atividade Bactericida do Sangue/imunologia , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitocôndrias/microbiologia , Fagócitos/microbiologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fagossomos/microbiologia , Proteína Quinase C-alfa/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Sepse/etiologia , Sepse/imunologia , Sepse/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Receptores Toll-Like/metabolismo , Ubiquitinação , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismoRESUMO
Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.
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Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Glucose/metabolismo , Glicólise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Pentose Fosfato , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Aminoácidos/deficiência , Aminoácidos/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Biomarcadores Tumorais/metabolismo , Morte Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Frutose-Bifosfatase/metabolismo , Genótipo , Células HCT116 , Hexoquinase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Masculino , Camundongos Knockout , Fenótipo , Fosfofrutoquinase-1/metabolismo , Fosfopiruvato Hidratase/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/metabolismoRESUMO
Targeted analysis of data-independent acquisition (DIA) data needs a spectral library, which is generated by data-dependent acquisition (DDA) experiments or directly from DIA data. A comparison of the DDA library and DIA library in analyzing DIA data has been reported. However, the effects of different spectral libraries on the analysis of diaPASEF data have not been investigated. Here, we generate different spectral libraries with varying proteome coverage to analyze parallel accumulation-serial fragmentation (diaPASEF) data. Besides, we also employ the library-free strategy. The library, constructed by extensive fractionation DDA experiments, produces the highest numbers of precursors and proteins but with a high percentage of missing values. The library-free strategy identifies 10-20% fewer proteins than the library-based method but with a high degree of data completeness. A further study shows that the library-free strategy, although it identifies fewer proteins than the library-based method, leads to similar biological conclusions as the library-based method.
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Proteoma , Proteômica , Biblioteca de Peptídeos , Proteômica/métodosRESUMO
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.
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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 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.
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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
The shift of carbon utilization from primarily glucose to other nutrients is a fundamental metabolic adaptation to cope with decreased blood glucose levels and the consequent decline in glucose oxidation. AMP-activated protein kinase (AMPK) plays crucial roles in this metabolic adaptation. However, the underlying mechanism is not fully understood. Here, we show that PDZ domain containing 8 (PDZD8), which we identify as a new substrate of AMPK activated in low glucose, is required for the low glucose-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527) and promotes the interaction of PDZD8 with and activation of glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis. In vivo, the AMPK-PDZD8-GLS1 axis is required for the enhancement of glutaminolysis as tested in the skeletal muscle tissues, which occurs earlier than the increase in fatty acid utilization during fasting. The enhanced glutaminolysis is also observed in macrophages in low glucose or under acute lipopolysaccharide (LPS) treatment. Consistent with a requirement of heightened glutaminolysis, the PDZD8-T527A mutation dampens the secretion of pro-inflammatory cytokines in macrophages in mice treated with LPS. Together, we have revealed an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis ahead of increased fatty acid utilization under glucose shortage.
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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.
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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
Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC) cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Serina/metabolismo , Linhagem Celular TumoralRESUMO
Aberrant activation of EGFR due to overexpression or mutation is associated with poor prognosis in many types of tumors. Here we show that blocking the sorting system that directs EGFR to plasma membrane is a potent strategy to treat EGFR-dependent tumors. We find that EGFR palmitoylation by DHHC13 is critical for its plasma membrane localization and identify ARF6 as a key factor in this process. N-myristoylated ARF6 recognizes palmitoylated EGFR via lipid-lipid interaction, recruits the exocyst complex to promote EGFR budding from Golgi, and facilitates EGFR transporting to plasma membrane in a GTP-bound form. To evaluate the therapeutic potential of this sorting system, we design a cell-permeable peptide, N-myristoylated GKVL-TAT, and find it effectively disrupts plasma membrane localization of EGFR and significantly inhibits progression of EGFR-dependent tumors. Our findings shed lights on the underlying mechanism of how palmitoylation directs protein sorting and provide an potential strategy to manage EGFR-dependent tumors.
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Fatores de Ribosilação do ADP , Neoplasias , Fatores de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , Receptores ErbB/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lipídeos , Neoplasias/metabolismo , Transporte ProteicoRESUMO
Dysfunction of protein trafficking has been intensively associated with neurological diseases, including neurodegeneration, but whether and how protein transport contributes to oligodendrocyte (OL) maturation and myelin repair in white matter injury remains unclear. ER-to-Golgi trafficking of newly synthesized proteins is mediated by coat protein complex II (COPII). Here, we demonstrate that the COPII component Sec13 was essential for OL differentiation and postnatal myelination. Ablation of Sec13 in the OL lineage prevented OPC differentiation and inhibited myelination and remyelination after demyelinating injury in the central nervous system (CNS), while improving protein trafficking by tauroursodeoxycholic acid (TUDCA) or ectopic expression of COPII components accelerated myelination. COPII components were upregulated in OL lineage cells after demyelinating injury. Loss of Sec13 altered the secretome of OLs and inhibited the secretion of pleiotrophin (PTN), which was found to function as an autocrine factor to promote OL differentiation and myelin repair. These data suggest that Sec13-dependent protein transport is essential for OL differentiation and that Sec13-mediated PTN autocrine signaling is required for proper myelination and remyelination.
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Doenças Desmielinizantes , Bainha de Mielina , Comunicação Autócrina , Proteínas de Transporte , Diferenciação Celular/fisiologia , Citocinas , Doenças Desmielinizantes/metabolismo , Humanos , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismoRESUMO
The activity of 5'-adenosine monophosphate-activated protein kinase (AMPK) is inversely correlated with the cellular availability of glucose. When glucose levels are low, the glycolytic enzyme aldolase is not bound to fructose-1,6-bisphosphate (FBP) and, instead, signals to activate lysosomal AMPK. Here, we show that blocking FBP binding to aldolase with the small molecule aldometanib selectively activates the lysosomal pool of AMPK and has beneficial metabolic effects in rodents. We identify aldometanib in a screen for aldolase inhibitors and show that it prevents FBP from binding to v-ATPase-associated aldolase and activates lysosomal AMPK, thereby mimicking a cellular state of glucose starvation. In male mice, aldometanib elicits an insulin-independent glucose-lowering effect, without causing hypoglycaemia. Aldometanib also alleviates fatty liver and nonalcoholic steatohepatitis in obese male rodents. Moreover, aldometanib extends lifespan and healthspan in both Caenorhabditis elegans and mice. Taken together, aldometanib mimics and adopts the lysosomal AMPK activation pathway associated with glucose starvation to exert physiological roles, and might have potential as a therapeutic for metabolic disorders in humans.
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Insulinas , Inanição , Humanos , Masculino , Camundongos , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Lisossomos/metabolismo , Inanição/metabolismo , Adenosina Trifosfatases/metabolismo , Caenorhabditis elegans , Monofosfato de Adenosina/metabolismo , Frutose/metabolismo , Insulinas/metabolismoRESUMO
TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.
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Infecções Bacterianas/imunologia , Imunidade Inata , Canais Iônicos/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Infecções por Escherichia coli/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Serina-Treonina Quinase 3 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways. By employing the SWATH-MS technique, we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes. Combining SWATH-MS-based network modeling and experimental validation, we found that when RIP1 level is below ~1000 molecules/cell (mpc), the cell solely undergoes TRADD-dependent apoptosis. When RIP1 is above ~1000 mpc, pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount, which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical observations that RIP1 is required for necroptosis but its increase down-regulates necroptosis. Higher amount of RIP1 (>~46,000 mpc) suppresses apoptosis, leading to necroptosis alone. The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic. Our study provides a resource for encoding the complexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes, enabling RIP1 to play diverse roles in governing cell fate decisions.
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Apoptose , Caspase 8/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Caspase 8/genética , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.
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Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/inervação , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sistema Nervoso Simpático/fisiologia , Termogênese , Proteína Desacopladora 1/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Metabolismo Energético , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Proteínas de Transferência de Fosfolipídeos/deficiência , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação , Transdução de Sinais , Proteína Desacopladora 1/deficiência , Proteína Desacopladora 1/genéticaRESUMO
Targeted SWATH-MS data analysis is critically dependent on the spectral library. Comprehensive spectral libraries of human or several other organisms have been published, but the extensive spectral library for mouse, a widely used model organism is not available. Here, we present a large murine spectral library covering more than 11,000 proteins and 240,000 proteotypic peptides, which included proteins derived from 9 murine tissue samples and one murine L929 cell line. This resource supports the quantification of 67% of all murine proteins annotated by UniProtKB/Swiss-Prot. Furthermore, we applied the spectral library to SWATH-MS data from murine tissue samples. Data are available via SWATHAtlas (PASS01441).
Assuntos
Biblioteca de Peptídeos , Proteoma/análise , Animais , Bases de Dados de Proteínas , Camundongos , ProteômicaRESUMO
Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1-/- mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy.