RESUMO
cGAS is an evolutionarily conserved enzyme that has a pivotal role in immune defence against infection1-3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP)4,5, which leads to the expression of antimicrobial genes6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signalling systems (CBASS) have been discovered8-11. These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria on phage infection, thereby stopping phage spread. Of the CBASS systems reported, approximately 39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating enzymes, respectively8,12. Although these proteins are required to prevent infection of some bacteriophages8, the mechanism by which the enzymatic activities exert an anti-phage effect is unknown. Here we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increases the production of cGAMP. Using a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signalling by binding tightly to cGAMP (dissociation constant of approximately 30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that was bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrates an arms race between bacteria and viruses through controlling CDN levels.
Assuntos
Bactérias , Proteínas de Bactérias , Bacteriófagos , Nucleotidiltransferases , Ubiquitina , Animais , Bactérias/enzimologia , Bactérias/imunologia , Bactérias/metabolismo , Bactérias/virologia , Bacteriófagos/imunologia , Nucleotídeos Cíclicos/biossíntese , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Ubiquitina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Virais/metabolismo , Interações entre Hospedeiro e MicrorganismosRESUMO
The programmed necrosis induced by TNF-α requires the activities of the receptor-interacting serine-threonine kinases RIP1 and RIP3 and their interaction with the mixed lineage kinase domain-like protein MLKL. We report the identification of RIP1- and RIP3-containing protein complexes that form specifically in response to necrosis induction. One component of these complexes is the mitochondrial protein phosphatase PGAM5, which presents as two splice variants, PGAM5L (long form) and PGAM5S (short form). Knockdown of either form attenuated necrosis induced by TNF-α as well as reactive oxygen species (ROS) and calcium ionophore, whereas knockdown of RIP3 and MLKL blocked only TNF-α-mediated necrosis. Upon necrosis induction, PGAM5S recruited the mitochondrial fission factor Drp1 and activated its GTPase activity by dephosphorylating the serine 637 site of Drp1. Drp1 activation caused mitochondrial fragmentation, an early and obligatory step for necrosis execution. These data defined PGAM5 as the convergent point for multiple necrosis pathways.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Necrose/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Animais , Dinaminas/metabolismo , Células HeLa , Humanos , Camundongos , Mitocôndrias/enzimologia , Fosfoproteínas Fosfatases , Isoformas de Proteínas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects infections or tissue damage by binding to microbial or self DNA in the cytoplasm1. Upon binding DNA, cGAS produces cGAMP that binds to and activates the adaptor protein STING, which then activates the kinases IKK and TBK1 to induce interferons and other cytokines2-6. Here we report that STING also activates autophagy through a mechanism that is independent of TBK1 activation and interferon induction. Upon binding cGAMP, STING translocates to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and the Golgi in a process that is dependent on the COP-II complex and ARF GTPases. STING-containing ERGIC serves as a membrane source for LC3 lipidation, which is a key step in autophagosome biogenesis. cGAMP induced LC3 lipidation through a pathway that is dependent on WIPI2 and ATG5 but independent of the ULK and VPS34-beclin kinase complexes. Furthermore, we show that cGAMP-induced autophagy is important for the clearance of DNA and viruses in the cytosol. Interestingly, STING from the sea anemone Nematostella vectensis induces autophagy but not interferons in response to stimulation by cGAMP, which suggests that induction of autophagy is a primordial function of the cGAS-STING pathway.
Assuntos
Autofagia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Animais , Autofagossomos/metabolismo , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/deficiência , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Citosol/virologia , Vírus de DNA/genética , Vírus de DNA/metabolismo , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Evolução Molecular , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Interferons/biossíntese , Interferons/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos Cíclicos/imunologia , Nucleotídeos Cíclicos/metabolismo , Proteínas de Ligação a Fosfato , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Anêmonas-do-Mar , Proteínas de Transporte Vesicular/metabolismoRESUMO
Smac mimetics induce apoptosis synergistically with TNF-alpha by triggering the formation of a caspase-8-activating complex containing receptor interacting protein kinase-1 (RIPK1). Caspase inhibitors block this form of apoptosis in many types of cells. However, in several other cell lines, caspase inhibitors switch the apoptotic response to necrosis. A genome wide siRNA screen revealed another member of the RIP kinase family, RIP3, to be required for necrosis. The expression of RIP3 in different cell lines correlates with their responsiveness to necrosis induction. The kinase activity of RIP3 is essential for necrosis execution. Upon induction of necrosis, RIP3 is recruited to RIPK1 to form a necrosis-inducing complex. Embryonic fibroblasts from RIP3 knockout mice are resistant to necrosis and RIP3 knockout animals are devoid of inflammation inflicted tissue damage in an acute pancreatitis model. These data indicate RIP3 as the determinant for cellular necrosis in response to TNF-alpha family of death-inducing cytokines.
Assuntos
Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Tumoral , Ceruletídeo , Humanos , Camundongos , Mutação , Pancreatite/induzido quimicamente , Pancreatite/fisiopatologia , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Ferroptosis, a newly discovered form of regulated cell death dependent on iron and reactive oxygen species, is mainly characterized by mitochondrial shrinkage, increased density of bilayer membranes and the accumulation of lipid peroxidation, causing membrane lipid peroxidation and eventually cell death. Similar with the most forms of regulated cell death, ferroptosis also participated in the pathological metabolism of myocardial infarction and myocardial ischemia/reperfusion injuries, which are still the leading causes of death worldwide. Given the crucial roles ferroptosis played in cardiovascular diseases, such as myocardial infarction and myocardial ischemia/reperfusion injuries, it is considerable to delve into the molecular mechanisms of ferroptosis contributing to the progress of cardiovascular diseases, which might offer the potential role of ferroptosis as a targeted treatment for a wide range of cardiovascular diseases. This review systematically summarizes the process and regulatory metabolisms of ferroptosis, discusses the relationship between ferroptosis and myocardial infarction as well as myocardial ischemia/reperfusion injuries, which might potentially provide novel insights for the pathological metabolism and original ideas for the prevention as well as treatment targeting ferroptosis of cardiovascular diseases such as myocardial infarction and myocardial ischemia/reperfusion injuries.
Assuntos
Ferroptose , Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Humanos , Traumatismo por Reperfusão Miocárdica/patologia , Apoptose , Peroxidação de LipídeosRESUMO
The inflammatory response of mammalian cells to TNF-alpha can be switched to apoptosis either by cotreatment with a protein synthesis inhibitor, cycloheximide, or Smac mimetic, a small molecule mimic of Smac/Diablo protein. Cycloheximide promotes caspase-8 activation by eliminating endogenous caspase-8 inhibitor, c-FLIP, while Smac mimetic does so by triggering autodegradation of cIAP1 and cIAP2 (cIAP1/2), leading to the release of receptor interacting protein kinase (RIPK1) from the activated TNF receptor complex to form a caspase-8-activating complex consisting of RIPK1, FADD, and caspase-8. This process also requires the action of CYLD, a RIPK1 K63 deubiquitinating enzyme. RIPK1 is critical for caspase-8 activation-induced by Smac mimetic but dispensable for that triggered by cycloheximide. Moreover, Smac mimetic-induced caspase-8 activation is not blocked by endogenous c-FLIP. These findings revealed that TNF-alpha is able to induce apoptosis via two distinct caspase-8 activation pathways that are differentially regulated by cIAP1/2 and c-FLIP.
Assuntos
Apoptose , Caspase 8/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Cicloeximida/farmacologia , Ativação Enzimática/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Mimetismo Molecular , Complexos Multiproteicos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de SinaisRESUMO
Soluble receptor for advanced glycation end-products (sRAGE) was reported to inhibit cardiac apoptosis through the mitochondrial pathway during myocardial ischemia/reperfusion (I/R) injury. Meanwhile, the proapoptotic protein Bcl2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3) was reported to mediate mitochondrial depolarization and be activated by the Forkhead box protein O3 (FoxO3a). Therefore, it is supposed that FoxO3a-Bnip3 pathway might be involved in the inhibiting effects of sRAGE on mitochondrial apoptosis during I/R. I/R surgery or glucose deprivation/reoxygenation was adopted to explore mitochondrial depolarization, apoptosis and related signaling pathways in mice hearts and cultured cardiomyocytes. The results showed that overexpression of sRAGE in cardiomyocytes dramatically improved cardiac function and reduced infarct areas in I/R treated mice. sRAGE inhibited mitochondrial depolarization and cardiac apoptosis during I/R, which correlated with reduced expression of Bnip3, Sirt2, phosphorylation of Akt and FoxO3a which translocated into nucleus in cultured cardiomyocytes. Either Sirt2 or FoxO3a silencing enhanced the inhibiting effects of sRAGE on mitochondrial depolarization induced by I/R in cultured cardiomyocytes. Meanwhile, overexpression or silencing of FoxO3a affected the inhibiting effects of sRAGE on Bnip3 and cleaved caspase-3 in cultured cardiomyocytes. Therefore, it is suggested that sRAGE inhibited I/R injuries via reducing mitochondrial apoptosis through the FoxO3a-Bnip3 pathway.
Assuntos
Traumatismo por Reperfusão Miocárdica , Animais , Apoptose , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Sirtuína 2/metabolismo , Sirtuína 2/farmacologiaRESUMO
Soluble receptor for advanced glycation end-product (sRAGE) was reported to protect myocardial ischemia/reperfusion (I/R) injuries via directly interacting with cardiomyocytes besides competing with RAGE for AGEs. However, the specific molecule for the interaction between sRAGE and cardiomyocytes are not clearly defined. Integrins which were reported to interact with RAGE on leukocytes were also expressed on myocardial cells, therefore it was supposed that sRAGE might interact with integrins on cardiomyocytes to protect hearts from ischemia/reperfusion injuries. The results showed that sRAGE increased the expression of integrinß3 but not integrinß1, ß2, ß4 or ß5 in cardiomyocytes during I/R injuries. Meanwhile, the suppressive effects of sRAGE on cardiac function, cardiac infraction size and apoptosis in mice were cancelled by inhibition of integrinß3 with cilengitide (CLG, 75 mg/kg). The results from cultured cardiomyocytes also proved that sRAGE attenuated myocardial apoptosis and autophagy through interacting with integrinß3 to activate Akt and STAT3 pathway during oxygen and glucose deprivation/reperfusion (OGD/R) treatment. Furthermore, the phosphorylation of STAT3 was significantly downregulated by the inhibition of Akt (LY294002, 10 µM) in OGD/R and sRAGE treated cardiomyocytes, which suggested that STAT3 pathway was induced by Akt in I/R and sRAGE treated cardiomyocytes. The present study contributes to the understanding of myocardial I/R pathogenesis and provided a novel integrinß3-dependent therapy strategy for sRAGE ameliorating I/R injuries.
Assuntos
Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Apoptose , Integrinas/genética , Isquemia , Camundongos , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Reperfusão , Transdução de SinaisRESUMO
RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here, we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.
Assuntos
RNA Helicases DEAD-box/fisiologia , Vírus da Encefalomiocardite/fisiologia , Poliubiquitina/metabolismo , Animais , Sistema Livre de Células , Proteína DEAD-box 58 , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Vírus da Encefalomiocardite/genética , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HEK293/metabolismo , Células HEK293/virologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/biossíntese , Interferon beta/genética , Camundongos , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Ligação Proteica , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Receptores Imunológicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
The novel coronavirus disease (COVID-19) has become a universally prevalent infectious disease. The causative virus of COVID-19 is severe acute respiratory syndrome coronavirus type 2. Recent retrospective clinical studies have established a significant association between the incidence of vascular thrombotic events and the severity of COVID-19. The enhancement in serum levels of markers that reflect a hypercoagulable state has been suggested to indicate a poor prognosis. Therefore, at present, it is crucial to understand the mechanisms that foster the hypercoagulable state in COVID-19. Over-activated inflammatory response, which is manifested as excessive cytokine release in COVID-19 patients, is also associated with COVID-19 severity. This review discusses the immuno-pathological basis of the excessive cytokine release in COVID-19. Besides, this article reviews the role of pro-inflammatory or anti-inflammatory cytokines, whose significant elevations in their serum levels have been consistently detected in multiple different clinical studies, in promoting the hypercoagulable state. Since the expression of angiotensin-converting enzyme 2 (ACE2) is potentially down-regulated in COVID-19, as proposed by a recent bio-informatic analysis, mechanisms through which reduced ACE2 expressions promote vascular thrombosis are summarized. In addition, the reciprocal-enhancing effects of the excessive cytokine release and the downregulated ACE2 expression on their pro-thrombotic activities are further discussed. Here, based on currently available evidence, we review the pathogenic mechanisms of the hypercoagulable state associated with severe cases of COVID-19 to give insights into prevention and treatment of the vascular thrombotic events in COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2/biossíntese , COVID-19/sangue , Citocinas/sangue , Regulação para Baixo , SARS-CoV-2/metabolismo , Trombofilia/sangue , Biomarcadores/sangue , Humanos , Índice de Gravidade de DoençaRESUMO
Soluble receptor for advanced glycation end-products (sRAGE), which exerts cardioprotective effect through inhibiting cardiomyocyte apoptosis and autophagy during ischemia/reperfusion (I/R) injury, is also known to enhance angiogenesis in post-ischemic reperfusion injury-critical limb ischemia (PIRI-CLI) mice. However, whether sRAGE protects the heart from myocardial I/R injury via promoting angiogenesis remains unclear. Myocardial model of I/R injury was conducted by left anterior descending (LAD) ligation for 30 min and reperfusion for 2 weeks in C57BL/6 mice. And I/R injury in cardiac microvascular endothelial cells (CMECs) was duplicated by oxygen and glucose deprivation. The results showed that I/R-induced cardiac dysfunction, inflammation and myocardial fibrosis were all reversed by sRAGE. CD31 immunohistochemistry staining showed that sRAGE increased the density of vessels after I/R injury. The results from cultured CMECs showed that sRAGE inhibited apoptosis and increased proliferation, migration, angiogenesis after exposure to I/R. These effects were dependent on signal transducer and activator of transcription 3 (STAT3) pathway. Together, the present study demonstrated that activation of STAT3 contributed to the protective effects of sRAGE on myocardial I/R injury via promoting angiogenesis.
Assuntos
Células Endoteliais/metabolismo , Produtos Finais de Glicação Avançada/genética , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Neovascularização Fisiológica , Receptor para Produtos Finais de Glicação Avançada/genética , Fator de Transcrição STAT3/genética , Animais , Apoptose/genética , Autofagia/genética , Débito Cardíaco/fisiologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Regulação da Expressão Gênica , Glucose/deficiência , Produtos Finais de Glicação Avançada/metabolismo , Frequência Cardíaca/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Oxigênio/farmacologia , Cultura Primária de Células , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Solubilidade , Volume Sistólico/fisiologiaRESUMO
BACKGROUND: Spontaneous isolated superior mesenteric artery dissection (SISMAD) is a rare vascular disorder, and the treatment strategies remain controversial. This study aimed to compare outcomes of conservative and endovascular treatments in symptomatic patients with SISMAD. METHODS: Forty-two consecutive SISMAD patients who were admitted to a single center between October 2009 and May 2018 were enrolled in this study. Based on their symptoms, 15 had conservative treatment, and 27 had endovascular treatment. The baseline characteristics, treatments, and follow-up results of the conservative group and endovascular group were analysed. RESULTS: The rates of symptom relief were 93.3% in the conservative group and 96.3% in the endovascular group. The procedure-related complications in the endovascular group included one case of pseudoaneurysm formation in the left brachial artery. During the follow-up period (median 28.5 months), a higher proportion of patients in the conservative group had symptom recurrence (42.9% in the conservative group versus 4.8% in the endovascular group, p < 0.001). Four patients in the conservative group and one patient in the endovascular group had additional endovascular intervention during follow-up. Compared with the conservative group, patients in the endovascular group had statistically significantly longer symptom-free survival (p = 0.014) and a higher rate of superior mesenteric artery (SMA) remodeling (p < 0.001). CONCLUSIONS: For symptomatic SISMAD, endovascularly treated patients had a lower rate of symptom recurrence and a higher rate of SMA remodeling in the long term. Prospective, multi-center studies are needed to confirm the long-term outcomes of both treatments.
Assuntos
Dissecção Aórtica/terapia , Tratamento Conservador , Procedimentos Endovasculares , Artéria Mesentérica Superior/cirurgia , Dissecção Aórtica/diagnóstico por imagem , Dissecção Aórtica/fisiopatologia , Tratamento Conservador/efeitos adversos , Procedimentos Endovasculares/efeitos adversos , Feminino , Humanos , Masculino , Artéria Mesentérica Superior/diagnóstico por imagem , Artéria Mesentérica Superior/fisiopatologia , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento , Remodelação VascularRESUMO
The ubiquitin-proteasome system (UPS) is essential for protein degradation and plays critical roles in myocardial ischemia/reperfusion (MI/R) injuries. Previous studies have demonstrated that the soluble receptor for advanced glycation end-product (sRAGE) inhibited MI/R-induced apoptosis by upregulating proteasome subunits. However, the mechanism remains unknown. An MI/R model was established by left anterior descending (LAD) coronary artery ligation in mice. Recombinant sRAGE protein or saline was injected intramyocardially with or without neutralizing interferon-γ (IFN-γ) antibody injected intraperitoneally before ligation. In cardiomyocytes, ischemia was simulated with "ischemia buffer" and sRAGE was overexpressed by adenovirus. Adenovirus expressing the interference RNA of ß5i was used to knockdown ß5i in cardiomyocytes. IFN-γ was induced by sRAGE both in sham and MI/R mice. Blockade of IFN-γ using IFN-γ antibody abolished the rescue effects of sRAGE for cardiac dysfunction, infarct size and apoptosis provoked by MI/R. Blockade of IFN-γ reversed the upregulation of ß1i and ß5i expression induced by sRAGE during MI/R in heart, accompanied by decreasing chymotrypsin-like proteasome activity. In addition, IFN-γ antibody abolished the suppressing effect of sRAGE on MI/R-induced p38 and c-Jun N-terminal kinase (JNK) activation, as well as p53 expression, both in vivo and in vitro. However, knockdown of ß5i abolished the antiapoptosis effect of sRAGE during hypoxia/reoxygenation (H/R) in vitro, accompanied by decreased degradation of p53. Our data suggest a novel mechanism for sRAGE in preventing MI/R-induced apoptosis in heart: sRAGE inhibits MI/R-induced apoptosis in cardiomyocytes by degrading p53 by ß5i subunit that is increased via upregulation of IFN-γ.
Assuntos
Interferon gama/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Anticorpos Neutralizantes/administração & dosagem , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada/antagonistas & inibidores , Receptor para Produtos Finais de Glicação Avançada/genética , Proteína Supressora de Tumor p53/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20's seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome.
Assuntos
Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , NF-kappa B/metabolismo , Proteínas Nucleares , Transdução de Sinais/genética , Dedos de Zinco/genética , Autoimunidade/genética , Proteínas de Ligação a DNA , Ativação Enzimática/genética , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Imunoprecipitação , Inflamação/genética , Interleucina-1beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Poliubiquitina , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , UbiquitinaçãoRESUMO
During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.
Assuntos
Apoptose , Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Caspase 9/metabolismo , Inibidores de Caspase , Proteína de Suscetibilidade a Apoptose Celular/antagonistas & inibidores , Proteína de Suscetibilidade a Apoptose Celular/genética , Ativação Enzimática , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares , Nucleotídeos/metabolismo , Interferência de RNA , Proteínas de Ligação a RNARESUMO
sRAGE can protect cardiomyocytes from apoptosis induced by ischemia/reperfusion (I/R). However, the signaling mechanisms in cardioprotection by sRAGE are currently unknown. We investigated the cardioprotective effect and potential molecular mechanisms of sRAGE inhibition on apoptosis in the mouse myocardial I/R as an in vivo model and neonatal rat cardiomyocyte subjected to ischemic buffer as an in vitro model. Cardiac function and myocardial infarct size following by I/R were evaluated with echocardiography and Evans blue/2,3,5-triphenyltetrazolium chloride. Apoptosis was detected by TUNEL staining and caspase-3 activity. Expression of the apoptosis-related proteins p53, Bax, Bcl-2, JAK2/p-JAK2, STAT3/p-STAT3, AKT/p-AKT, ERK/p-ERK, STAT5A/p-STAT5A and STAT6/p-STAT6 were detected by western blot analysis in the presence and absence of the JAK2 inhibitor AG 490. sRAGE (100 µg/day) improved the heart function in mice with I/R: the left ventricular ejection fraction and fractional shortening were increased by 42 and 57%, respectively; the infarct size was decreased by 52%, the TUNEL-positive myocytes by 66%, and activity of caspase-3 by 24%, the protein expression of p53 and ratio of Bax to Bcl-2 by 29 and 88%, respectively; protein expression of the p-JAK2, p-STAT3 and p-AKT were increased by 92, 280 and 31%, respectively. sRAGE have no effect on protein expression of p-ERK1/2, p-STAT5A and p-STAT6 following by I/R. sRAGE (900 nmol/L) exhibited anti-apoptotic effects in cardiomyocytes by decreasing TUNEL-positive myocytes by 67% and caspase-3 activity by 20%, p53 protein level and the Bax/Bcl-2 ratio by 58 and 86%, respectively; increasing protein expression of the p-JAK2 and p-STAT3 by 26 and 156%, respectively, p-AKT protein level by 33%. The anti-apoptotic effects of sRAGE following I/R were blocked by JAK2 inhibitor AG 490. The effect of sRAGE reduction on TUNEL-positive myocytes and caspase-3 activity were abolished by PI3K inhibitor LY294002, but not ERK 1/2 inhibitor PD98059. These results suggest that sRAGE protects cardiomyocytes from apoptosis induced by I/R in vitro and in vivo by activating the JAK2/STAT3 signaling pathway.
Assuntos
Apoptose , Miocárdio/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose/metabolismo , Cromonas/farmacologia , Flavonoides/farmacologia , Expressão Gênica/efeitos dos fármacos , Janus Quinase 2/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Morfolinas/farmacologia , Miócitos Cardíacos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Tirfostinas/farmacologiaRESUMO
Angiogenesis plays an important role in myocardial infarction. Apelin and its natural receptor (angiotensin II receptor-like 1, AGTRL-1 or APLNR) induce sprouting of endothelial cells in an autocrine or paracrine manner. The aim of this study is to investigate whether apelin can improve the cardiac function after myocardial infarction by increasing angiogenesis in infarcted myocardium. Left ventricular end-diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), left ventricular developed pressure (LVDP), maximal left ventricular pressure development (±LVdp/dtmax), infarct size, and angiogenesis were evaluated to analyze the cardioprotective effects of apelin on ischemic myocardium. Assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2'-deoxyuridine incorporation, wound healing, transwells, and tube formation were used to detect the effects of apelin on proliferation, migration, and chemotaxis of cardiac microvascular endothelial cells. Fluorescein isothiocyanate-labeled bovine serum albumin penetrating through monolayered cardiac microvascular endothelial cells was measured to evaluate the effects of apelin on permeability of microvascular endothelial cells. In vivo results showed that apelin increased ±LV dp/dtmax and LVESP values, decreased LVEDP values (all p < 0.05), and promoted angiogenesis in rat heart after ligation of the left anterior descending coronary artery. In vitro results showed that apelin dose-dependently enhanced proliferation, migration, chemotaxis, and tube formation, but not permeability of cardiac microvascular endothelial cells. Apelin also increased the expression of vascular endothelial growth factor receptors-2 (VEGFR2) and the endothelium-specific receptor tyrosine kinase (Tie-2) in cardiac microvascular endothelial cells. These results indicated that apelin played a protective role in myocardial infarction through promoting angiogenesis and decreasing permeability of microvascular endothelial cells via upregulating the expression of VEGFR2 and Tie-2 in cardiac microvascular endothelial cells.
Assuntos
Indutores da Angiogênese/farmacologia , Cardiotônicos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Wistar , Receptor TIE-2/efeitos dos fármacos , Receptor TIE-2/metabolismo , Recuperação de Função Fisiológica , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
Macroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells adapt to starvation and other stress responses. Upon starvation, autophagy is induced, providing cells with needed nutrient supplies. We report here that Unc-51-like kinase 1 (Ulk1), a key initiator for mammalian autophagy, undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. Phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and adenosine monophosphate activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner. Proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to the wild-type ULK1, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at an early time point, but does not alter the maximum capacity of autophagy when starvation prolongs. This study therefore revealed previously unnoticed acute autophagy response to environmental changes.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Substituição de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Linhagem Celular , Camundongos , Mutação de Sentido Incorreto , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genéticaRESUMO
BACKGROUND: The association between blood pressure (BP) variability and stroke outcome is controversial, and there are few studies that have focused on the impact of BP variability in diabetic patients with stroke. Therefore, we aimed to examine the impact of BP variability on cardiovascular outcome in diabetic and nondiabetic patients with stroke. METHODS: A total of 373 ischemic stroke patients with large artery atherosclerosis were recruited and followed up. Ambulatory BP monitoring was performed in all patients and divided according to the 25th and 75th percentiles interval of SD of daytime systolic BP (SBP). Kaplan-Meier analysis and Cox regression were used to assess the relationship between BP variability and cardiovascular outcomes including stroke recurrence, vascular events and cardiovascular death. RESULTS: The 339 patients were included in the final analysis. During an average follow-up of 19.0 ± 5.1 months (.6-26.8 months), 69 (20.4%) cardiovascular events occurred in all patients. Kaplan-Meier analysis found that there were no differences in cardiovascular events-free survival among the different BP variability groups in diabetic patients (P = .995); however, nondiabetic patients with greater BP variability showed a lesser cardiovascular events-free survival (P = .039). Through Cox regression we found the SD of daytime SBP (hazard ratio 1.103; 95% CI 1.011-1.203) was associated with cardiovascular outcomes in nondiabetic patients with stroke. CONCLUSIONS: We show that SBP variability is associated with cardiovascular outcomes in stroke patients without diabetes, but we didn't find a correlation between SBP variability and cardiovascular outcomes in stroke patients with diabetes.
Assuntos
Pressão Sanguínea , Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Complicações do Diabetes/fisiopatologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologia , Idoso , Isquemia Encefálica/mortalidade , Doenças Cardiovasculares/mortalidade , Complicações do Diabetes/mortalidade , Feminino , Seguimentos , Testes de Função Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Acidente Vascular Cerebral/mortalidade , Análise de SobrevidaRESUMO
BACKGROUND AND OBJECTIVE: The diagnostic value of ST-segment deviation detected by ambulatory electrocardiography (AECG) is controversial in identifying coronary artery disease (CAD) referred for coronary angiography (CAG). Recently, many parameters which evaluate CAD can be derived from AECG. Therefore, we aimed to investigate the diagnostic value of AECG in screening CAD referred for CAG when several parameters were combined. METHODS: We studied the 104 chest pain inpatients. All patients received the CAG and AECG. A lumen diameter reduction of ≥ 50% was considered CAD according to CAG. The parameters derived from AECG included ST-segment deviation, apnea hypopnea index (AHI), QT interval dispersion (QTd) and heart rate variability (HRV). The diagnostic value of AECG in screening CAD was evaluated. RESULTS: Of the 104 patients, 57 (54.8%) had CAD according to CAG. The sensitivity of ST-segment deviation in screening CAD was 64.9%; the specificity was 89.4%; and the Kappa value was 0.528. The sensitivity of at least three combined parameters including ST-segment deviation, AHI, QTd and HRV was 89.5%; the specificity was 87.2%; and the Kappa value was 0.767. CONCLUSION: AECG is very useful in screening CAD referred for CAG, especially while several parameters including ST-segment deviation, AHI, HRV and QTd are combined.