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Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.
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Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Caspase 3/uso terapêutico , Células Endoteliais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genéticaRESUMO
We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Apoptose , Caspase 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).
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Enzima de Conversão de Angiotensina 2 , COVID-19 , Autoanticorpos , Células Endoteliais , Humanos , Imunoglobulina M , SARS-CoV-2RESUMO
Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.
Assuntos
Dipiridamol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
SARS-CoV-2 infection induces severe disease in a subpopulation of patients, but the underlying mechanisms remain unclear. We demonstrate robust IgM autoantibodies that recognize angiotensin converting enzyme-2 (ACE2) in 18/66 (27%) patients with severe COVID-19, which are rare (2/52; 3.8%) in hospitalized patients who are not ventilated. The antibodies do not undergo class-switching to IgG, suggesting a T-independent antibody response. Purified IgM from anti-ACE2 patients activates complement. Pathological analysis of lung obtained at autopsy shows endothelial cell staining for IgM in blood vessels in some patients. We propose that vascular endothelial ACE2 expression focuses the pathogenic effects of these autoantibodies on blood vessels, and contributes to the angiocentric pathology observed in some severe COVID-19 patients. These findings may have predictive and therapeutic implications.
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Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro The E protein from avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.
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Coronavirus/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Bronquite/imunologia , Bronquite/virologia , Membrana Celular/metabolismo , Chlorocebus aethiops , Coronavirus/patogenicidade , Proteínas do Envelope de Coronavírus , Infecções por Coronavirus/virologia , Complexo de Golgi/fisiologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Vírus da Bronquite Infecciosa/imunologia , Via Secretória , Células Vero , Proteínas do Envelope Viral/fisiologia , Vírion/metabolismo , Montagem de Vírus , Viroses/metabolismoRESUMO
Noncanonical roles for caspase-3 are emerging in the fields of cancer and developmental biology. However, little is known of nonapoptotic functions of caspase-3 in most cell types. We have recently demonstrated a disassociation between caspase-3 activation and execution of apoptosis with accompanying cytoplasmic caspase-3 sequestration and preserved endothelial barrier function. Therefore, we tested the hypothesis that nonapoptotic caspase-3 activation promotes endothelial barrier integrity. Human lung microvascular endothelial cells were exposed to thrombin, a nonapoptotic stimulus, and endothelial barrier function was assessed using electric cell-substrate impedance sensing. Actin cytoskeletal rearrangement and paracellular gap formation were assessed using phalloidin staining. Cell stiffness was evaluated using magnetic twisting cytometry. In addition, cell lysates were harvested for protein analyses. Caspase-3 was inhibited pharmacologically with pan-caspase and a caspase-3-specific inhibitor. Molecular inhibition of caspase-3 was achieved using RNA interference. Cells exposed to thrombin exhibited a cytoplasmic activation of caspase-3 with transient and nonapoptotic decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery. Inhibition of caspases led to a more pronounced and rapid drop in thrombin-induced endothelial barrier function, accompanied by increased endothelial cell stiffness and paracellular gaps. Caspase-3-specific inhibition and caspase-3 knockdown both resulted in more pronounced thrombin-induced endothelial barrier disruption. Taken together, our results suggest cytoplasmic caspase-3 has nonapoptotic functions in human endothelium and can promote endothelial barrier integrity.
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Caspase 3/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Mucosa Respiratória/citologia , Junções Íntimas/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Caspase 3/genética , Células Cultivadas , Impedância Elétrica , Endotélio Vascular/citologia , Humanos , Pulmão/citologia , Interferência de RNA , RNA Interferente Pequeno/genética , Trombina/farmacologiaRESUMO
Enveloped viruses gain entry into host cells by fusing with cellular membranes, a step that is required for virus replication. Coronaviruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and infectious bronchitis virus (IBV), fuse at the plasma membrane or use receptor-mediated endocytosis and fuse with endosomes, depending on the cell or tissue type. The virus spike (S) protein mediates fusion with the host cell membrane. We have shown previously that an Abelson (Abl) kinase inhibitor, imatinib, significantly reduces SARS-CoV and MERS-CoV viral titres and prevents endosomal entry by HIV SARS S and MERS S pseudotyped virions. SARS-CoV and MERS-CoV are classified as BSL-3 viruses, which makes experimentation into the cellular mechanisms involved in infection more challenging. Here, we use IBV, a BSL-2 virus, as a model for studying the role of Abl kinase activity during coronavirus infection. We found that imatinib and two specific Abl kinase inhibitors, GNF2 and GNF5, reduce IBV titres by blocking the first round of virus infection. Additionally, all three drugs prevented IBV S-induced syncytia formation prior to the hemifusion step. Our results indicate that membrane fusion (both virus-cell and cell-cell) is blocked in the presence of Abl kinase inhibitors. Studying the effects of Abl kinase inhibitors on IBV will be useful in identifying the host cell pathways required for coronavirus infection. This will provide an insight into possible therapeutic targets to treat infections by current as well as newly emerging coronaviruses.
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Endossomos/virologia , Vírus da Bronquite Infecciosa/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Animais , Antivirais/farmacologia , Benzamidas/farmacologia , Membrana Celular , Chlorocebus aethiops , Mesilato de Imatinib/farmacologia , Vírus da Bronquite Infecciosa/metabolismo , Pirimidinas/farmacologia , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Replicação ViralRESUMO
Cold temperature blocks used to synchronize protein trafficking inhibit GBF1 function, leading to a decrease in ARF1-GTP levels and mislocalization of the ARF1 effector golgin-160. Several other, but not all, Golgi proteins including ARL1 also mislocalize. ARF1 activity and golgin-160 localization require more than 30 min to recover from these blocks.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Transporte Proteico , Rede trans-Golgi/metabolismo , Fator 1 de Ribosilação do ADP/genética , Autoantígenos/genética , Autoantígenos/metabolismo , Temperatura Baixa , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , HumanosRESUMO
Sterol regulatory element-binding protein (SREBP) transcription factors are central regulators of cellular lipid homeostasis and activate expression of genes required for fatty acid, triglyceride, and cholesterol synthesis and uptake. SREBP cleavage activating protein (SCAP) plays an essential role in SREBP activation by mediating endoplasmic reticulum (ER)-to-Golgi transport of SREBP. In the Golgi, membrane-bound SREBPs are cleaved sequentially by the site-1 and site-2 proteases. Recent studies have shown a requirement for the SREBP pathway in the development of fatty liver disease and tumor growth, making SCAP a target for drug development. Fatostatin is a chemical inhibitor of the SREBP pathway that directly binds SCAP and blocks its ER-to-Golgi transport. In this study, we determined that fatostatin blocks ER exit of SCAP and showed that inhibition is independent of insulin-induced gene proteins, which function to retain the SCAP-SREBP complex in the ER. Fatostatin potently inhibited cell growth, but unexpectedly exogenous lipids failed to rescue proliferation of fatostatin-treated cells. Furthermore, fatostatin inhibited growth of cells lacking SCAP Using a vesicular stomatitis virus glycoprotein (VSVG) trafficking assay, we demonstrated that fatostatin delays ER-to-Golgi transport of VSVG. In summary, fatostatin inhibited SREBP activation, but fatostatin additionally inhibited cell proliferation through both lipid-independent and SCAP-independent mechanisms, possibly by general inhibition of ER-to-Golgi transport.
Assuntos
Retículo Endoplasmático/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Piridinas/farmacologia , Tiazóis/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
The Golgi complex is a central organelle of the secretory pathway where sorting and processing of cargo occurs. While Golgi structure is important for the efficient processing of secretory cargo, the unusual organization suggests additional potential functions. The Golgi is disassembled after various cellular stresses, and we hypothesize that Golgi disassembly activates a stress signaling pathway. This pathway would function to correct the stress if possible, with irreparable stress resulting in apoptosis. Neurons appear to be particularly sensitive to Golgi stress; early disassembly of the organelle correlates with many neurodegenerative diseases. Here, Golgi stress and potential signaling pathways to the nucleus are reviewed.
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UNLABELLED: Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE: CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics.
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Multimerização Proteica/fisiologia , Síndrome Respiratória Aguda Grave/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus/fisiologia , Animais , Chlorocebus aethiops , Células HeLa , Humanos , Camundongos , Estrutura Quaternária de Proteína , Síndrome Respiratória Aguda Grave/genética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas ViroporinasRESUMO
Golgin-160 is a member of the golgin family of proteins, which have been implicated in the maintenance of Golgi structure and in vesicle tethering. Golgin-160 is atypical; it promotes post-Golgi trafficking of specific cargo proteins, including the ß-1 adrenergic receptor (ß1AR), a G protein-coupled receptor. Here we show that golgin-160 binds directly to the third intracellular loop of ß1AR and that this binding depends on three basic residues in this loop. Mutation of the basic residues does not affect trafficking of ß1AR from the endoplasmic reticulum through the Golgi complex, but results in reduced steady-state levels at the plasma membrane. We hypothesize that golgin-160 promotes incorporation of ß1AR into specific transport carriers at the trans-Golgi network to ensure efficient delivery to the cell surface. These results add to our understanding of the biogenesis of ß1AR, and suggest a novel point of regulation for its delivery to the plasma membrane.
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Autoantígenos/metabolismo , Proteínas de Membrana/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Receptores Adrenérgicos beta 1/químicaRESUMO
Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPK's immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.
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Apoptose/fisiologia , Caspase 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/irrigação sanguínea , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Permeabilidade Capilar , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poli(ADP-Ribose) PolimerasesRESUMO
Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease caused by the expansion of polyglutamine repeats in the gene for huntingtin (Htt). In HD, the corpus striatum selectively degenerates despite the uniform expression of mutant huntingtin (mHtt) throughout the brain and body. Striatal selectivity reflects the binding of the striatal-selective protein Rhes to mHtt to augment cytotoxicity, but molecular mechanisms underlying the toxicity have been elusive. Here, we report that the Golgi protein acyl-CoA binding domain containing 3 (ACBD3) mediates mHtt cytotoxicity via a Rhes/mHtt/ACBD3 complex. ACBD3 levels are markedly elevated in the striatum of HD patients, in a striatal cell line harboring polyglutamine repeats, and in the brains of HD mice. Moreover, ACBD3 deletion abolishes HD neurotoxicity, which is increased by ACBD3 overexpression. Enhanced levels of ACBD3 elicited by endoplasmic reticulum, mitochondrial, and Golgi stresses may account for HD-associated augmentation of ACBD3 and neurodegeneration.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Huntington/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/virologia , RNA Interferente Pequeno/genética , Transfecção , Regulação para CimaRESUMO
In mammalian cells, the Golgi complex has an elaborate structure consisting of stacked, flattened cisternal membranes collected into a ribbon in the center of the cell. Amazingly, the flattened cisternae can rapidly dilate to accommodate large cargo as it traffics through the organelle. The mechanism by which this occurs is unknown. Exocytosis of large cargo is essential for many physiological processes, including collagen and lipoprotein secretion, and defects in the process lead to disease. In addition, enveloped viruses that bud into the endoplasmic reticulum or Golgi complex must also be transported through Golgi cisternae for secretion from the infected cell. This review summarizes our understanding of intra-Golgi transport of large cargo, and outlines current questions open for experimentation.
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Complexo de Golgi/química , Complexo de Golgi/metabolismo , Animais , Transporte Biológico , Retículo Endoplasmático/metabolismo , HumanosRESUMO
The coronavirus E protein is a small membrane protein with a single predicted hydrophobic domain (HD), and has a poorly defined role in infection. The E protein is thought to promote virion assembly, which occurs in the Golgi region of infected cells. It has also been implicated in the release of infectious particles after budding. The E protein has ion channel activity in vitro, although a role for channel activity in infection has not been established. Furthermore, the membrane topology of the E protein is of considerable debate, and the protein may adopt more than one topology during infection. We previously showed that the HD of the infectious bronchitis virus (IBV) E protein is required for the efficient release of infectious virus, an activity that correlated with disruption of the secretory pathway. Here we report that a single residue within the hydrophobic domain, Thr16, is required for secretory pathway disruption. Substitutions of other residues for Thr16 were not tolerated. Mutations of Thr16 did not impact virus assembly as judged by virus-like particle production, suggesting that alteration of secretory pathway and assembly are independent activities. We also examined how the membrane topology of IBV E affected its function by generating mutant versions that adopted either a transmembrane or membrane hairpin topology. We found that a transmembrane topology was required for disrupting the secretory pathway, but was less efficient for virus-like particle production. The hairpin version of E was unable to disrupt the secretory pathway or produce particles. The findings reported here identify properties of the E protein that are important for its function, and provide insight into how the E protein may perform multiple roles during infection.
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Infecções por Coronavirus/virologia , Vírus da Bronquite Infecciosa/química , Vírus da Bronquite Infecciosa/metabolismo , Via Secretória , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Linhagem Celular Tumoral , Infecções por Coronavirus/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Mutação , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência , Proteínas do Envelope Viral/química , Montagem de VírusRESUMO
The coronavirus E protein is a small membrane protein that has an important role in the assembly of virions. Recent studies have indicated that the E protein has functions during infection beyond assembly, including in virus egress and in the host stress response. Additionally, the E protein has ion channel activity, interacts with host proteins, and may have multiple membrane topologies. The goal of this review is to highlight the properties and functions of the E protein, and speculate on how they may be related.
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Coronavirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Liberação de Vírus , Sequência de Aminoácidos , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Proteínas do Envelope Viral/químicaRESUMO
The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, trans-Golgi membranes come in close apposition with ER (endoplasmic reticulum) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to SM (sphingomyelin) synthase at the trans-Golgi via CERT (ceramide transfer protein). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. In the present study, we show that Golgi disassembly during pro-apoptotic stress induced by TNFα (tumour necrosis factor α) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases 2, 3 and 9. Truncated versions of CERT corresponding to fragments generated by caspase 2 cleavage at Asp213 were mislocalized and did not promote efficient de novo SM synthesis. Thus it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to pro-apoptotic stress.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Substituição de Aminoácidos , Ácido Aspártico/química , Sítios de Ligação , Caspase 2/metabolismo , Ceramidas/metabolismo , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esfingomielinas/metabolismo , Estresse Fisiológico , Especificidade por SubstratoRESUMO
S-Palmitoylation of G protein-coupled receptors (GPCRs) is a prevalent modification, contributing to the regulation of receptor function. Despite its importance, the palmitoylation status of the ß(1)-adrenergic receptor, a GPCR critical for heart function, has never been determined. We report here that the ß(1)-adrenergic receptor is palmitoylated on three cysteine residues at two sites in the C-terminal tail. One site (proximal) is adjacent to the seventh transmembrane domain and is a consensus site for GPCRs, and the other (distal) is downstream. These sites are modified in different cellular compartments, and the distal palmitoylation site contributes to efficient internalization of the receptor following agonist stimulation. Using a bioorthogonal palmitate reporter to quantify palmitoylation accurately, we found that the rates of palmitate turnover at each site are dramatically different. Although palmitoylation at the proximal site is remarkably stable, palmitoylation at the distal site is rapidly turned over. This is the first report documenting differential dynamics of palmitoylation sites in a GPCR. Our results have important implications for function and regulation of the clinically important ß(1)-adrenergic receptor.