RESUMO
BACKGROUND: KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. RESULTS: We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. CONCLUSIONS: These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Complexo de Golgi , Receptores de Peptídeos , Membrana Celular , Proteínas Quinases Dependentes de AMP CíclicoRESUMO
Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease, contains a variety of oxidative components that were implicated in the regulation of Src homology domain 2-containing protein tyrosine phosphatase 2 (Shp2) activity. However, the contribution of Shp2 enzyme to chronic obstructive pulmonary disease pathogenesis remains unclear. We investigated the role of Shp2 enzyme in blockading CS-induced pulmonary inflammation. Shp2 levels were assessed in vivo and in vitro. Mice (C57BL/6) or pulmonary epithelial cells (NCI-H292) were exposed to CS or cigarette smoke extract (CSE) to induce acute injury and inflammation. Lungs of smoking mice showed increased levels of Shp2, compared with those of controls. Treatment of lung epithelial cells with CSE showed elevated levels of Shp2 associated with the increased release of IL-8. Selective inhibition or knockdown of Shp2 resulted in decreased IL-8 release in response to CSE treatment in pulmonary epithelial cells. In comparison with CS-exposed wild-type mice, selective inhibition or conditional knockout of Shp2 in lung epithelia reduced IL-8 release and pulmonary inflammation in CS-exposed mice. In vitro biochemical data correlate CSE-mediated IL-8 release with Shp2-regulated epidermal growth factor receptor/Grb-2-associated binders/MAPK signaling. Our data suggest an important role for Shp2 in the pathological alteration associated with CS-mediated inflammation. Shp2 may be a potential target for therapeutic intervention for inflammation in CS-induced pulmonary diseases.
Assuntos
Pneumonia/imunologia , Pneumonia/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/fisiologia , Fumar/efeitos adversos , Fumar/patologia , Produtos do Tabaco/toxicidade , Doença Aguda , Animais , Linhagem Celular , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Interleucina-8/metabolismo , Interleucina-8/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pneumonia/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiência , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Fumar/metabolismoRESUMO
Microtubules (MTs) are built from α-/ß-tubulin dimers and used as tracks by kinesin and dynein motors to transport a variety of cargos, such as mRNAs, proteins, and organelles, within the cell. Tubulins are subjected to several post-translational modifications (PTMs). Glutamylation is one of them, and it is responsible for adding one or more glutamic acid residues as branched peptide chains to the C-terminal tails of both α- and ß-tubulin. However, very little is known about the specific modifications found on the different tubulin isotypes in vivo and the role of these PTMs in MT transport and other cellular processes in vivo. In this study, we found that in Drosophila ovaries, glutamylation of α-tubulin isotypes occurred clearly on the C-terminal ends of αTub84B and αTub84D (αTub84B/D). In contrast, the ovarian α-tubulin, αTub67C, is not glutamylated. The C-terminal ends of αTub84B/D are glutamylated at several glutamyl sidechains in various combinations. Drosophila TTLL5 is required for the mono- and poly-glutamylation of ovarian αTub84B/D and with this for the proper localization of glutamylated microtubules. Similarly, the normal distribution of kinesin-1 in the germline relies on TTLL5. Next, two kinesin-1-dependent processes, the precise localization of Staufen and the fast, bidirectional ooplasmic streaming, depend on TTLL5, too, suggesting a causative pathway. In the nervous system, a mutation of TTLL5 that inactivates its enzymatic activity decreases the pausing of anterograde axonal transport of mitochondria. Our results demonstrate in vivo roles of TTLL5 in differential glutamylation of α-tubulins and point to the in vivo importance of α-tubulin glutamylation for cellular functions involving microtubule transport.
Cells are brimming with many different proteins, compartments, and other cell components that all play specific roles, often at very precise locations in a cell at particular moments in time. Human cells, like those of other animals and plants, contain long tracks called microtubules that are able to transport such components to wherever they are needed. Microtubules consist of chains of proteins known as tubulins that the cell can modify with small molecule tags at specific locations. For example, an enzyme called TTLL5 attaches molecules of glutamic acid to multiple positions on one of the tubulin proteins (known as α-tubulin). However, it remains unclear what role such modifications have on the ability of microtubules to move components around the cell. Fruit flies are often used as models of animal biology in research studies. Three different versions of α-tubulin are found within the ovaries of fruit flies. Two of these are 'general' α-tubulins that are expressed in almost all tissues around the body, but the third is exclusively made in the ovaries. Bao et al. studied the effect of TTLL5 activity on microtubules in fruit flies. The experiments revealed that TTLL5 played a crucial role in adding glutamic acid marks to the two general α-tubulin proteins. These modifications were needed for microtubules to successfully distribute a transporting motor protein named kinesin-1 to where it was needed for cargo transport within the egg cells. On the other hand, glutamic acid tags were not added to the oocyte α-tubulin protein. Further experiments studied nerve cells, called neurons, in the wings of the flies. In mutant fruit flies with inactive TTLL5 enzymes, cell compartments known as mitochondria moved along microtubules to one end of the neurons with fewer pauses than those in normal cells. This work shows that glutamic acid tags play important roles in regulating the transport of cell components along microtubules in fruit flies. In the future, these findings may support efforts to develop new treatments for human neurodegenerative diseases that are linked to defects in microtubules.
Assuntos
Cinesinas , Tubulina (Proteína) , Animais , Tubulina (Proteína)/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Ácido Glutâmico/metabolismo , Processamento de Proteína Pós-Traducional , Drosophila/metabolismoRESUMO
Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Imunofluorescência , Proteínas Ativadoras de GTPase/química , Células HeLa , Humanos , Immunoblotting , Proteínas de Membrana/química , Microscopia Confocal , Ligação Proteica , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/químicaRESUMO
OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a complex chronic inflammatory disease involving oxidative stress as well as a wide variety of cells activated from smoking cigarettes. There have been disappointingly few therapeutic advances in drug therapy for COPD. Plant polyphenols have been the topic of much research regarding their antioxidant activities and antiinflammatory and immunomodulatory effects. In the present study, we ask whether apple polyphenol provides protection against cigarette smoke (CS)-induced acute lung injury. METHODS: ICR mice were exposed to CS for 4 d with increasing exposure time for up to 6 h per day to elicit epithelial cells injury. One hour before smoke exposure, mice were treated with apple polyphenol (APP) by gavage; all examinations were performed 18 h after the last CS exposure. RESULTS: APP at 30, 100, or 300 mg not only significantly dose-dependently reduced the CS-induced accumulation of inflammatory cells and gene/protein expression of proinflammatory factors both in the lung and in bronchoalveolar lavage fluid, but also significantly reversed oxidative stress in the lungs. Additionally, treatment with APP also significantly regulated the CS-induced imbalance of matrix metalloproteinases-9/tissue inhibitor of metalloproteinase-1 expression in the lungs. To investigate further the possible signaling pathway of APP effects, we examined protein expression of p-P38 MAPK by immunohistochemistry that found treatment with APP significantly decreased the CS-induced increases of p-P38 expression in the lungs. CONCLUSION: Taken together, APP may be a potential dietary nutrient supplement agent to improve quality of life of COPD patients by inhibiting CS-exposed acute lung injury via P38 MAPK signaling pathway.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Malus , Polifenóis/administração & dosagem , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Quimiocinas/genética , Citocinas/genética , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Malus/química , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cigarette smoking is associated with an increased incidence of chronic obstructive pulmonary disease (COPD). In this study, we hypothesized that liquiritin apioside (LA), a main flavonoid component from Glycyrrhiza uralensis, had antioxidant properties by inducing glutathione (GSH) biosynthesis via the inhibition of cytokines and protected lung epithelial cells against cigarette smoke-mediated oxidative stress. A549 cells were treated with cigarette smoke extract (CSE) and/or LA. ICR mice were exposed to cigarette smoke (CS) for four days with increasing exposure time for up to 6 h per day to elicit epithelial cells injury. One hour before smoke exposure, mice were treated with LA by gavage; 18 h after the last CS exposure all examinations were performed. Treatment with LA concentration-dependently prevented CSE-induced cytotoxicity, increase of TGF-ß and TNF-α mRNA expression, depletion of GSH and apoptosis in A549 cells. LA at doses 3, 10 and 30 mg/kg dose-dependently inhibited pulmonary neutrophil and macrophage inflammation. Lung sections of the CS-exposed LA treated mice showed an apparently reduced pulmonary inflammation and a significant inhibitory effect on mucus containing goblet cells in the large airways. Furthermore, the CS-induced pulmonary release of TGF-ß, TNF-α and myeloperoxidase activity was reduced, and superoxide dismutase activity was enhanced.These results indicate that protective roles of LA on CS-induced the lung epithelial cell injury are mediated by inhibiting TGF-ß and TNF-α expression and increasing anti-oxidative levels of GSH, suggesting that LA might be effective as protective agent against epithelial injury in COPD.
Assuntos
Células Epiteliais/efeitos dos fármacos , Flavanonas/farmacologia , Glucosídeos/farmacologia , Lesão Pulmonar/tratamento farmacológico , Fumar/efeitos adversos , Animais , Antioxidantes/farmacologia , Citocinas/metabolismo , Células Epiteliais/patologia , Feminino , Flavonoides/farmacologia , Glutationa/metabolismo , Glycyrrhiza uralensis/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Lesão Pulmonar/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos ICR , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/patologia , Superóxido Dismutase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Clinically, sublingual immunotherapy (SLIT) using allergen extracts effectively alleviates the symptoms of allergic rhinitis and asthma. We hypothesized that oral administration of a high-dose of allergen extracts imitates SLIT, which may prevent IgE-related responses in allergic diseases. In the present study, we investigated the effects of oral administration of allergen extracts from mugwort pollen (MP) on allergen-induced inflammation and airway hyperresponsiveness (AHR) in an allergic mouse model. After administration of MPdrop containing Art v 1 and Art v 4 extracts derived from MP specifically in MP-sensitized mice, the effects of MPdrop on AHR, inflammatory cell accumulation, cytokine production in the bronchoalveolar lavage fluid and lung tissue, and serum IgE and IgG levels were investigated. The results indicated that MPdrop not only prevented the AHR in response to methacholine in a dose-dependent manner but also significantly reduced the total serum and allergen-specific IgE levels. All of the maximal effects were achieved at a dose of 100µg/(kgd) and were comparable to the effects of dexamethasone at a dose of 0.5mg/(kgd). Furthermore, oral administration of MPdrop dose-dependently elevated allergen-specific serum IgG2a levels, reduced total and allergen-specific IgE levels and normalized the imbalance between the Th1 cytokine IL-12 and Th2 cytokines IL-4 and IL-5. Finally, oral administration of MPdrop significantly reduced goblet cell hyperplasia and eosinophilia in the MP-sensitized allergic mouse model. These data suggest that MPdrop effectively improves specific allergen-induced inflammation and AHR in MP-sensitized and -challenged mice and provides the rationale for clinical use of MPdrop in the specific allergen-induced asthma.
Assuntos
Alérgenos/administração & dosagem , Alérgenos/isolamento & purificação , Artemisia/química , Dessensibilização Imunológica/métodos , Hipersensibilidade/terapia , Pólen/química , Administração Oral , Animais , Artemisia/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/patologia , Hipersensibilidade/prevenção & controle , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Inflamação/imunologia , Inflamação/patologia , Inflamação/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Pólen/imunologia , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Resultado do TratamentoRESUMO
M(3) muscarinic receptors are localized on inflammatory cells, airway smooth muscle, and submucosal glands, known to mediate bronchoconstriction, mucus secretion, and airway remodeling. It is hypothesized bencycloquidium bromide (BCQB), a novel M(3) receptor antagonist, might have potential effects on airway hyperresponsiveness, inflammation and airway remodeling in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was examined to determine the total and differential cell counts, and cytokine levels. Lung tissues were evaluated for cell infiltration, mucus hypersecretion, airway remodeling, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Inhalation administration of BCQB significantly not only reduced ovalbumin-induced airway hyperresponsiveness comparing to methacholine, and prevented the ovalbumin-induced increase in total cell counts and eosinophil counts. Reverse transcriptase polymerase chain reaction analysis of whole lung lysates revealed that BCQB markedly suppressed ovalbumin-induced mRNA expression of eotaxin, IL-5, IL-4 and MMP-9, and increased mRNA expression of IFN-γ and TIMP-1 in a dose-dependent manner. Substantial IFN-γ/IL-4 (Th1/Th2) levels were recovered in bronchoalveolar lavage fluid after BCQB treatment. In addition, histological studies showed that BCQB dramatically inhibited ovalbumin-induced lung tissue eosinophil infiltration, airway mucus production and collagen deposition in lung tissues. Results reported in current paper suggest that M(3) receptors antagonist may provide a novel therapeutic approach to treat airway inflammation, hyperresponsiveness and remodeling.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Hipersensibilidade/complicações , Hipersensibilidade/tratamento farmacológico , Receptor Muscarínico M3/antagonistas & inibidores , Sistema Respiratório/efeitos dos fármacos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Líquido da Lavagem Broncoalveolar , Quimiocinas/genética , Eosinofilia/tratamento farmacológico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipersensibilidade/genética , Hipersensibilidade/patologia , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Metaloproteinases da Matriz/genética , Cloreto de Metacolina/farmacologia , Camundongos , Pneumonia/tratamento farmacológico , Pneumonia/genética , Pneumonia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Sistema Respiratório/fisiopatologiaRESUMO
In this study we have investigated the antagonist affinity, efficacy and duration of action of bencycloquidium bromide (BCQB), a selective muscarinic M(3) receptor antagonist, as a possible clinical bronchodilator for the treatment of chronic obstructive pulmonary disease (COPD) and asthma. In competition studies, BCQB showed high affinity toward the M(3) receptor in Chinese hamster ovary (CHO) cells (M(3) pKi=8.21, M(2) pKi=7.21, and M(1) pKi=7.86); pA(2)=8.85, 8.71 and 8.57 in methacholine-induced contraction of trachea, ileum and urinary bladder, 8.19 in methacholine-induced bradycardia of right atrium in vitro, respectively. In function studies, duration of inhibition of carbachol-induced tonic contraction, BCQB and ipratropium had a very similar onset and offset of action, but onset faster and offset slower than that of tiotropium. After treatment with intratracheally instilled or the inhalation route, BCQB protects against methacholine or antigen-induced bronchoconstriction in a dose-dependent manner in the normal and sensitized guinea pigs in vivo. BCQB and ipratropium-induced inhibitory activity was short lasting, as it declined quickly when compared to tiotropium. These results suggest that BCQB bind muscarinic M(3) receptors with high affinity. On this basis we speculate that a putative BCQB-based therapy for COPD might require more than once-a-day administration to be as effective as the currently employed once-daily therapy with tiotropium. Nevertheless, Inhalable M(3)-selective compounds may spare M(2)-cardiac receptors and reduce the risks of cardiovascular events associated with the long-term treatment of these agents.