Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.527
Filtrar
1.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
2.
Nat Commun ; 11(1): 4285, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855390

RESUMO

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Citocininas/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meristema/citologia , Meristema/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
PLoS One ; 15(6): e0233993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484843

RESUMO

Multidrug resistance (MDR) to chemotherapeutic drugs remains one of the major impediments to the treatment of cancer. Discovery and development of drugs that can prevent and reverse the acquisition of multidrug resistance constitute a foremost challenge in cancer therapeutics. In this work, we screened a library of 1,127 compounds with known targets for their ability to overcome Pgp-mediated multidrug resistance in cancer cell lines. We identified four compounds (CHIR-124, Elesclomol, Tyrphostin-9 and Brefeldin A) that inhibited the growth of two pairs of parental and Pgp-overexpressing multidrug-resistant cell lines with similar potency irrespective of their Pgp status. Mechanistically, CHIR-124 (a potent inhibitor of Chk1 kinase) inhibited Pgp activity in both multidrug-resistant cell lines (KB-V1 and A2780-Pac-Res) as determined through cell-based Pgp-efflux assays. Other three inhibitors on the contrary, were effective in Pgp-overexpressing resistant cells without increasing the cellular accumulation of a Pgp substrate, indicating that they overcome resistance by avoiding efflux through Pgp. None of these compounds modulated the expression of Pgp in resistant cell lines. PIK-75, a PI3 Kinase inhibitor, was also determined to inhibit Pgp activity, despite being equally potent in only one of the two pairs of resistant and parental cell lines. Strong binding of both CHIR-124 and PIK-75 to Pgp was predicted through docking studies and both compounds inhibited Pgp in a biochemical assay. The inhibition of Pgp causes accumulation of these compounds in the cells where they can modulate the function of their target proteins and thereby inhibit cell proliferation. In conclusion, we have identified compounds with various cellular targets that overcome multidrug resistance in Pgp-overexpressing cell lines through mechanisms that include Pgp inhibition and efflux evasion. These compounds, therefore, can avoid challenges associated with the co-administration of Pgp inhibitors with chemotherapeutic or targeted drugs such as additive toxicities and differing pharmacokinetic properties.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Brefeldina A/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazinas/farmacologia , Hidrazonas/farmacologia , Neoplasias/genética , Neoplasias/patologia , Quinolinas/farmacologia , Quinuclidinas/farmacologia , Sulfonamidas/farmacologia , Tirfostinas/farmacologia
4.
Allergol Immunopathol (Madr) ; 48(1): 8-17, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31883622

RESUMO

INTRODUCTION AND OBJECTIVES: LRBA deficiency is caused by loss of LRBA protein expression, due to either homozygous or compounds heterozygous mutations in LRBA. LRBA deficiency has been shown to affect vesicular trafficking and autophagy. To date, LRBA has been observed in the cytosol, Golgi apparatus and some lysosomes in LPS-stimulated murine macrophages. The objectives of the present study were to study the LRBA localization in organelles involved in vesicular traffic, phagocytosis, and autophagy in mononuclear phagocytes (MP). MATERIALS AND METHODS: We analyzed LRBA colocalization with different endosomes markets using confocal microscopy in MP. We used the autophagy inhibitors to determine the role of LRBA in formation, maturation or degradation of the autophagosome. RESULTS: LRBA intracellular trafficking depends on the activity of the GTPase ADP ribosylation factor-1 (ARF) in MP. LRBA was identified in early, late endosomes but did not colocalize strongly with lysosomal markers. Although LRBA appears not to be recruited during the phagocytic cargo uptake, it greatly colocalized with the microtubule-associated protein 1A/1B-light chain 3 (LC3) under a steady state and this decreased after the induction of autophagy flux. Although the use of inhibitors of lysosome fusion did not restore the LRBA/LC3 colocalization, inhibitors of either early to late endosomes trafficking or PI3K pathway did. CONCLUSIONS: Taken together, our results show that LRBA is located in endomembrane system vesicles, mainly in the early and late endosomes. Although LRBA appears not to be involved in the phagocytic uptake, it is recruited in the early steps of the autophagy flux.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Fatores de Ribosilação do ADP/antagonistas & inibidores , Fatores de Ribosilação do ADP/metabolismo , Autofagia/efeitos dos fármacos , Brefeldina A/farmacologia , Endossomos/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Fagócitos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia
5.
Physiol Plant ; 169(1): 122-134, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31816092

RESUMO

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic streaming. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indicated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive electrodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and distribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the aggregation of organelles into BFA compartments within 30-120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting formation of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illuminated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.


Assuntos
Brefeldina A/farmacologia , Membrana Celular/metabolismo , Chara/metabolismo , Cloroplastos/metabolismo , Endossomos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Concentração de Íons de Hidrogênio
6.
Cells ; 8(12)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847122

RESUMO

BACKGROUND: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. METHODS: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. RESULTS: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. CONCLUSION: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.


Assuntos
Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Brefeldina A/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Imunoprecipitação , Masculino , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica , Microscopia Confocal , Neoplasias da Próstata/metabolismo , Ligação Proteica
7.
Am J Physiol Renal Physiol ; 317(6): F1695-F1706, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31630542

RESUMO

Transient receptor potential vanilloid family member 4 (TRPV4) transcript and protein expression increased in the urinary bladder and lumbosacral dorsal root ganglia of transgenic mice with chronic urothelial overexpression of nerve growth factor (NGF-OE). We evaluated the functional role of TRPV4 in bladder function with open-outlet cystometry, void spot assays, and natural voiding (Urovoid) assays with the TRPV4 antagonist HC-067047 (1 µM) or vehicle in NGF-OE and littermate wild-type (WT) mice. Blockade of TRPV4 at the level of the urinary bladder significantly (P ≤ 0.01) increased the intercontraction interval (2.2-fold) and void volume (2.6-fold) and decreased nonvoiding contractions (3.0-fold) in NGF-OE mice, with lesser effects (1.3-fold increase in the intercontraction interval and 1.3-fold increase in the void volume) in WT mice. Similar effects of TRPV4 blockade on bladder function in NGF-OE mice were demonstrated with natural voiding assays. Intravesical administration of HC-067047 (1 µM) significantly (P ≤ 0.01) reduced pelvic sensitivity in NGF-OE mice but was without effect in littermate WT mice. Blockade of urinary bladder TRPV4 or intravesical infusion of brefeldin A significantly (P ≤ 0.01) reduced (2-fold) luminal ATP release from the urinary bladder in NGF-OE and littermate WT mice. The results of the present study suggest that TRPV4 contributes to luminal ATP release from the urinary bladder and increased voiding frequency and pelvic sensitivity in NGF-OE mice.


Assuntos
Trifosfato de Adenosina/urina , Morfolinas/farmacologia , Fator de Crescimento Neural/biossíntese , Pelve , Pirróis/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Micção/efeitos dos fármacos , Urotélio/metabolismo , Animais , Brefeldina A/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Crescimento Neural/genética , Estimulação Física , Inibidores da Síntese de Proteínas/farmacologia , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiopatologia , Bexiga Urinária Hiperativa/fisiopatologia , Urotélio/efeitos dos fármacos
8.
J Biol Chem ; 294(48): 18475-18487, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31628189

RESUMO

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.


Assuntos
Ameloblastos/metabolismo , Esmalte Dentário/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Queratina-6/metabolismo , Amelogenina/genética , Amelogenina/metabolismo , Animais , Antibacterianos , Brefeldina A/farmacologia , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Expressão Gênica , Humanos , Queratina-6/genética , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley
9.
Biochem Biophys Res Commun ; 520(3): 526-531, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31610914

RESUMO

Rab18 is a small GTPase associated with lipid droplets and other membranes. While it likely has multiple functions on lipid droplets, one proposed function is regulation of lipolysis. Previous work has concentrated on regulation of autophagy; however, in this study, we provide evidence that Rab18 plays a role upstream of the cytosolic lipolytic enzyme adipose triglyceride lipase (ATGL) and that recruitment of ATGL by Rab18 is mediated by elements of the Arf/GBF1 machinery. We find that Arf4-GFP is accumulated on the subset of lipid droplets associated with Rab18, and that this association is lost within 5 min upon treatment with 5 µg/ml of the drug brefeldin A, which targets GBF1 and other Sec7-domain containing Arf exchange factors. ATGL-GFP is also recruited to lipid droplets, but is lost more slowly after treatment with 5 µg/ml brefeldin A, with significant loss from lipid droplets after 1 h treatment, and almost complete loss from lipid droplets 4 h after brefeldin A treatment. Upon overexpression of the dominant negative GDP-locked cerulean-Rab18-S22 N, GFP-ATGL and Arf4 are lost from the surface of lipid droplets similarly to BFA treatment. This study establishes, for the first time, an essential role for Rab18 in recruiting ATGL to lipid droplets.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lipase/metabolismo , Lipólise/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Brefeldina A/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo
10.
J Vis Exp ; (149)2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31380855

RESUMO

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.


Assuntos
Interferon gama/biossíntese , Listeriose/metabolismo , Listeriose/microbiologia , Baço/metabolismo , Transferência Adotiva , Animais , Brefeldina A/farmacologia , Microambiente Celular , Citocinas/biossíntese , Imunofluorescência , Camundongos , Microscopia Confocal , Inibidores da Síntese de Proteínas/farmacologia , Transdução de Sinais , Baço/microbiologia , Fixação de Tecidos
11.
Blood Adv ; 3(16): 2436-2447, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31416821

RESUMO

Disseminated intravascular coagulation is a frequent manifestation during bacterial infections and is associated with negative clinical outcomes. Imbalanced expression and activity of intravascular tissue factor (TF) is central to the development of infection-associated coagulopathies. Recently, we showed that anthrax peptidoglycan (PGN) induces disseminated intravascular coagulation in a nonhuman primate model of anthrax sepsis. We hypothesized that immune recognition of PGN by monocytes is critical for procoagulant responses to PGN and investigated whether and how PGN induces TF expression in primary human monocytes. We found that PGN induced monocyte TF expression in a large cohort of healthy volunteers similar to lipopolysaccharide stimulation. Both immune and procoagulant responses to PGN involve intracellular recognition after PGN internalization, as well as surface signaling through immune Fcγ receptors (FcγRs). In line with our hypothesis, blocking immune receptor function, both signaling and FcγR-mediated phagocytosis, significantly reduced but did not abolish PGN-induced monocyte TF expression, indicating that FcγR-independent internalization contributes to intracellular recognition of PGN. Conversely, when intracellular PGN recognition is abolished, TF expression was sensitive to inhibitors of FcγR signaling, indicating that surface engagement of monocyte immune receptors can promote TF expression. The primary procoagulant responses to PGN were further amplified by proinflammatory cytokines through paracrine and autocrine signaling. Despite intersubject variability in the study cohort, dual neutralization of tumor necrosis factor-α and interleukin-1ß provided the most robust inhibition of the procoagulant amplification loop and may prove useful for reducing coagulopathies in gram-positive sepsis.


Assuntos
Antraz/imunologia , Coagulação Sanguínea/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Peptidoglicano/imunologia , Transdução de Sinais , Biomarcadores , Coagulação Sanguínea/efeitos dos fármacos , Brefeldina A/farmacologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/imunologia , Monócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Tromboplastina/metabolismo
12.
J Biochem Mol Toxicol ; 33(10): e22380, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31339623

RESUMO

Lung endothelial barrier dysfunction leads to severe pathologies, including the lethal Acute Respiratory Distress Syndrome. P53 has been associated with anti-inflammatory activities. The current study employs a variety of unfolded protein response (UPR) activators and inhibitors to investigate the regulation of P53 by UPR in lung cells. The bovine cells that were exposed to the UPR inductors brefeldin A, dithiothreitol, and thapsigargin; demonstrated elevated expression levels of P53 compared to the vehicle-treated cells. On the contrary, the UPR inhibitors N-acetyl cysteine, kifunensine, and ATP-competitive IRE1α kinase-inhibiting RNase attenuator; produced the opposite effects. The outcomes of the present study reveal a positive regulation between UPR and P53. Since it has been shown that a mild induction of the unfolded protein response opposes inflammation, we suggest that P53 is involved in those protective activities in the lung.


Assuntos
Genes p53 , Artéria Pulmonar/metabolismo , Resposta a Proteínas não Dobradas , Acetilcisteína/farmacologia , Alcaloides/farmacologia , Animais , Brefeldina A/farmacologia , Bovinos , Células Cultivadas , Ditiotreitol/farmacologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
13.
IET Syst Biol ; 13(4): 169-179, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31318334

RESUMO

Developing a model for a signalling pathway requires several iterations of experimentation and model refinement to obtain an accurate model. However, the implementation of such an approach to model a signalling pathway induced by a poorly-known stimulus can become labour intensive because only limited information on the pathway is available beforehand to formulate an initial model. Therefore, a large number of iterations are required since the initial model is likely to be erroneous. In this work, a numerical scheme is proposed to construct a time-varying model for a signalling pathway induced by a poorly-known stimulus when its nominal model is available in the literature. Here, the nominal model refers to one that describes the signalling dynamics under a well-characterised stimulus. First, global sensitivity analysis is implemented on the nominal model to identify the most important parameters, which are assumed to be piecewise constants. Second, measurement data are clustered to determine temporal subdomains where the parameters take different values. Finally, a least-squares problem is solved to estimate the parameter values in each temporal subdomain. The effectiveness of this approach is illustrated by developing a time-varying model for NF-[inline-formula removed]B signalling dynamics induced by lipopolysaccharide in the presence of brefeldin A.


Assuntos
Brefeldina A/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Modelos Biológicos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise por Conglomerados , Interações Medicamentosas , Fatores de Tempo
14.
Chin Med J (Engl) ; 132(15): 1823-1832, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31306228

RESUMO

BACKGROUND: Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury. METHODS: Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology. RESULTS: Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group. CONCLUSIONS: We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation.


Assuntos
Autoantígenos/genética , Colágeno Tipo IV/genética , Estresse do Retículo Endoplasmático/fisiologia , Mutação/genética , Podócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Brefeldina A/farmacologia , Estresse do Retículo Endoplasmático/genética , Mutação da Fase de Leitura/genética , Humanos , Lentivirus/genética , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto/genética , Podócitos/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/genética
15.
FEBS Lett ; 593(19): 2771-2778, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291699

RESUMO

CREB3 is a transcription factor localized to the ER. Here, we investigated endogenous CREB3 expression in HEK293 cells using pharmacological and genome editing approaches. Full-length CREB3 detected under resting conditions disappeared following treatment with tunicamycin, brefeldin A and nigericin. Treatment with cycloheximide and MG132 indicated that endogenous CREB3 is a proteasome substrate. Using cells deficient for the ER-associated protein degradation (ERAD) factors SEL1L and Herp, we demonstrate that SEL1L, but not Herp, plays a crucial role in the posttranslational regulation of full-length CREB3 expression. In addition, kifunensine, an α-mannosidase inhibitor, remarkably increased full-length CREB3 expression. Our study suggests that endogenous full-length CREB3 is a novel substrate for ERAD and identifies unique cellular signals distinct from those in canonical ER stress.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Brefeldina A/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Leupeptinas/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nigericina/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas/genética , Proteínas/metabolismo , Tunicamicina/farmacologia
16.
Sci Rep ; 9(1): 9790, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278300

RESUMO

Tumor protein D52 (TPD52) is amplified and overexpressed in breast and prostate cancers which are frequently characterised by dysregulated lipid storage and metabolism. TPD52 expression increases lipid storage in mouse 3T3 fibroblasts, and co-distributes with the Golgi marker GM130 and lipid droplets (LDs). We examined the effects of Brefeldin A (BFA), a fungal metabolite known to disrupt the Golgi structure, in TPD52-expressing 3T3 cells, and in human AU565 and HMC-1-8 breast cancer cells that endogenously express TPD52. Five-hour BFA treatment reduced median LD numbers, but increased LD sizes. TPD52 knockdown decreased both LD sizes and numbers, and blunted BFA's effects on LD numbers. Following BFA treatment for 1-3 hours, TPD52 co-localised with the trans-Golgi network protein syntaxin 6, but after 5 hours BFA treatment, TPD52 showed increased co-localisation with LDs, which was disrupted by microtubule depolymerising agent nocodazole. BFA treatment also increased perilipin (PLIN) family protein PLIN3 but reduced PLIN2 detection at LDs in TPD52-expressing 3T3 cells, with PLIN3 recruitment to LDs preceding that of TPD52. An N-terminally deleted HA-TPD52 mutant (residues 40-184) almost exclusively targeted to LDs in both vehicle and BFA treated cells. In summary, delayed recruitment of TPD52 to LDs suggests that TPD52 participates in a temporal hierarchy of LD-associated proteins that responds to altered LD packaging requirements induced by BFA treatment.


Assuntos
Brefeldina A/farmacologia , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Imunofluorescência , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Camundongos , Mutação , Proteínas de Neoplasias/genética , Perilipina-3/metabolismo , Transporte Proteico
17.
J Clin Invest ; 129(9): 3894-3908, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31219804

RESUMO

Induction of memory CD8 T cells is important for controlling infections such as malaria HIV/AIDS, and for cancer immunotherapy. Accurate assessment of antigen (Ag)-specific CD8 T-cells is critical for vaccine optimization and defining correlates of protection. However, conditions for determining Ag-specific CD8 T-cell responses ex-vivo using ICS may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influences the magnitude of Ag-specific and bystander T cell responses. Indeed, following immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ producing memory CD8 T-cells comprised of antigen specific and bystander responses were detected by increasing the duration of Ag-stimulation prior to addition of BFA. Mechanistic analyses of virus-specific CD8 T-cells in mice revealed that the increase in IFNg producing CD8 T-cells was due to bystander activation of Ag-experienced memory CD8 T-cells, and correlated with the proportion of Ag-experienced CD8 T-cells in the stimulated populations. Incubation with anti-cytokine antibodies (ex. IL-12) improved accuracy in detecting bona-fide memory CD8 T-cell responses suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8 T-cells.


Assuntos
Antígenos/imunologia , Efeito Espectador , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Animais , Brefeldina A/farmacologia , Citocinas/metabolismo , Feminino , Humanos , Imunização , Memória Imunológica , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Malária/prevenção & controle , Vacinas Antimaláricas , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Transdução de Sinais , Baço/citologia , Vacinas Atenuadas/imunologia
18.
Mol Cell Endocrinol ; 493: 110470, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158417

RESUMO

Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of ß-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay ß-cell failure caused by proinsulin misfolding and ER stress.


Assuntos
Retículo Endoplasmático/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/química , Proinsulina/metabolismo , Adulto , Animais , Brefeldina A/farmacologia , Células Cultivadas , Retículo Endoplasmático/química , Estresse do Retículo Endoplasmático , Feminino , Humanos , Células Secretoras de Insulina/citologia , Camundongos , Pessoa de Meia-Idade , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico
19.
J Cell Biochem ; 120(9): 15604-15615, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31111546

RESUMO

ß-amyloid peptide (Aß) deposition derived from sequential cleavage of the amyloid precursor protein (APP) through the amyloidogenic pathway is an important characteristic feature of Alzheimer's disease (AD). During this process, cellular trafficking plays a crucial role. A large Sec7-domain containing ADP-ribosylation factor guanine nucleotide exchange factor (ARF-GEF), Golgi brefeldin A resistance factor 1 (GBF1) has been reported to initiate the ADP-ribosylation factor (Arf) activation cascade at trans-Golgi network, which plays a crucial function at the endoplasmic reticulum-Golgi interface. In this study, we investigated the role of GBF1 in APP transmembrane transport and Aß formation. Using APP/PS1 (presenilin 1) overexpressing transgenic mice, we demonstrate that GBF1 has upregulated the expression of APP, indicating a role for GBF1 in APP physiological process. Knocking down of GBF1 using small interfering has significantly increased the intracellular but not the surface expression of APP. In contrast, overexpression of wild-type (WT) and guanine nucleotide exchange factor (GEF) in the activated form but not the GEF deficient mutation induced continuous activation of GBF1, which subsequently increased the surface level of APP. Interestingly, inhibition of GBF1 by c(BFA) also impaired APP trafficking and induced endoplasmic reticulum (ER) stress in SH-SY5Y cells. Our results thus for identified the role of GBF1 in APP trafficking and cleavage, and provide evidence for GBF1 as a possible therapeutic target in AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/genética , Complexo de Golgi/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Brefeldina A/efeitos adversos , Brefeldina A/farmacologia , Movimento Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Células HeLa , Humanos , Camundongos , Transporte Proteico/genética
20.
J Cell Physiol ; 234(11): 20111-20117, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30950061

RESUMO

Brefeldin A (BFA) is a lactone antibiotic synthesized from palmitic acid by several fungi that could block anterograde transport of proteins from endoplasmic reticulum to Golgi apparatus by reversible disruption of the Golgi complex. Previous investigations have shown that BFA induces the apoptosis of cancer cells in mitosis and impairs asymmetric spindle positioning in meiosis. Here, we document that exposure to BFA in porcine oocytes compromises the meiotic maturation via disrupting both nuclear and cytoplasmic maturation. We found that BFA exposure collapsed the cytoskeleton assembly by showing the aberrant spindle organization with misaligned chromosomes and defective actin dynamics. Furthermore, the distribution of both mitochondria and cortical granules (CGs), two important indexes of cytoplasmic maturation of oocytes, was disturbed following BFA exposure. We finally validated that the localization of ovastacin, a component of CGs that is essential for the postfertilization removal of sperm-binding sites in the zona pellucida, was also perturbed in BFA-exposed oocytes, which might weaken their fertilization capacity. Collectively, these findings indicate that Golgi-mediated protein transport is indispensable for the porcine oocyte meiotic maturation.


Assuntos
Brefeldina A/farmacologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Actinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Feminino , Fertilização/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitose/efeitos dos fármacos , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Suínos , Zona Pelúcida/efeitos dos fármacos , Zona Pelúcida/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA