RESUMO
INTRODUCTION AND OBJECTIVES: Liver cancer, with high recurrence and metastasis rate, is a common malignant tumor. Circular RNA_0078710 (circ_0078710) has been shown to be take part in the advance of hepatocellular carcinoma. However, the interaction between circ_0091579 and microRNA-431-5p (miR-431-5p) in liver cancer has not been studied. MATERIALS AND METHODS: The expressions of circ_0078710, miR-431-5p and Thioredoxin domain-containing 5 (TXNDC5) in liver cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of cric_0078710 in liver cancer cells was assessed by Cell Counting Kit-8 (CCK-8) assay, Transwell, flow cytometry and Dual-luciferase reporter assay. Glycolysis metabolism was examined by lactate production, glucose uptake and ATP level. The protein levels of ki-67, bax and TXNEC5 were tested by western blot. The role of circ_0078710 in vivo was determined by animal study. RESULTS: Circ_0078710 and TXNDC5 were notably expressed in liver cancer tissues and cells. Circ_0078710 knockdown diminished proliferation, migration, invasion and glycolytic metabolism of huh7 and Hep3B cells, and accelerated cell apoptosis. MiR-431-5p is the target of circ_0078710, and silence circ_0078710 can inhibit the malignant behavior and glycolysis of hepatocellular carcinoma (HCC) cells by releasing miR-431-5p. In addition, TXNDC5 was a target of miR-431-5p, and overexpression of TXNDC5 restored cell proliferation and glycolysis inhibition due to miR-431-5p. Animal experiments made clear the anti-tumor effect of circ_0078710 knockdown. CONCLUSION: Circ_0078710 promotes the progression of liver cancer by regulating TXNDC5 expression by targeting miR-431-5p. These results demonstrate that circ_0078710 could be a remedy target for liver cancer.
Assuntos
Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fígado/patologia , MicroRNAs/genética , Isomerases de Dissulfetos de Proteínas/genética , Regulação para Cima , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Isomerases de Dissulfetos de Proteínas/biossínteseRESUMO
Glycosylation is a common posttranslational modification of proteins, which plays a role in the malignant transformation, growth, progression, chemoresistance, and immune response of tumors. Disulfide isomerase family A3 (PDIA3) specifically acts on newly synthesized glycoproteins to promote the correct folding of sugar chains. Studies have shown that PDIA3 participates in multidrug-resistant gastric cancer (MDR-GC). In this study, we performed western blot analysis and immunohistochemistry to identify PDIA3 expression. Cell proliferation was assessed by CCK-8 assay. Transwell assays were used to detect the migration and invasion abilities of cells. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis was employed to identify PDIA3-interacting proteins and the associated pathways in MDR-GC cells. Glycoprotein interactions and translocation were detected by immunofluorescence assay. The results showed that PDIA3 knockdown significantly inhibited the proliferation, invasion, and migration abilities of MDR-GC cells. Kyoto Encyclopedia of Genes and Genomes analysis of the IP-MS results showed that PDIA3 was closely associated with focal adhesion pathways in MDR-GC cells. Additionally, important components of focal adhesion pathways, including fibronectin-1 (FN1) and integrin α5 (ITGA5), were identified as pivotal PDIA3-binding glycoproteins. Knockdown of PDIA3 altered the cellular locations of FN1 and ITGA5, leading to abnormal accumulation. In conclusion, our results suggest that knockdown of PDIA3 inhibited the malignant behaviors of MDR-GC cells and influenced the translocation of FN1 and ITGA5.
Assuntos
Proliferação de Células , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Retículo Endoplasmático/enzimologia , Inativação Gênica , Proteínas de Neoplasias/biossíntese , Isomerases de Dissulfetos de Proteínas/biossíntese , Neoplasias Gástricas/enzimologia , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Retículo Endoplasmático/patologia , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Isomerases de Dissulfetos de Proteínas/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells - suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of ß-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of ß-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.
Assuntos
Antígeno B7-H1/metabolismo , Complexo do Signalossomo COP9/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Pancreáticas/metabolismo , Peptídeo Hidrolases/biossíntese , Isomerases de Dissulfetos de Proteínas/biossíntese , Evasão Tumoral/fisiologia , beta Catenina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno B7-H1/genética , Complexo do Signalossomo COP9/genética , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Progressão da Doença , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Gradação de Tumores/métodos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Peptídeo Hidrolases/genética , Isomerases de Dissulfetos de Proteínas/genética , beta Catenina/genéticaRESUMO
Endoplasmic reticulum (ER) stress has been reported to play a role in acute lung injury (ALI), yet the in-depth mechanism remains elusive. This study aims to investigate the effect of ER stress-induced autophagy of alveolar macrophage (AM) on acute lung injury (ALI) and airway inflammation using mouse models. ALI models were induced by intranasal instillation of lipopolysaccharide (LPS). The lung weight/body weight (LW/BW) ratio and excised lung gas volume (ELGV) in each group were measured. Mouse bronchoalveolar lavage fluid (BALF) was collected for cell sorting and protein concentration determination. Expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in lung tissues and BALF was also detected. Mouse AMs were isolated to observe the autophagy. Expression of GRP78, PERK, LC3I, LC3II and Beclin1 was further determined. The results indicated that tunicamycin (TM) elevated GRP78 and PERK expression of AMs in ALI mice in a dose-dependent manner. Low dosage of TM abated LC3I expression, increased LC3II and Beclin1 expression, triggered ER stress and AM autophagy, and alleviated pathological changes of AMs in ALI mice. Also, in ALI mice, low dosage of TM attenuated goblet cell proliferation of tracheal wall, and declined LW/BW ratio, ELGV, total cells and neutrophils, protein concentrations in BALF, and IL-6 and TNF-α expression in lung tissues and BALF. Collectively, this study suggests that a low dosage of TM-induced ER stress can enhance the autophagy of AM in ALI mice models, thus attenuating the progression of ALI and airway inflammation.
Assuntos
Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Autofagia , Estresse do Retículo Endoplasmático , Inflamação/patologia , Inflamação/prevenção & controle , Macrófagos Alveolares/citologia , Lesão Pulmonar Aguda/imunologia , Animais , Autofagia/efeitos dos fármacos , Proteína Beclina-1/biossíntese , Proteína Beclina-1/metabolismo , Peso Corporal , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Células Caliciformes/citologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Inflamação/imunologia , Interleucina-6/genética , Pulmão/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos , Neutrófilos/citologia , Tamanho do Órgão , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/metabolismo , Traqueia/citologia , Traqueia/efeitos dos fármacos , Traqueia/patologia , Fator de Necrose Tumoral alfa/genética , Tunicamicina/farmacologia , eIF-2 Quinase/biossíntese , eIF-2 Quinase/metabolismoRESUMO
Prolyl 4-hydroxylase, beta polypeptide (P4HB) protein has been found to be associated with tumorigenesis in many types of tumor, However, the relationship between P4HB and clear cell renal cell carcinoma (ccRCC) has not been clarified. In this study, we focus on the correlation between P4HB expression and ccRCC. Through the Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) database, our database and immunohistochemical (IHC) staining. Compared with adjacent normal tissues, both the mRNA and protein levels of P4HB in ccRCC tissues were enhanced. The Kaplan-Meier survival analysis showed that high expression of P4HB is correlated with poor prognosis in both TCGA database and our own database. Multivariate survival analysis and Univariate analysis showed that P4HB expression and age are significantly correlative with poor prognose. All the results indicated that P4HB is correlated with poor prognosis in human clear cell renal cell carcinoma.
Assuntos
Carcinoma de Células Renais/enzimologia , Neoplasias Renais/enzimologia , Pró-Colágeno-Prolina Dioxigenase/biossíntese , Isomerases de Dissulfetos de Proteínas/biossíntese , Carcinogênese , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prognóstico , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Transfecção , Regulação para CimaRESUMO
The human parasites Schistosoma mansoni and Leishmania major are co-endemic and a major threat to human health. Though displaying different tissue tropisms, they excrete/secrete similar subsets of intracellular proteins that, interacting with the host extracellular matrix (ECM), help the parasites invading the host. We selected one of the most abundant proteins found in the secretomes of both parasites, protein disulfide isomerase (PDI), and performed a comparative screening with surface plasmon resonance imaging (SPRi), looking for ECM binding partners. Both PDIs bind heparan sulfate; none of them binds collagens; each of them binds further ECM components, possibly linked to the different tropisms. We investigated by small-angle X-ray scattering both PDIs structures and those of a few complexes with host partners, in order to better understand the differences within this conserved family fold. Furthermore, we highlighted a previously undisclosed moonlighting behaviour of both PDIs, namely a concentration-dependent switch of function from thiol-oxidoreductase to holdase. Finally, we have tried to exploit the differences to look for possible compounds able to interfere with the redox activity of both PDI.
Assuntos
Leishmania major/enzimologia , Isomerases de Dissulfetos de Proteínas/química , Schistosoma mansoni/enzimologia , Sequência de Aminoácidos , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fenômenos Químicos , Descoberta de Drogas , Ativação Enzimática , Matriz Extracelular , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Estrutura Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/biossínteseRESUMO
Endoplasmic reticulum resident protein 44 (ERp44) is a protein disulfide isomerase (PDI) and a member of thioredoxin family, which is involved in several functions such as oxidative folding and polymerization of molecules, carrier protein activity, regulation of the Ca2+ ion levels in the endoplasmic reticulum (ER), cellular signaling, and maintenance of lumen redox homeostasis. In this study, ERp44 from Hippocampus abdominalis, commonly known as the big belly seahorse, was characterized. ERp44 possessed three PDI-like domains and one thioredoxin fold with a CXXC conserved motif. The open reading frame consisted of 1233â¯bp encoding 410 amino acids. Additionally, it contained a C-terminal RDEL motif, which suggests a localization of ERp44 to the endoplasmic reticulum. ShERp44 showed highest mRNA expression in the ovary, brain, and gills. Temporal expression of ShERp44 in blood showed significant upregulation against bacterial, LPS, and PolyI:C stimuli at 24 and 72â¯h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24â¯h in response to lipopolysaccharides and Streptococcus iniae and at 72â¯h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122⯱â¯0.0075⯵mol/s protein/µg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5â¯min. Moreover, rShERp44-treated fathead minnow cells showed significant cell survival against 2-hydroxyethyl disulfide and thus exhibited capability to resist oxidative stress. Taken together, these findings provide insight into teleost defense mechanisms and functional properties of ERp44 in controlling redox homeostasis at the molecular level.
Assuntos
Retículo Endoplasmático , Proteínas de Peixes , Estresse Oxidativo/fisiologia , Isomerases de Dissulfetos de Proteínas , Smegmamorpha , Motivos de Aminoácidos , Animais , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Regulação da Expressão Gênica/fisiologia , Rim/enzimologia , Especificidade de Órgãos/fisiologia , Oxirredução , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Domínios Proteicos , Smegmamorpha/genética , Smegmamorpha/metabolismoRESUMO
Osteoporosis is a serious public health problem and icariin (ICA) is the active component of the Epimedium sagittatum, a traditional Chinese medicinal herb. The present study aimed to investigate the effects and underlying mechanisms of ICA as a potential therapy for osteoporosis. Calvaria osteoblasts were isolated from newborn rats and treated with ICA. Cell viability, apoptosis, alkaline phosphatase activity and calcium deposition were analyzed. Bioinformatics analyses were performed to identify differentially expressed proteins (DEPs) in response to ICA treatment. Western blot analysis was performed to validate the expression of DEPs. ICA administration promoted osteoblast viability, alkaline phosphatase activity, calcium deposition and inhibited osteoblast apoptosis. Secretome analysis of ICAtreated cells was performed using twodimensional gel electrophoresis and matrixassisted laser desorption/ionization timeofflight mass spectrometry. A total of 56 DEPs were identified, including serpin family F member 1 (PEDF), protein disulfide isomerase family A, member 3 (PDIA3), nuclear protein, coactivator of histone transcription (NPAT), cMyc and heat shock protein 70 (HSP70). These proteins were associated with signaling pathways, including Fas and p53. Bioinformatics and western blot analyses confirmed that the expression levels of the six DEPs were upregulated following ICA treatment. These genes may be directly or indirectly involved in ICAmediated osteogenic differentiation and osteogenesis. It was demonstrated that ICA treatment promoted osteogenesis by modulating the expression of PEDF, PDIA3, NPAT and HSP70 through signaling pathways, including Fas and p53.
Assuntos
Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas do Olho/biossíntese , Proteínas de Choque Térmico HSP70/biossíntese , Masculino , Fatores de Crescimento Neural/biossíntese , Proteínas Nucleares/biossíntese , Osteoblastos/citologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Ratos , Ratos Sprague-Dawley , Serpinas/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Receptor fas/biossínteseRESUMO
Accumulated evidence indicates that circadian genes regulate cell damage and senescence in most mammals. Endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) regulate longevity in many organisms. However, the specific mechanisms of the relationship between the circadian clock and the two stress processes in organisms are poorly understood. Here, we show that Clock-mediated Pdia3 expression is required to sustain reactive oxidative reagents and ER stress. First, ER stress and ROS are strongly activated in the liver tissue of Clock∆19 mutant mice, which exhibit a significant aging phenotype. Next, transcription of Pdia3 is mediated by the circadian gene Clock, but this process is affected by the Clock∆19 mutant due to the low affinity of the E-box motif in the promoter. Finally, ablation of Pdia3 with siRNA causes ER stress with sustained phosphorylation of PERK and eIF1α, resulting in exaggerated up-regulation of UPR target genes and increased apoptosis as well as ROS. Moreover, the combined effects result in an imbalance of cell homeostasis and ultimately lead to cell damage and senescence. Taken together, this study identified the circadian gene Clock as a regulator of ER stress and senescence, which will provide a reference for the clinical prevention of aging.
Assuntos
Proteínas CLOCK/genética , Senescência Celular/genética , Estresse do Retículo Endoplasmático/genética , Isomerases de Dissulfetos de Proteínas/genética , Animais , Feminino , Regulação da Expressão Gênica/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Isomerases de Dissulfetos de Proteínas/biossínteseRESUMO
Protein disulfide isomerase (PDI) is a chaperone protein located in the endoplasmic reticulum (ER). Nitric oxide-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to protein accumulation and activation of the unfolded protein response. Protein disulfide isomerase P5 (P5) is a member of the PDI family that mostly localizes to the ER lumen. Both S-nitrosylated PDI and S-nitrosylated P5 are found in Alzheimer's disease (AD) brain. Previously, we showed that expression of the ER stress marker, growth arrest, and DNA damage protein (GADD34) was significantly increased in neurons and oligodendrocytes in AD brain. In the present study, we showed that PDI and P5 levels were significantly decreased in oligodendrocytes in the brains of AD patients and an AD mouse model. Interestingly, these decreases were evident before the animals displayed typical AD pathology. Because we previously showed that small short interfering RNA knockdown of PDI or P5 could affect the viability of neuronal cells under ER stress, dysfunction of PDI and P5 under ER stress could cause apoptosis of neuronal cells. In summary, we showed that the levels of PDI and P5 were significantly decreased in the oligodendrocytes of AD patients. This phenomenon was also found in an AD mouse model before the animals displayed AD pathology. The overall findings suggest that oligodendrocytes may play important roles in AD pathogenesis.
Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Oligodendroglia/enzimologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Masculino , Camundongos , Isomerases de Dissulfetos de Proteínas/análiseRESUMO
Aim. ERp57 is involved in virus induced endoplasmic reticulum stress (ERS) and plays an important role in tumorigenesis. This study aimed to find whether HBV infection altered ERp57 expression and whether ERp57 regulation was involved in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) genesis. Materials and Methods. HBV-HCC tissues, chronic hepatitis B (CHB) liver tissues, and normal liver tissues were acquired. ERp57 expressions in these tissues were detected through immunohistochemistry (IHC). And ERp57 expression in liver cell line L02, HBV replicative liver cell line L02-pHBV4.1, and HCC cell lines were detected through western blot for verification. Then medical data on patients providing HCC tissues were collected and analyzed along with ERp57 expression. Results. Higher ERp57 expression was found in HCC and CHB tissues (p < 0.001). And HCC cell lines and L02-pHBV4.1 presented higher ERp57 expression as well. In patients, ERp57 expression showed significant differences between death and survival groups (p = 0.037). And cumulative survival in patients with higher ERp57 (score ⩾ 8.75) is significantly lower (p = 0.009). Conclusion. Our study found increased expression of ERp57 in HBV-HCC. Such altered expression could be related to HBV infection and high ERp57 expression may lead to poor prognosis of HBV-HCC patients.
Assuntos
Carcinoma Hepatocelular/genética , Hepatite B Crônica/genética , Neoplasias Hepáticas/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Idoso , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Transformação Celular Neoplásica , Estresse do Retículo Endoplasmático , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/patologia , Hepatite B Crônica/virologia , Humanos , Fígado/patologia , Fígado/virologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Isomerases de Dissulfetos de Proteínas/genéticaRESUMO
Despite the recent identification of over 40 missense heterozygous Reelin gene (RELN) mutations in autism spectrum disorder (ASD), none of these has been functionally characterized. Reelin is an integral signaling ligand for proper brain development and post-natal synapse function - properties likely disrupted in ASD patients. We find that the R2290C mutation, which arose de novo in an affected ASD proband, and other analogous mutations in arginine-amino acid-arginine domains reduce protein secretion. Closer analysis of RELN R2290C heterozygous neurospheres reveals up-regulation of Protein Disulfide Isomerase A1, best known as an endoplasmic reticulum-chaperone protein, which has been linked to neuronal pathology. This effect is recapitulated in a heterozygous RELN mouse mutant that is characterized by defective Reelin secretion. These findings suggest that both a deficiency in Reelin signaling and pathologic impairment of Reelin secretion may contribute to ASD risk.
Assuntos
Transtorno do Espectro Autista/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Diferenciação Celular/genética , Cerebelo/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Edição de RNA , Proteína Reelina , Receptores X de Retinoides/biossíntese , Receptores X de Retinoides/genéticaRESUMO
Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together ß-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond.
Assuntos
Clonagem Molecular/métodos , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/biossíntese , Periplasma/metabolismo , Anticorpos de Domínio Único/biossíntese , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Dissulfetos/química , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Peptidilprolil Isomerase/biossíntese , Peptidilprolil Isomerase/genética , Plasmídeos/genética , Conformação Proteica em Folha beta , Desnaturação Proteica , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Estabilidade Proteica , Desdobramento de Proteína , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Relação Estrutura-Atividade , TemperaturaRESUMO
CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca2+ was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A).
Assuntos
Clonagem Molecular/métodos , Citoplasma/metabolismo , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Glicoproteínas de Membrana/biossíntese , Receptores de Complemento/biossíntese , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Dissulfetos/química , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Oxirredutases/biossíntese , Oxirredutases/genética , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética , Domínios Proteicos , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes/biossíntese , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Escherichia coli continues to be a popular expression host for the production of proteins, yet successful recombinant expression of active proteins to high yields remains a trial and error process. This is mainly due to decoupling of the folding factors of a protein from its native host, when expressed recombinantly in E. coli. Failure to fold could be due to many reasons but is often due to lack of post-translational modifications that are absent in E. coli. One such post-translational modification is the formation of disulfide bonds, a common feature of secreted proteins. The genetically engineered SHuffle cells offer an expression solution to proteins that require disulfide bonds for their folding and activity. The purpose of this protocol unit is to familiarize the researcher with the biology of SHuffle cells and guide the experimental design in order to optimize and increase the chances of successful expression of their desired protein of choice. Example of the expression and purification of a model disulfide-bonded protein DsbC is described in detail. © 2016 by John Wiley & Sons, Inc.
Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Dissulfetos/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Periplasma/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/isolamento & purificação , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
OBJECTIVE: Thiol isomerases facilitate protein folding in the endoplasmic reticulum, and several of these enzymes, including protein disulfide isomerase and ERp57, are mobilized to the surface of activated platelets, where they influence platelet aggregation, blood coagulation, and thrombus formation. In this study, we examined the synthesis and trafficking of thiol isomerases in megakaryocytes, determined their subcellular localization in platelets, and identified the cellular events responsible for their movement to the platelet surface on activation. APPROACH AND RESULTS: Immunofluorescence microscopy imaging was used to localize protein disulfide isomerase and ERp57 in murine and human megakaryocytes at various developmental stages. Immunofluorescence microscopy and subcellular fractionation analysis were used to localize these proteins in platelets to a compartment distinct from known secretory vesicles that overlaps with an inner cell-surface membrane region defined by the endoplasmic/sarcoplasmic reticulum proteins calnexin and sarco/endoplasmic reticulum calcium ATPase 3. Immunofluorescence microscopy and flow cytometry were used to monitor thiol isomerase mobilization in activated platelets in the presence and absence of actin polymerization (inhibited by latrunculin) and in the presence or absence of membrane fusion mediated by Munc13-4 (absent in platelets from Unc13d(Jinx) mice). CONCLUSIONS: Platelet-borne thiol isomerases are trafficked independently of secretory granule contents in megakaryocytes and become concentrated in a subcellular compartment near the inner surface of the platelet outer membrane corresponding to the sarco/endoplasmic reticulum of these cells. Thiol isomerases are mobilized to the surface of activated platelets via a process that requires actin polymerization but not soluble N-ethylmaleimide-sensitive fusion protein attachment receptor/Munc13-4-dependent vesicular-plasma membrane fusion.
Assuntos
Plaquetas/enzimologia , Membrana Celular/enzimologia , Megacariócitos/enzimologia , Ativação Plaquetária , Isomerases de Dissulfetos de Proteínas/sangue , Actinas/sangue , Animais , Plaquetas/efeitos dos fármacos , Proteínas Sanguíneas/deficiência , Proteínas Sanguíneas/genética , Calnexina/sangue , Membrana Celular/efeitos dos fármacos , Genótipo , Humanos , Megacariócitos/efeitos dos fármacos , Fusão de Membrana , Proteínas de Membrana/sangue , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Ativação Plaquetária/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/biossíntese , Transporte Proteico , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/sangueRESUMO
Whole-vessel remodeling critically determines lumen caliber in vascular (patho)physiology, and it is reportedly redox-dependent. We hypothesized that the cell-surface pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1 (peri/epicellular=pecPDI), which is known to support thrombosis, also regulates disease-associated vascular architecture. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling and plaque stability. In a rabbit iliac artery overdistension model, there was unusually high PDI upregulation (≈25-fold versus basal, 14 days postinjury), involving both intracellular and pecPDI. PecPDI neutralization with distinct anti-PDI antibodies did not enhance endoplasmic reticulum stress or apoptosis. In vivo pecPDI neutralization with PDI antibody-containing perivascular gel from days 12 to 14 post injury promoted 25% decrease in the maximally dilated arteriographic vascular caliber. There was corresponding whole-vessel circumference loss using optical coherence tomography without change in neointima, which indicates constrictive remodeling. This was accompanied by decreased hydrogen peroxide generation. Constrictive remodeling was corroborated by marked changes in collagen organization, that is, switching from circumferential to radial fiber orientation and to a more rigid fiber type. The cytoskeleton architecture was also disrupted; there was a loss of stress fiber coherent organization and a switch from thin to medium thickness actin fibers, all leading to impaired viscoelastic ductility. Total and PDI-associated expressions of ß1-integrin, and levels of reduced cell-surface ß1-integrin, were diminished after PDI antibody treatment, implicating ß1-integrin as a likely pecPDI target during vessel repair. Indeed, focal adhesion kinase phosphorylation, a downstream ß1-integrin effector, was decreased by PDI antibody. Thus, the upregulated pecPDI pool tunes matrix/cytoskeleton reshaping to counteract inward remodeling in vascular pathophysiology.
Assuntos
Estenose Coronária/genética , Vasos Coronários/patologia , Isomerases de Dissulfetos de Proteínas/genética , RNA/genética , Remodelação Vascular , Animais , Membrana Celular/metabolismo , Células Cultivadas , Estenose Coronária/metabolismo , Estenose Coronária/patologia , Vasos Coronários/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Masculino , Fosforilação , Isomerases de Dissulfetos de Proteínas/biossíntese , CoelhosRESUMO
Rheumatoid arthritis (RA) is a systemic autoimmune disease whose main symptom is a heightened inflammatory response in synovial tissues. To verify the anti-arthritic activities of Achyranthes aspera and its possible therapy-related factors on the pathogenesis of RA, the saponins in A. aspera root were isolated and identified to treat the collagen-induced arthritis (CIA) rats. Phytochemical analysis isolated and identified methyl caffeate, 25-S-inokosterone, 25-S-inokosterone ß-D-glucopyranosyl 3-(O-ß-D-glucopyranosyloxy)-oleanolate, and ß-D-glucopyranosyl 3-(O-ß-D-galactopyranosyl (1â2)(O-ß-D-glucopyranosyloxy)-oleanolate as main compounds in the root of A. aspera. Proteomics was performed to determine the differentially expressed proteins in either inflamed or drug-treated synovium of CIA rats. Treatment resulted in dramatically decreased paw swelling, proliferation of inflammatory cells, and bone degradation. Fibrinogen, procollagen, protein disulfide-isomerase A3, and apolipoprotein A-I were all increased in inflamed synovial tissues and were found to decrease when administered drug therapy. Furthermore, Alpha-1-antiproteinase and manganese superoxide dismutase were both increased in drug-treated synovial tissues. The inhibition of RA progression shows that A. aspera is a promising candidate for future treatment of human arthritis. Importantly, the total saponins found within A. aspera are the active component. Finally, autoantigens such as fibrinogen and collagen could act as inducers of RA due to their aggravation of inflammation. Given this, it is possible that the vimentin and PDIA3 could be the candidate biomarkers specific to Achyranthes saponin therapy for rheumatoid arthritis in synovial membrane.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/biossíntese , Achyranthes/química , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Biomarcadores/metabolismo , Ácidos Cafeicos/administração & dosagem , Colestenos/administração & dosagem , Colágeno/toxicidade , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Ratos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologiaRESUMO
Regulation of the endoplasmic reticulum (ER) stress-response pathway during the course of diabetes specifically in renal tubules is unclear. Since tubule cell dysfunction is critical to progression of diabetic nephropathy, this study analyzed markers of ER stress response and ER chaperones at different stages of diabetes and in different renal tubule subtypes of OVE26 type-1 diabetic mice. ER stress-responseinduced chaperones GRP78, GRP94, and protein disulfide isomerase (PDI) were increased in isolated cortical tubules of older diabetic mice, while PDI was decreased in tubules of young diabetic mice. Immunofluorescence staining of kidneys from older mice showed GRP78 and PDI upregulation in all cortical tubule segments, with substantial induction of PDI in distal tubules. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation was increased in cortical tubules of young diabetic mice, with no differences between older diabetic and control mice. Expression of ER stress-induced PERK inhibitor p58IPK was decreased and then increased in all tubule subtypes of young and older mice, respectively. Knockdown of PERK by small interfering RNA (siRNA) increased fibronectin secretion in cultured proximal tubule cells. Tubules of older diabetic mice had significantly more apoptotic cells, and ER stress-induced proapoptotic transcription factor C/EBP homologous protein (CHOP) was increased in proximal and distal tubules of diabetic mice and diabetic humans. CHOP induction in OVE26 mice was not altered by severity of proteinuria. Overexpression of CHOP in cultured proximal tubule cells increased expression of fibronectin. These findings demonstrate differential ER stress-response signaling in tubule subtypes of diabetic mice and implicate a role for PERK and CHOP in tubule cell matrix protein production.
Assuntos
Diabetes Mellitus/patologia , Estresse do Retículo Endoplasmático/fisiologia , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Fatores Etários , Animais , Apoptose/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Feminino , Fibronectinas/metabolismo , Proteínas de Choque Térmico HSP40/biossíntese , Proteínas de Choque Térmico/biossíntese , Humanos , Túbulos Renais Distais/citologia , Túbulos Renais Proximais/citologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Transgênicos , Fosforilação , Isomerases de Dissulfetos de Proteínas/biossíntese , Proteinúria/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição CHOP/biossíntese , Regulação para Cima , eIF-2 Quinase/genéticaRESUMO
Reactive oxygen species (ROS) in plants, arising from various environmental stresses, impair the thiol-contained proteins that are susceptible to irregular oxidative formation of disulfide bonds, which might be alleviated by a relatively specific modifier called protein disulfide isomerase (PDI). From our previous data of the transcriptome and digital gene expression of cold-hardened Jatropha curcas, a PDI gene was proposed to be cold-relevant. In this study, its full-length cDNA (JcPDI) was cloned, with the size of 1649 bp containing the entire open reading frame (ORF) of 1515 bp. This ORF encodes a polypeptide of 504 amino acids with theoretical molecular weight of 56.6 kDa and pI value of 4.85. One N-terminal signal peptide (-MASKGSIWSCMFLFSLI VAISAGEG-) and the C-terminal anchoring sequence motif (-KDEL-) specific to the endoplasmic reticulum, as well as two thioredoxin domains (-CGHC-), are also found by predictions. Through semi-quantitative RT-PCR, the expression of JcPDI was characterized to be tissue-differential strongly in leaves and roots, but weakly in stems, and of cold-induced alternations. Furthermore, JcPDI overexpression in yeast could notably enhance the cold resistance of host cells. Conclusively, these results explicitly suggested a considerable association of JcPDI to cold response and a putative application potential for its correlated genetic engineering.