ABSTRACT
Em condições inflamatórias do sistema vascular, altas concentrações de mieloperoxidase somada à presença do ácido úrico, sugerem a formação local do oxidante hidroperóxido de urato. A ação desse peróxido já foi demonstrada sobre glutationa e peroxirredoxinas, tornando plausível a possibilidade de que outras proteínas tiólicas também pudessem ser alvo de oxidação. A proteína dissulfeto isomerase é uma ditiol-dissulfeto oxidoredutase e chaperona, localizada principalmente no retículo endoplasmático, onde participa do enovelamento de proteínas nascentes. Além disso, um pool dessas enzimas foi identificado na superfície da célula e no meio extracelular (secretada) e parece ser especialmente importante em eventos vasculares como ativação e agregação de plaquetas, trombose e remodelamento vascular. Primeiramente, foi investigado se o hidroperóxido de urato era capaz de oxidar a PDI. Pelo ensaio do DTNB foi verificado que os tióis livres da proteína eram consumidos após reação com o peróxido e, em seguida, por nLC-MS/MS os resíduos de cisteínas dos sítios catalíticos foram identificados como os principais alvos de oxidação. Embora não tenham sido verificadas outras modificações além de dissulfetos, foi observado que o tratamento com hidroperóxido promoveu agregação e inativação da proteína. Os estudos subsequentes envolveram uma linhagem de células endoteliais (HUVECs). Análises preliminares de citotoxicidade (detecção da atividade da enzima lactato desidrogenase no sobrenadante e incorporação de sondas fluorescentes ao DNA) mostraram que tratamentos com concentrações de até 400 µM de hidroperóxido de urato não são letais às células em cultura. Usando alquilantes impermeáveis à membrana celular foi mostrado que o hidroperóxido de urato oxida não só a proteína dissulfeto isomerase, mas também proteínas tiólicas totais expressas na superfície das HUVECs. Experimentos de wound healing foram feitos para avaliação da capacidade de migração das células mediante o tratamento com hidroperóxido de urato, mas nenhuma diferença foi observada. Contudo, a incubação das células com os agentes oxidantes hidroperóxido de urato e diamida, inibidores de PDI e integrina e um alquilante de tiol, resultaram, pelo menos nos trinta primeiros minutos, em menor capacidade de adesão das células à fibronectina. Além disso, as células tratadas com hidroperóxido de urato se tornaram mais sensíveis ao destacamento da placa de cultura e apresentaram alteração na morfologia. O tratamento com o peróxido também afetou a homeostase redox das HUVECs, observado pela diminuição da razão GSH/GSSG. Finalmente foram apresentadas evidênciasindiretas de que o ácido úrico é substrato da peroxidasina, uma heme peroxidase abundantemente expressa no sistema vascular. Primeiro, pelo ensaio do Amplex Red foi observado que a presença de ácido úrico na mistura reacional resultou em menor taxa de oxidação do reagente. Depois, por LC-MS/MS, também em amostra na qual o ácido úrico estava presente, foi identificado o hidroxiisourato, álcool resultante da decomposição do hidroperóxido de urato. Todo o conjunto de dados deverá contribuir para o maior entendimento da participação do hidroperóxido de urato em processos oxidativos vasculares − especialmente a oxidação de proteínas − que pode ser um dos mecanismos responsáveis pela alteração da função endotelial e da homeostase vascular
During vascular inflammatory conditions, high amounts of myeloperoxidase added to the presence of uric acid, suggest the local formation of urate hydroperoxide. Its oxidative action has already been demonstrated on glutathione and peroxiredoxins, making plausible the possibility that other thiol proteins could also be a target for oxidation. The protein disulfide isomerase is a dithiol-disulfide oxidoreductase and chaperone, located mainly in the endoplasmic reticulum, where it is involved in the correct folding of nascent proteins. Also, a pool of these enzymes has been identified in cell surface and the extracellular (secreted) milieu and appears to be important in vascular events, such as platelet activation and aggregation, thrombosis and vascular remodeling. First, it was investigated whether urate hydroperoxide was capable of oxidizing PDI. By the DTNB assay, it was found that the free thiols of the protein were consumed after reaction with the peroxide and then, by nLC-MS / MS, the active redox cysteine residues were identified as the main oxidation targets. Although no modifications other than disulfides have been found, hydroperoxide treatment has been shown to promote protein aggregation and inactivation. Subsequent studies involved an endothelial cell line (HUVECs). Preliminary cytotoxicity analyzes (detection of lactate dehydrogenase enzyme activity in the supernatant and incorporation of fluorescent probes into DNA) have shown that treatments with concentrations up to 400 µM are not lethal to cells in culture. Then, using alkylating agents impermeable to the cell membrane, urate hydroperoxide was shown to oxidize not only PDI but also total thiol proteins expressed on HUVECs surface. Wound healing experiments were performed to evaluate cell migration after treatment with urate hydroperoxide, but no difference was observed. However, incubation of the cells with the oxidizing agents urate hydroperoxide and diamide, inhibitors of both PDI and integrin and a thiol alkylator, resulted, at least for the first thirty minutes, in reduced cell adhesion to fibronectin. In addition, cells treated with urate hydroperoxide became more sensitive to detachment from the culture dish and exhibited alterations in morphology. Treatment with the peroxide also affected the redox homeostasis of the HUVECs, observed by a decrease in the GSH / GSSG ratio. Finally, indirect evidence was presented that uric acid is a substrate of peroxidasin, a heme peroxidase abundantly expressed in the vascular system. First, with the Amplex Red assay it was observed that the presence of uric acid in the reaction mixture resulted in lower oxidation rates of the reagent. Then, by LC-MS / MS, hydroxyisourate, which is the alcohol derived from urate hydroperoxide decomposition, was also identified in samples containing uric acid. Taken together, the data presented should contribute to a better understanding of the involvement of urate hydroperoxide in vascular oxidative processes − especially protein oxidation − that may be one mechanism associated to disturbances in endothelial function and vascular homeostasis
Subject(s)
Endothelium, Vascular , Oxidation/adverse effects , Uric Acid/agonists , In Vitro Techniques/instrumentation , Protein Disulfide-Isomerases/analysisABSTRACT
The protein encoded by TXNDC5 is a member the protein disulfide isomerase family, which has disulfide isomerase activity and can act as the molecular chaperone to reduce the synthesis of abnormal proteins. Its biological functions include anti-oxidation, promoting angiogenesis, taking part in cellular inflammation, and energy metabolism, etc. Studies have demonstrated that the expression of TXNDC5 is increased in many types of tumors including cervical carcinoma, gastric carcinoma and colorectal cancer. Moreover, TXNDC5 is also closely associated with rheumatoid arthritis, diabetes, hepatic steatosis and vitiligo. This paper aims to summarize the latest progress in research on TXNDC5 in terms of biochemical function, relationship with diseases and the underlying mechanism.
Subject(s)
Animals , Humans , Arthritis, Rheumatoid , Genetics , Diabetes Mellitus , Genetics , Neoplasms , Genetics , Protein Disulfide-Isomerases , GeneticsABSTRACT
Background: A thermostable lipase MAS1 from marine Streptomyces sp. strain was considered as a potential biocatalyst for industrial application, but its production level was relatively low. Here, the effect of chaperones co-expression on the secretory expression of lipase MAS1 in Pichia pastoris was investigated. Result: Co-expression of protein disulfide isomerase (PDI), HAC1 and immunoglobulin binding protein could increase the expression level of lipase MAS1, whereas co-expression of Vitreoscilla hemoglobin showed a negative effect to the lipase MAS1 production. Among them, PDI co-expression increased lipase MAS1 expression level by 1.7-fold compared to the control strain harboring only the MAS1 gene. Furthermore, optimizing production of lipase MAS1 with Pichia pastoris strain X-33/MAS1-PDI in a 30-L bioreactor were conducted. Lower induction temperature was found to have a benefit effect for lipase MAS1 production. Lipase activity at 24 and 22°C showed 1.7 and 2.1-fold to that at 30°C, respectively. Among the induction pH tested, the highest lipase activity was obtained at pH 6.0 with activity of 440 U/mL after 144 h fermentation. Conclusion: Our work showed a good example for improving the production of recombinant enzymes in Pichia pastoris via chaperon co-expression and fermentation condition optimization.
Subject(s)
Streptomyces/metabolism , Lipase/biosynthesis , Pichia , Temperature , Cell Survival , Molecular Chaperones , Bioreactors , Protein Disulfide-Isomerases , Vitreoscilla , Fermentation , Batch Cell Culture Techniques , Hydrogen-Ion ConcentrationABSTRACT
OBJECTIVE: Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. METHODS: U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. RESULTS: Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). CONCLUSION: Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.
Subject(s)
Bacitracin , Blotting, Western , Caspase 3 , Cell Adhesion , Cell Movement , Focal Adhesion Protein-Tyrosine Kinases , Gelatin , Glioblastoma , Glioma , Immunohistochemistry , Matrix Metalloproteinase 2 , Polymerase Chain Reaction , Protein Disulfide-IsomerasesABSTRACT
<p><b>OBJECTIVE</b>To study the predicting effect of proly 4-hydroxylase beta polypeptide (P4HB) in treating non-small cell lung cancer (NSCLC) patients by Yiqi Chutan Recipe (YCR).</p><p><b>METHODS</b>Totally 46 stage III and IV NSCLC patients were treated by YCR for 4 therapeutic courses. Effect was assessed by RECIST of solid tumor. P4HB expression was detected in the lung cancer tissue by immunohistochemical assay. Factors affecting disease control rates (DCR) of YCR were analyzed by Logistic regression analysis. The correlation between P4HB expression and the effect of YCR was analyzed.</p><p><b>RESULTS</b>The DCR of advanced NSCLC treated by YCR was 36.96% (17/46 cases). P4HB was high expressed in advanced lung cancer tissue (6/15 cases). Gender, initial treatment, and retreatment are independent factors for affecting DCR of treating lung cancer by YCR.</p><p><b>CONCLUSION</b>P4HB might be taken as a factor for predicting the effect of YCR in treating NSCLC.</p>
Subject(s)
Humans , Male , Carcinoma, Non-Small-Cell Lung , Drug Therapy , Metabolism , Drugs, Chinese Herbal , Therapeutic Uses , Lung , Lung Neoplasms , Drug Therapy , Metabolism , Procollagen-Proline Dioxygenase , Metabolism , Protein Disulfide-Isomerases , MetabolismABSTRACT
<p><b>OBJECTIVE</b>This study was aimed to investigate the regulatory effect of protein disulfide isomerase (PDI) on platelet GPIbα ectodomain shedding.</p><p><b>METHODS</b>The washed platelets were obtained from healthy volunteers. Platelets were incubated with PDI inhibitor bacitracin before stimulation with PMA (Phorbol-12-myristate-13-acetate), dibucaine and collagen. The N-terminal domain of GPIbα in supernatant was detected by Western blot, the GPIbα expression and the intraplatelet ROS levels were measured by flow cytometry.</p><p><b>RESULTS</b>neither GC content nor GPIbα expression was changed after the washed platelets from the healthy donors were incubated only with PDI inhibitor. The washed platelets were incubated with PDI inhibitor before stimulation with different stimulin, PMA, dibucaine or collagen, and then GPIbα was cleaved and ROS levels were elevated more than that in the controls.</p><p><b>CONCLUSION</b>PDI participates in the induced GPIbα ectodomein shedding, and the effect of PDI in this process maybe depend on the change of ROS level inside platelets. These results might provide a new point of view for the platelet drug development.</p>
Subject(s)
Humans , Blood Platelets , Collagen , Flow Cytometry , Platelet Glycoprotein GPIb-IX Complex , Protein Disulfide-IsomerasesABSTRACT
The 1,095 bp gene encoding peroxidase from Coprinus cinereus was synthesized and integrated into the genome of Pichia pastoris with a highly inducible alcohol oxidase. The recombinant CiP (rCiP) fused with the a-mating factor per-pro leader sequence derived from Saccharomyces cerevisiae was secreted into the culture medium and identified as the target protein by mass spectrometry, confirming that a C. cinereus peroxidase (CiP) was successfully expressed in P. pastoris. The endoplasmic reticulum oxidoreductase 1 (Ero1) and protein disulfide isomerase (PDI) were co-expressed with rCiP separately and simultaneously. Compared with the wild type, overexpression of PDI and Erol-PDI increaseed Cip activity in 2.43 and 2.6 fold and their activity reached 316 U/mL and 340 U/mL respectively. The strains co-expressed with Erol-PDI was used to high density fermentation, and their activity reached 3,379 U/mL, which was higher than previously reported of 1,200 U/mL.
Subject(s)
Coprinus , Culture Media , Cytoplasm , Fermentation , Glycoproteins , Metabolism , Mass Spectrometry , Mating Factor , Oxidoreductases Acting on Sulfur Group Donors , Metabolism , Peptides , Peroxidases , Pichia , Metabolism , Protein Disulfide-Isomerases , Metabolism , Protein Folding , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins , MetabolismABSTRACT
Dissulfeto isomerase protéica (PDIA1 ou PDI) é uma chaperona e ditiol-dissulfeto oxido-redutase residente do reticulo endoplasmático (RE). PDI é essencial à regulação da proteostase por ter função no enovelamento oxidativo de proteínas e na via de degradação associada ao RE (ERAD). Além disso, PDI interage fisicamente e regula a atividade de NADPH oxidases, e fora da célula é um regulador redox essencial à atividade de proteínas extracelulares. Este pool epi/pericelular da PDI (pecPDI) regula função de proteínas de membrana/secretadas, como integrinas, glicoproteínas gp120 do virus HIV e outras, com múltiplas funções que incluem: trombose, ativação plaquetária, adesão celular, infecção viral e remodelamento vascular. A rota de externalização da PDI permanece obscura, e seu conhecimento pode indicar mecanismos dos efeitos (fisio)patológicos da PDI. A secreção da PDI pela rota RE-Golgi foi sugerida em células endoteliais infectadas pelo vírus da dengue, células pancreáticas e tireoideanas. No entanto, uma varredura sistemática das possíveis rotas de externalização da PDI não foi previamente realizada. Neste estudo, mostramos que células endoteliais (EC) externalizam constitutivamente, por rotas distintas, dois pools de PDI, de superfície celular e solúvel, enquanto na EC não estimulada PDI não foi detectada significativamente em micropartículas. PDI externalizada corresponde a ca.1,4% do pool total de PDI celular. Tanto a PDI de superfície celular como a solúvel foram majoritariamente secretadas pela via de secreção não-convencional do tipo IV independente de GRASP. Contudo, a via de secreção clássica também contribui para externalização basal da PDI de superfície celular, mas não da solúvel basal ou estimulada por PMA, ATP e trombina indicando que todas envolvem escape do Golgi. Além disso, a externalização constitutiva da PDI de superfície em célula muscular lisa vascular também ocorre por via independente de Golgi. Externalização...
Protein disulfide isomerase (PDIA1 or PDI) is dithiol-disulfide oxireductase chaperone resident in the endoplasmic reticulum (ER). PDI is essential for proteostasis, due to its support of oxidative protein folding and ER-associated protein degradation (ERAD). In addition, PDI associates with NADPH oxidase(s) and regulate its activity, while outside of the cell, PDI redox-dependently modulates extracellular proteins. This epi/pericellular PDI (pecPDI) pool is known to regulate membrane/secreted proteins such as integrins, HIV glycoprotein gp120 and others, with functions that involve thrombosis, platelet function, cell adhesion, viral infection and vascular remodeling. PDI externalization route remains enigmatic and its elucidation can help understand some (patho)physiological PDI effects. An ER-Golgi route for PDI secretion has been as described on dengue virus-infected endothelial cells pancreatic and thyroid) cells. However, none of these papers addressed PDI secretion routes in a systematic fashion. Here, we show that endothelial cells (EC) constitutively externalize, through different routes, two PDI pools, a cell-surface and a secreted one, while in nonstimulated ECs PDI was not significantly detected in microparticles. Externalized PDI corresponds to < 2% of total cellular PDI pool. Both cell-surface and soluble PDI were predominantly externalized through unconventional type IV GRASP-independent pathway(s). However, the classical secretory pathway also contributes to basal cell-surface, but not soluble, PDI externalization, as PMA, ATP or thrombin-stimulated secretion also involve Golgi bypass. Furthermore, constitutive cell-surface PDI externalization in vascular smooth muscle cells also occurs in a Golgi-independent way. PDI externalization was not detectably mediated by non-conventional type I, II and III secretion routes, secretory lysosomes, recycling endosomes and ATP dependent active transport in EC. Since chaperones are essential for cellular...
Subject(s)
Animals , Male , Rabbits , Rats , Cell Biology , Endoplasmic Reticulum , Endothelial Cells , Extracellular Space , Muscle, Smooth, Vascular , Protein Disulfide-IsomerasesABSTRACT
<p><b>OBJECTIVE</b>To investigate the role of TXNDC5 in serum starvation-induced proliferation inhibition of HeLa cell.</p><p><b>METHODS</b>TXNDC5 was either over-expressed or knocked down by small interfering RNA (siRNA) in HeLa cells which were then cultured in conventional medium or serum starvation medium. The protein level of TXNDC5 was evaluated by Western blot analysis. The mRNA level of TXNDC5 was measured by quantitative real-time PCR. Cell growth rate was determined by cell proliferation assay kit (MTS method). Cell cycle distribution and apoptosis were detected by flow cytometry.</p><p><b>RESULTS</b>Serum starvation mildly reduced the mRNA level of TXNDC5 (P<0.05), but dramatically increased the protein level of TXNDC5 in HeLa cells. The stability of TXNDC5 mRNA remained unchanged. Cycloheximide abolished the serum starvation-induced up-regulation of TXNDC5 protein. Over-expression of TXNDC5 had no effect on cell proliferation. However, suppression of TXNDC5 attenuated the proliferation inhibition of HeLa cell induced by serum starvation (P<0.05), increased the proportion of cells in S phase (P<0.05), but had no effect on cell apoptosis.</p><p><b>CONCLUSION</b>TXNDC5 mediates serum starvation-induced proliferation inhibition of HeLa cell.</p>
Subject(s)
Humans , Apoptosis , Cell Cycle , Cell Proliferation , Culture Media , Chemistry , Gene Knockdown Techniques , HeLa Cells , Protein Disulfide-Isomerases , Genetics , Metabolism , Serum , ChemistryABSTRACT
BACKGROUND/AIMS: Protein disulfide isomerase (PDI) has been implicated in the survival and progression of some cancer cells, by compensating for endoplasmic reticulum stress by upregulating the protein-folding capacity. However, its prognostic role in patients with hepatocellular carcinoma (HCC) has not been investigated. METHODS: We collected HCC tissues from 83 HCC patients who underwent surgical resection for an immunohistochemical study of PDI. Overall survival (OS) was measured from the date of surgical resection until the date of death from any cause. Radiological progression was evaluated using the modified Response Evaluation Criteria in Solid Tumors in an independent radiological assessment. RESULTS: PDI expression was found to be increased in human HCC compared to adjacent nontumor tissues. Increased immunopositivity for PDI was associated with a high Edmondson-Steiner grade (p = 0.028). Univariate analysis of patients who had undergone surgical resection for HCC showed that tumor PDI upregulation is a significant risk factor for poor OS (p = 0.016; hazard ratio [HR], 1.980) and time to progression (TTP; p = 0.007; HR, 1.971). Multivariate analyses revealed that high PDI expression was an independent predictor of a shorter TTP (p = 0.015; HR, 1.865) and poor OS (p = 0.012; HR, 2.069). CONCLUSIONS: Upregulated PDI expression is associated with aggressive clinicopathological features of HCC; thus, PDI might serve as an independent prognostic factor and a potential therapeutic target for HCC patients.
Subject(s)
Female , Humans , Male , Middle Aged , Carcinoma, Hepatocellular/enzymology , Kaplan-Meier Estimate , Liver Neoplasms/enzymology , Prognosis , Protein Disulfide-Isomerases/metabolism , Retrospective Studies , Biomarkers, Tumor/metabolismABSTRACT
Live attenuated vaccines have recently been introduced for preventing rotavirus disease in children. However, alternative strategies for prevention and treatment of rotavirus infection are needed mainly in developing countries where low vaccine coverage occurs. In the present work, N-acetylcysteine (NAC), ascorbic acid (AA), some nonsteroidal anti-inflammatory drugs (NSAIDs) and peroxisome proliferator-activated receptor gamma (PPARγ) agonists were tested for their ability to interfere with rotavirus ECwt infectivity as detected by the percentage of viral antigen-positive cells of small intestinal villi isolated from ECwt-infected ICR mice. Administration of 6 mg NAC/kg every 8 h for three days following the first diarrhoeal episode reduced viral infectivity by about 90%. Administration of AA, ibuprofen, diclofenac, pioglitazone or rosiglitazone decreased viral infectivity by about 55%, 90%, 35%, 32% and 25%, respectively. ECwt infection of mice increased expression of cyclooxygenase-2, ERp57, Hsc70, NF-κB, Hsp70, protein disulphide isomerase (PDI) and PPARγ in intestinal villus cells. NAC treatment of ECwt-infected mice reduced Hsc70 and PDI expression to levels similar to those observed in villi from uninfected control mice. The present results suggest that the drugs tested in the present work could be assayed in preventing or treating rotaviral diarrhoea in children and young animals.
Subject(s)
Animals , Mice , Acetylcysteine/pharmacology , /pharmacology , Diarrhea/drug therapy , PPAR gamma/agonists , Rotavirus , Rotavirus Infections/drug therapy , Antioxidants/therapeutic use , Cyclooxygenase Inhibitors/therapeutic use , Diarrhea/virology , /metabolism , /metabolism , Intestines/virology , Mice, Inbred ICR , NF-kappa B/metabolism , Protein Disulfide-Isomerases/metabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the effects of gastrin on the expression of 1,25(OH)2D3-membrane associated rapid response steroid (1,25D3-MARRS) binding protein in rat intestinal epithelium.</p><p><b>METHODS</b>SD rats received intraperitoneal injections of gastrin, omeprazole or physiological saline. The protein expression of 1,25D3-MARRS binding protein in SD rat intestinal was determined with Western blotting and immunohistochemistry, and its mRNA levels determined by RT-PCR. The serum calcium and phosphate levels in the rats were also detected.</p><p><b>RESULTS</b>Immunohistochemistry showed that 1,25D3-MARRS binding protein was expressed mainly in the nuclei, cytoplasm and membrane of the intestinal epithelial cells. Both the protein and mRNA expression levels of 1,25D3-MARRS binding protein were up-regulated after treatments with gastrin and omeprazole (P<0.05), but the serum calcium and phosphate concentrations showed no obvious increase.</p><p><b>CONCLUSION</b>1,25D3-MARRS binding protein, which is widely expressed with versatile functionalities, is regulated by gastrin and shows high potentials in the study of gastrointestinal diseases.</p>
Subject(s)
Animals , Male , Rats , Calcitriol , Metabolism , Epithelial Cells , Metabolism , Gastrins , Pharmacology , Intestines , Cell Biology , Protein Disulfide-Isomerases , Metabolism , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To investigate the relationship and significance between endoplasmic reticulum protein 57 (ERp57) gene promoter region methylation with the pathogenesis of cervical lesions in Uighur women.</p><p><b>METHODS</b>The special software was used to design specific primers of CpG island fragments of ERp57 gene promoter and bisulfite-modified SiHa cancer cell DNA for PCR amplification, cloning and sequencing the target fragments to obtain relevant information of CpG methylation in the gene base sequencs. Seventy-eight fresh tissues of CIN, CSCC and normal control were collected, and the methylation level of ERp57 gene promoter regions in different cervical lesions were identified using Sequenom MassARRAY(DNA) technology.</p><p><b>RESULTS</b>ERp57 gene corresponding target fragment contained the 18 CpG sites. All of the CpG sites methylation occurred in SiHa cervical cancer cell genomic DNA. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between three CpG sites (CpG_1, CpG_5 and CpG_7) from CpG_1, CpG_2, CpG_3.4, CpG_5, CpG_6, CpG_7, CpG_8 and CpG_ 9 had significant differences in the CSCC, CIN or control groups.</p><p><b>CONCLUSIONS</b>Although the global methylation level of the ERp57 gene promoter is higher in CSCC than that in CIN and normal control tissues in Uighur women, hypermethylation occurs only in certain CpG islands and sites. This indicates that the regulation of expression by DNA methylation is not CpG island-specific, but varies for individual CpG sites, and may explain to a certain extent the epigenetic mechanisms regulated by Erp57 gene expression.</p>
Subject(s)
Adult , Aged , Female , Humans , Middle Aged , Asian People , Ethnology , Genetics , Carcinoma, Squamous Cell , Genetics , Cell Line, Tumor , Uterine Cervical Dysplasia , Genetics , CpG Islands , Genetics , DNA Methylation , Promoter Regions, Genetic , Genetics , Protein Disulfide-Isomerases , Genetics , Uterine Cervical Neoplasms , GeneticsABSTRACT
Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of the extracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichia coli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.
Subject(s)
Amino Acid Sequence , Bacterial Proteins , Chemistry , Metabolism , Catalytic Domain , Crystallography, X-Ray , Disulfides , Chemistry , Escherichia coli , Metabolism , Escherichia coli Proteins , Chemistry , Metabolism , Molecular Docking Simulation , Molecular Sequence Data , Mycobacterium tuberculosis , Metabolism , Oxidation-Reduction , Protein Disulfide-Isomerases , Chemistry , Metabolism , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Static ElectricityABSTRACT
O remodelamento vascular é um determinante fundamental do lúmen em doenças vasculares, porém os mecanismos envolvidos não estão completamente elucidados. Nós investigamos o papel da chaperona redox residente do retículo endoplasmático Dissulfeto Isomerase Proteica (PDI) e sua fração localizada na superfície celular (peri/epicelular=pecPDI) no calibre e arquitetura vascular durante reparação à lesão. Em artérias ilíacas de coelho submetidas à lesão in vivo, houve importante aumento do mRNA e expressão proteica (~25x aumento 14 dias pós-lesão vs. controle) da PDI. O silenciamento da PDI por siRNA (cultura de órgãos) acentuou o estresse do retículo e apoptose, diferentemente da inibição da pecPDI com anticorpo neutralizante (PDI Ab). Bloqueio in vivo da pecPDI por aplicação de gel perivascular contendo PDI Ab no 12° dia após lesão, com análise após 48 h, promoveu ca.25% redução no calibre vascular analisado por arteriografia e diminuição similar na área total do vaso detectada por tomografia de coerência óptica. Neste processo, não ocorreu alteração no tamanho da neoíntima, indicando assim, que PDI Ab acentuou remodelamento constrictivo. Neutralização da pecPDI promoveu importantes alterações na arquitetura da matriz de colágeno e citoesqueleto, resultando em fibras com orientação invertida e desorganizadas. Diminuição na produção de espécies reativas de oxigênio e óxidos de nitrogênio também ocorreu. Análise de propriedades viscoelásticas nas artérias indicou redução na ductilidade vascular, evidenciada pela menor distância para ruptura. As alterações subcelulares no citoesqueleto observadas in vivo após PDI Ab foram recapituladas em um modelo de estiramento cíclico em células musculares lisas vasculares, com importante redução na formação das fibras de estresse. Em modelo de migração randômica de células musculares lisas, a exposição a PDI Ab reduziu a resiliência de regulação da polaridade. Embora a neutralização da pecPDI não tenha afetado a atividade...
Whole-vessel remodeling is a critical lumen caliber determinant in vascular disease, but underlying mechanisms are poorly understood. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI(peri/epicellular=pecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and pecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis, contrarily to pecPDI neutralization with PDI-antibody(PDI Ab). PecPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. PecPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and disorganization. Decreased ROS and nitrogen oxide production also occurred. Viscoelastic artery properties assessment showed decreased ductility, evidenced by decreased distance to rupture. Subcellular cytoskeletal disruption by PDI Ab was recapitulated in vascular smooth muscle cell stretch model, with marked decrease in stress fiber buildup. Also, PDI Ab incubation promoted decreased regulation resilience of vascular smooth muscle migration properties. While pecPDI neutralization did not affect global RhoA activity, there was altered RhoA redistribution to the cell surface and association with caveolin-containing clusters, which mislocalized after stretch. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an...
Subject(s)
Humans , Animals , Male , Rabbits , Angioplasty, Balloon , Endoplasmic Reticulum Stress , Extracellular Space , Reactive Oxygen Species , Muscle, Smooth, Vascular , Neointima , Oxidative Stress , Protein Disulfide-Isomerases , Vascular System InjuriesABSTRACT
OBJECTIVE@#To determine the screening of the expression of membrane proteins in androgen-dependent prostate cancer (ADPC) and androgen-independent prostate cancer (AIPC) and to explore the mechanism of membrane proteins in these two cancers.@*METHODS@#Serum samples were collected from 3 patients with ADPC and another 3 patients with AIPC. The serum was incubated with ADPC cell line LNCaP and/or AIPC cell line PC-3 and detected by immunoprecipitation and Western blot. Differentially expressed proteins between ADPC and AIPC identified by mass spectrometry were compared and their expression level and location were analyzed by immunofluorescence.@*RESULTS@#Altogether 11 membrane proteins were identifited, such as the Neural-Cadherin precursor, ER60 precursor, Claudin-4, and so on. Immunofluorescence revealed that the expression level of Claudin-4 in PC-3 cells was higher than in LNCaP cells.@*CONCLUSION@#We can use the screening method to study membrane proteins in prostate cancer. Claudin-4 may play an important role in the pathogenesis and the development of AIPC.
Subject(s)
Aged , Humans , Male , Androgens , Genetics , Metabolism , Cell Line, Tumor , Claudin-4 , Genetics , Metabolism , Gene Expression Profiling , Membrane Glycoproteins , Genetics , Metabolism , Prostatic Neoplasms , Genetics , Metabolism , Pathology , Protein Disulfide-Isomerases , Genetics , MetabolismABSTRACT
<p><b>BACKGROUND</b>It has been confirmed that defective expression of human leukocyte antigen class I (HLA-I) molecules can contribute to the immune evasion of cancer cells in some types of cancer. The aim of this study was to examine the expression of HLA class I antigen and the antigen-processing machinery (APM) components in esophageal squamous cell carcinoma (ESCC) and their role in high risk human papillomavirus (HPV) infection, and to analyze their association with histopathological characteristics in the Kazak ethnic group.</p><p><b>METHODS</b>A total of 50 formalin-fixed, paraffin-embedded ESCC lesions were collected from the First Affiliated Hospital of Xinjiang Medical University, China. The expression levels of HLA-I antigen and APM components were determined by immunohistochemistry; the HPV DNA were detected using polymerase chain reaction (PCR).</p><p><b>RESULTS</b>A high frequency of down-regulation or loss of expression of HLA and APM components were found in esophageal cancer in Kazak people. HLA-I, TAP1, CNX, LMP7, Erp57, Tapasin and ERAP1 were down-regulated in 68%, 44%, 48%, 40%, 52%, 32% and 20% of ESCC lesions then, respectively. The loss of expression of HLA-I antigen was significantly correlated with part of the APM components and positively correlated with high risk HPV16 infection. TAP1, CNX, LMP7, Erp57 and Tapasin loss were significantly associated with tumor grading, lymph node metastasis and depth of invasion (P < 0.05).</p><p><b>CONCLUSION</b>Our results suggest that APM component defects are a mechanism underlying HLA-I antigen down-regulation in ESCC lesions, and indicate that the loss expression of HLA-I and APM components will become an important marker of ESCC and analysis of HLA-I and APM component expression can provide useful prognostic information for patients with ESCC from the Kazak ethnic group.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters , Genetics , Metabolism , Aminopeptidases , Genetics , Metabolism , Antigen Presentation , Genetics , Physiology , Calnexin , Genetics , Metabolism , Esophageal Neoplasms , Metabolism , Histocompatibility Antigens Class I , Genetics , Metabolism , Human papillomavirus 16 , Genetics , Immunohistochemistry , In Vitro Techniques , Membrane Transport Proteins , Genetics , Metabolism , Minor Histocompatibility Antigens , Polymerase Chain Reaction , Proteasome Endopeptidase Complex , Genetics , Metabolism , Protein Disulfide-Isomerases , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To determine the effect of butylphthalide on the expressions of protein disulfide isomerase (PDI) and P53 in the brain tissue of rats with Alzheimer's disease (AD).</p><p><b>METHODS</b>Sixty male adult rats were randomly divided into AD model group, butylphthalide group and control group (n = 20). AD models were established by injecting beta-amyloid protein 1-42 into the hippocampus of rats. Sixty days later, the learning and memory abilities of the rats were evaluated using Y-maze test, and the expressions of PDI and P53 in the brain tissue of the rats were measured by immunohistochemistry.</p><p><b>RESULTS</b>Compared with the control group, the rats in AD model group exhibited significantly reduced learning and memory abilities, lowered expressions of PDI in the hippocampus and increased expression of P53 in the cortex (P > 0.01). In comparison with the model group, the rats in the butylphthalide group showed significantly increased PDI-positive cells in the hippocampus and decreased expression of P53 in the cortex (P < 0.01).</p><p><b>CONCLUSION</b>Butylphthalide improves the learning and memory abilities of rats with experimental AD, the mechanism of which may involve inhibition of P53 expression and enhancement of PDI expression in the brain tissues.</p>
Subject(s)
Animals , Male , Rats , Alzheimer Disease , Apoptosis , Benzofurans , Pharmacology , Brain , Metabolism , Disease Models, Animal , Learning , Memory , Protein Disulfide-Isomerases , Metabolism , Random Allocation , Rats, Sprague-Dawley , Tumor Suppressor Protein p53 , MetabolismABSTRACT
BACKGROUND/AIMS: Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells. METHODS: Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed. RESULTS: Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, gamma-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement. CONCLUSIONS: Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar cells.
Subject(s)
Humans , Acinar Cells , Actins , Ceruletide , Cytoplasm , Cytoskeleton , Edema , Electrophoresis , Heat-Shock Proteins , Isocitrate Dehydrogenase , Isocitrates , Mass Spectrometry , Membrane Proteins , Membranes , Oxidative Stress , Pancreatitis , Protein Disulfide-Isomerases , Proteins , Proteome , Proton-Motive Force , SerineABSTRACT
<p><b>OBJECTIVE</b>To investigate the association between the pregnancy rate of intrauterine insemination (IUI) and the ERp57 expression level in donor sperm.</p><p><b>METHODS</b>Forty-two sperm samples were divided into 3 groups according to the pregnancy rate: Group A (n = 16, pregnancy rate = 0), B (n = 13, pregnancy rate 10%-20%) and C (n = 13, pregnancy rate > or = 20%). The optical density (OD) was normalized to the beta-tubulin band for the evaluation of the ERp57 level.</p><p><b>RESULTS</b>OD (ERp57/Tubulin) was 0.95 +/- 0.24 in Group A, 1.33 +/- 0.43 in Group B and 1.33 +/- 0.39 in Group C. The ERp57 expression level was significantly lower in Group A than in B and C (P < 0.05), with no significant differences between the latter two groups.</p><p><b>CONCLUSION</b>The ERp57 expression level in donor sperm could be used as an index to predict the pregnancy rate of IUI and to avoid IUI failure by removing low-level donor sperm.</p>