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1.
Artigo em Inglês | MEDLINE | ID: mdl-30797055

RESUMO

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 1233 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 72 h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24 h in response to lipopolysaccharides and Streptococcus iniae and at 72 h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122 ±â€¯0.0075 µmol/s protein/µg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5 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/metabolismo
2.
Mol Med Rep ; 17(5): 6515-6525, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29532868

RESUMO

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/biossíntese
3.
Neuropathology ; 37(6): 495-501, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28731225

RESUMO

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/análise
4.
J Neurochem ; 142(1): 89-102, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28419454

RESUMO

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 , Receptores X Retinoide/biossíntese , Receptores X Retinoide/genética
5.
Biomed Res Int ; 2017: 1252647, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28373975

RESUMO

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/genética
6.
J Immunol Methods ; 443: 64-67, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28131818

RESUMO

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 , Temperatura Ambiente
7.
J Immunol Methods ; 439: 67-73, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27742562

RESUMO

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/metabolismo
8.
Curr Protoc Protein Sci ; 85: 5.26.1-5.26.21, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27479507

RESUMO

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 & purificação
9.
Arterioscler Thromb Vasc Biol ; 36(6): 1164-73, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27079884

RESUMO

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/sangue
10.
Hypertension ; 67(3): 613-22, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26781284

RESUMO

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 , Coelhos
11.
Biochim Biophys Acta ; 1864(3): 308-316, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26724776

RESUMO

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 (1→2)(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/patologia
12.
Cell Stress Chaperones ; 21(1): 155-66, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26483256

RESUMO

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 , 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/genética
13.
Appl Biochem Biotechnol ; 176(2): 428-39, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25825250

RESUMO

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.


Assuntos
Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Jatropha , Proteínas de Plantas , Isomerases de Dissulfetos de Proteínas , Jatropha/enzimologia , Jatropha/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Isomerases de Dissulfetos de Proteínas/genética
14.
Biochimie ; 112: 121-8, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25765953

RESUMO

Although the early human embryo is capable of covering its cholesterol demand by endogenous synthesis, during later stages of development the fetus may become dependent on transplacental cholesterol transport. On one hand, this conclusion is based on the severe developmental abnormalities of embryos with mutations in the gene specifying the enzyme catalyzing the last step of cholesterol synthesis, 7-dehydrocholesterol reductase, causing Smith-Lemli-Opitz Syndrome. On the other hand, increased total maternal plasma cholesterol levels may reflect the requirement by the growing fetus and/or the placenta for cholesterol. Various molecules and complexes must cross the placental barrier consisting of trophoblasts and fetal endothelial cells to reach the fetal circulation. The de novo synthesis of apolipoprotein B (apoB)-containing lipoproteins coupled to secretion from trophoblasts towards the fetal side is one efficient pathway for cholesterol supply. ApoB and the microsomal triglyceride transfer protein (MTP) are essential components for the assembly of apoB-containing lipoproteins. The aim of this study was to evaluate functional properties of the human placental cell line BeWo as an in vitro model for placental synthesis of apoB-containing lipoproteins by focusing on components required for lipoprotein assembly and secretion. We demonstrate mRNA and protein production of apoB-100, MTP, and protein disulfide isomerase (PDI) in BeWo cells. In addition, metabolic radiolabeling and apoB-immunoprecipitation of cell extracts and media revealed that synthesis and secretion of apoB-containing lipoproteins are enhanced by estrogen. The expression of apoB-100, MTP, and PDI, and the estrogen-stimulated lipoprotein secretion by BeWo cells suggest that these cells are a useful system to study aspects of lipoprotein metabolism at the placental barrier.


Assuntos
Apolipoproteína B-100/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Placenta/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Gravidez , Isomerases de Dissulfetos de Proteínas/biossíntese
15.
Oncogene ; 34(36): 4735-45, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-25500540

RESUMO

Castration-resistant prostate cancer (CRPC) continues to be a major clinical problem and the mechanisms behind it remain unclear. Thioredoxin domain-containing protein 5 (TXNDC5) is involved in protein folding and chaperone activity, and its overexpression has been reported in multiple malignancies. In the current study, we demonstrated that TXNDC5 is up-regulated following long-term androgen-deprivation treatment (ADT) and is highly overexpressed in CRPC tumors compared with hormone-naive prostate cancer (PCa) cases. Functionally, in vitro and in vivo studies demonstrated that TXNDC5 overexpression promotes the growth of both androgen-dependent and castration-resistant PCa xenografts. Mechanistically, TXNDC5 directly interacts with the AR protein to increase its stability and thus enhances its transcriptional activity. TXDNC5-mediated CRPC growth can be fully abolished by AR inhibition, suggesting TXDNC5 up-regulation as an escape pathway for aberrant AR re-activation and CRPC growth in the milieu of low androgen. Indeed, we found that TXNDC5 is increased by ADT-induced hypoxia through HIF-1α in an miR-200b-dependent manner. Overall, we defined an important role of TXNDC5 in CRPC and further investigations are needed to screen TXNDC5 antagonists as a novel therapeutic approaches to treat PCa patients with CRPC.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Isomerases de Dissulfetos de Proteínas/biossíntese , Receptores Androgênicos/genética , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , MicroRNAs/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Isomerases de Dissulfetos de Proteínas/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais
16.
Acta Physiol (Oxf) ; 213(3): 664-75, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25389050

RESUMO

AIMS: Endothelium-derived protein disulphide isomerase (PDI) is required for thrombus formation in vivo. But, how to control PDI overproduction in oxidized low-density lipoprotein (oxLDL)-activated vascular endothelial cells (VECs) is not well understood. In this study, we try to answer this question using our newly identified activator of mTOC1 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2 (3H)-one (3BDO) that has been shown to protect VECs. METHODS: First, we performed a proteomics analysis on the oxLDL-activated vascular VECs in the presence or absence of 3BDO. Next, we constructed the heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) mutants at Ser43 and used the RNA-ChIP technique to investigate the relationship between hnRNP E1 and PDI production. Furthermore, we examined the effect of 3BDO on oxLDL-altered phosphorylation of Akt1 and Akt2. Finally, we studied the effect of 3BDO on oxLDL-altered PDI protein level in apolipoprotein E(-/-) mice with advanced atherosclerosis. RESULTS: In VECs, oxLDL-increased PDI protein level, induced hnRNP E1 phosphorylation at Ser43, suppressed the binding of hnRNP E1 to PDI 5'UTR and induced the phosphorylation of Akt2 but not Akt1. All of these processes were blocked by 3BDO. Importantly, Ser43 mutant of hnRNP E1 inhibited the increase of PDI protein level and the decrease of the binding of hnRNP E1 and PDI 5'UTR induced by oxLDL. Furthermore, 3BDO suppressed oxLDL-induced PDI protein increase in the serum and plaque endothelium of apolipoprotein E(-/-) mice. CONCLUSION: hnRNP E1 is a new regulator of PDI translation in oxLDL-activated VECs, and 3BDO is a powerful agent for controlling PDI overproduction.


Assuntos
Aterosclerose/enzimologia , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Células Endoteliais da Veia Umbilical Humana/enzimologia , Lipoproteínas LDL/metabolismo , Isomerases de Dissulfetos de Proteínas/biossíntese , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Regiões 5' não Traduzidas , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Sítios de Ligação , Células Cultivadas , Modelos Animais de Doenças , Ribonucleoproteínas Nucleares Heterogêneas/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fosforilação , Isomerases de Dissulfetos de Proteínas/genética , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transfecção
17.
Int J Clin Exp Pathol ; 7(6): 3305-11, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25031753

RESUMO

Pancreatic cancer (PC) is an aggressive and devastating disease with a dismal prognosis. The study aimed to investigate the role of HSP90α and PDIA3 in patients with PC. Immunohistochemistry was performed on tissue microarrays containing 186 pairs of PC and normal pancreatic tissues to assess the expression levels of HSP90α and PDIA3. The expression levels of cytoplasmic HSP90α (P = 0.032) and PDIA3 (P = 0.043) in PCs were significantly higher than those in normal pancreas tissues, but nuclear HSP90α showed lower expression in PC tissues (P = 0.002). In addition, cytoplasmic expression of HSP90α and PDIA3 was significantly associated with perineural invasion (PNI) (P = 0.004) and sex (P = 0.014), respectively. These results indicate that cytoplasmic HSP90α may serve as a biomarker for PNI in PCs.


Assuntos
Biomarcadores Tumorais/análise , Proteínas de Choque Térmico HSP90/biossíntese , Invasividade Neoplásica/patologia , Neoplasias Pancreáticas/patologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Citoplasma/metabolismo , Feminino , Proteínas de Choque Térmico HSP90/análise , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Nervos Periféricos/patologia , Isomerases de Dissulfetos de Proteínas/análise , Análise Serial de Tecidos
18.
FASEB J ; 28(8): 3720-33, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24823364

RESUMO

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.


Assuntos
Senescência Celular/genética , Enzimas Reparadoras do DNA/biossíntese , Proteínas de Ligação a DNA/biossíntese , Fibroblastos/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/biossíntese , Ribonucleoproteínas Nucleares Heterogêneas/biossíntese , MicroRNAs/fisiologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Linhagem Celular , Senescência Celular/fisiologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Humanos , Espectrometria de Massas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/fisiologia , Proteoma , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Regulação para Cima
19.
Cell Biol Toxicol ; 30(3): 127-36, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24687431

RESUMO

We previously demonstrated that cysteine-rich with EGF-like domains 2 (CRELD2), a novel ER stress-inducible factor, is a secretory glycoprotein; however, the stimuli that induce CRELD2 secretion have not yet been characterized. In this study, we found that the perturbation of intravesicular acidification of cytoplasmic organelles in HEK293 cells stably expressing wild-type (wt) CRELD2 induced its secretion. In particular, Concanamycin A (CMA) and Bafilomycin A1 (Baf), inhibitors of vacuolar ATPase (V-ATPase), increased the secretion of CRELD2 without relying on its C-terminal structure. The levels of secretion of EGFP-fused CRELD2 (SP-EGFP-CRELD2), which consists of EGFP following the putative signal peptide (SP) sequence of CRELD2, from COS7 cells transiently transfected with this construct were also increased after each of the treatments, but their intracellular localization was barely affected by CMA treatment. Transient overexpression of 78-kDa glucose-regulated protein (GRP78) and protein disulfide isomerase (PDI) also increased the secretion of CRELD2 from HEK293 cells expressing wt CRELD2, whereas the perturbation of intravesicular acidification did not alter the expression of GRP78 and PDI in the HEK293 cells. We further studied the roles of intracellular calcium ions and the Golgi apparatus in the secretion of CRELD2 from HEK293 cells in which intravesicular acidification was perturbed. The treatment with calcium ionophore increased the secretion of wt CRELD2, while that with BAPTA-AM, an intracellular calcium chelator, did not reduce the CMA-induced CRELD2 secretion. By contrast, treatment with brefeldin A (BFA), which inhibits the transportation of proteins from the ER to the Golgi apparatus, almost completely abolished the secretion of wt CRELD2 from the HEK293 cells. In conclusion, we demonstrated that the intravesicular acidification by V-ATPase regulates the secretion of CRELD2 without relying on the balance of intracellular calcium ions and the expression of ER chaperones such as GRP78 and PDI. These findings concerning the role of V-ATPases in modulating the secretion of CRELD2, a novel ER stress-inducible secretory factor, may provide new insights into the prevention and treatment of certain ER stress-related diseases.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Transporte Proteico/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Células COS , Moléculas de Adesão Celular/genética , Linhagem Celular , Cercopithecus aethiops , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Proteínas de Choque Térmico/biossíntese , Humanos , Macrolídeos/farmacologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Transfecção , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores
20.
Exp Biol Med (Maywood) ; 239(6): 707-14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24676904

RESUMO

Protein disulfide isomerase (PDI) is a multifunctional protein and plays important roles in protein folding, triglyceride transfer, insulin degradation, and thyroid hormone transportation. This study examined the modulation of PDI expression by soy consumption using rat as a model. Sprague-Dawley male and female rats at 50 days (d) of age were fed diets containing either 20% casein or alcohol-washed soy protein isolate (SPI, containing 50 mg isoflavones (ISFs)/kg diet) or SPI plus ISF (250 mg/kg diet) and mated at age of 120 d. The offspring (F1) were fed the same diets as their parents. Addition of ISF to SPI diet markedly increased PDI protein content in the liver and testis of the adult rats compared with the casein or SPI diet. PDI mRNA abundance in the liver and protein content in the brain, thyroid, heart, and uterus were unchanged by the diets. Two-dimensional Western blot showed that the rats fed diets containing SPI had a diminished hepatic PDI protein with an isoelectric point (pI) of 6.12, a dephosphorylated form, compared with the rats fed diets containing either casein or SPI with supplemental ISF. Soy ISF added into SPI diet remarkably suppressed hepatic PDI activity of the rats compared with the casein diet. Moreover, soy ISF dose-dependently increased PDI and thyroid hormone receptor (TR) ß protein content, whereas reduced TR DNA binding ability in human hepatocytes. Overall, this study shows that soy ISF increased hepatic PDI protein content, but addition of ISF into SPI diet inhibited its enzymatic activities and this effect may be mediated through a post-transcriptional mechanism.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Isoflavonas/farmacologia , Fígado/enzimologia , Proteínas de Vegetais Comestíveis/farmacologia , Isomerases de Dissulfetos de Proteínas/biossíntese , Proteínas de Soja/farmacologia , Soja/química , Animais , Feminino , Humanos , Isoflavonas/química , Masculino , Proteínas de Vegetais Comestíveis/química , Ratos , Ratos Sprague-Dawley , Receptores dos Hormônios Tireóideos/biossíntese , Proteínas de Soja/química
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