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Protein disulfide isomerase regulates endoplasmic reticulum stress and the apoptotic process during prion infection and PrP mutant-induced cytotoxicity.

Wang SB, Shi Q, Xu Y, Xie WL, Zhang J, Tian C, Guo Y, Wang K, Zhang BY, Chen C, Gao C, Dong XP - PLoS ONE (2012)

Bottom Line: Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15.Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL.A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.

ABSTRACT

Background: Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases.

Methodology/principal findings: To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active S-nitrosylated modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins.

Conclusion/significance: Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO-PDI may feed as an accomplice for PDI apoptosis.

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Detections of SNO-PDI in the brains of scrapie infected hamsters and in the cell models of misfolded prion proteins.A. Western blots for the PDI and SNO-PDI signals in hamsters’ brains. The biotinylated and immunoprecipitated by streptavidin-agarose beads proteins from brain homogenates of 263K infected or normal hamsters were separated in 15% SDS-PAGE and blotted with respective specific antibodies. B. Western blots for the PDI and SNO-PDI signals in the cell models in the presence (+) or absence (-) of L-NMMA. All cells were maintained for 48 h after transfection and then treated with 1 nM L-NMMA for 6 h. C. Evaluation of the influence of L-NMMA on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and blotted with respective antibodies. D. Cell viability. The results are calculated from three independent tests indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05.
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pone-0038221-g007: Detections of SNO-PDI in the brains of scrapie infected hamsters and in the cell models of misfolded prion proteins.A. Western blots for the PDI and SNO-PDI signals in hamsters’ brains. The biotinylated and immunoprecipitated by streptavidin-agarose beads proteins from brain homogenates of 263K infected or normal hamsters were separated in 15% SDS-PAGE and blotted with respective specific antibodies. B. Western blots for the PDI and SNO-PDI signals in the cell models in the presence (+) or absence (-) of L-NMMA. All cells were maintained for 48 h after transfection and then treated with 1 nM L-NMMA for 6 h. C. Evaluation of the influence of L-NMMA on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and blotted with respective antibodies. D. Cell viability. The results are calculated from three independent tests indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05.

Mentions: It has been suggested that PDIs can be modified by an S-nitrosylation process under continuous stress, which is involved in the pathogenesis of neurodegenerative disease [11]. To probe into the situation and the contribution of SNO-PDI in prion disease, levels of SNO-PDI in brain tissues of scrapie-263K-infected hamsters were screened with a biotin-switch assay. Accompanying the increases in PDI, a clear SNO-PDI signal was found in two infected brains but not in normal brains (Fig 7A), suggesting a disease-related phenomenon. To see this abnormal phenotype in cell models expressing PrP mutants, cell lysates transfected with different PrP constructs were subjected to SNO-PDI tests. Obvious SNO-PDI signals were detected in preparations of PrPKDEL and PrPPG15, but not in that of control and PrPWT (Fig 7B). In the presence of L-NMMA, an inhibitor for NO synthase (NOS), no SNO-PDI signal was detected in all preparations; meanwhile, neither the expression level of PDI (Fig 7B) nor that of PrP constructs showed remarkable change compared with those in the absence of L-NMMA (Fig 7C). It highlights that accumulations of PrPKDEL and PrPPG15 in cytoplasm result in the formation of SNO-PDI possibly through activating cellular NOS. Further, the potential protective effect of reduction of SNO-PDI against the cytotoxicity of those two PrP mutants was evaluated. CCK-8 assays identified that decreased cell viabilities due to misfolded PrPs were partially, but significantly, recovered by the presence of L-NMMA (Fig. 7D). It seems that cytotoxicity of misfolded PrPs is partially due to inducement of SNO-PDI.


Protein disulfide isomerase regulates endoplasmic reticulum stress and the apoptotic process during prion infection and PrP mutant-induced cytotoxicity.

Wang SB, Shi Q, Xu Y, Xie WL, Zhang J, Tian C, Guo Y, Wang K, Zhang BY, Chen C, Gao C, Dong XP - PLoS ONE (2012)

Detections of SNO-PDI in the brains of scrapie infected hamsters and in the cell models of misfolded prion proteins.A. Western blots for the PDI and SNO-PDI signals in hamsters’ brains. The biotinylated and immunoprecipitated by streptavidin-agarose beads proteins from brain homogenates of 263K infected or normal hamsters were separated in 15% SDS-PAGE and blotted with respective specific antibodies. B. Western blots for the PDI and SNO-PDI signals in the cell models in the presence (+) or absence (-) of L-NMMA. All cells were maintained for 48 h after transfection and then treated with 1 nM L-NMMA for 6 h. C. Evaluation of the influence of L-NMMA on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and blotted with respective antibodies. D. Cell viability. The results are calculated from three independent tests indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05.
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Related In: Results  -  Collection

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pone-0038221-g007: Detections of SNO-PDI in the brains of scrapie infected hamsters and in the cell models of misfolded prion proteins.A. Western blots for the PDI and SNO-PDI signals in hamsters’ brains. The biotinylated and immunoprecipitated by streptavidin-agarose beads proteins from brain homogenates of 263K infected or normal hamsters were separated in 15% SDS-PAGE and blotted with respective specific antibodies. B. Western blots for the PDI and SNO-PDI signals in the cell models in the presence (+) or absence (-) of L-NMMA. All cells were maintained for 48 h after transfection and then treated with 1 nM L-NMMA for 6 h. C. Evaluation of the influence of L-NMMA on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and blotted with respective antibodies. D. Cell viability. The results are calculated from three independent tests indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05.
Mentions: It has been suggested that PDIs can be modified by an S-nitrosylation process under continuous stress, which is involved in the pathogenesis of neurodegenerative disease [11]. To probe into the situation and the contribution of SNO-PDI in prion disease, levels of SNO-PDI in brain tissues of scrapie-263K-infected hamsters were screened with a biotin-switch assay. Accompanying the increases in PDI, a clear SNO-PDI signal was found in two infected brains but not in normal brains (Fig 7A), suggesting a disease-related phenomenon. To see this abnormal phenotype in cell models expressing PrP mutants, cell lysates transfected with different PrP constructs were subjected to SNO-PDI tests. Obvious SNO-PDI signals were detected in preparations of PrPKDEL and PrPPG15, but not in that of control and PrPWT (Fig 7B). In the presence of L-NMMA, an inhibitor for NO synthase (NOS), no SNO-PDI signal was detected in all preparations; meanwhile, neither the expression level of PDI (Fig 7B) nor that of PrP constructs showed remarkable change compared with those in the absence of L-NMMA (Fig 7C). It highlights that accumulations of PrPKDEL and PrPPG15 in cytoplasm result in the formation of SNO-PDI possibly through activating cellular NOS. Further, the potential protective effect of reduction of SNO-PDI against the cytotoxicity of those two PrP mutants was evaluated. CCK-8 assays identified that decreased cell viabilities due to misfolded PrPs were partially, but significantly, recovered by the presence of L-NMMA (Fig. 7D). It seems that cytotoxicity of misfolded PrPs is partially due to inducement of SNO-PDI.

Bottom Line: Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15.Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL.A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.

ABSTRACT

Background: Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases.

Methodology/principal findings: To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active S-nitrosylated modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins.

Conclusion/significance: Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO-PDI may feed as an accomplice for PDI apoptosis.

Show MeSH
Related in: MedlinePlus