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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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Related in: MedlinePlus

Influences of expression of various PrP constructs on the cell viability and the relevant cellular factors.A. Cell viabilities assayed by CCK-8 kit. Each group is assigned to three repeating parallel units and measured with a spectrophotometer under 450 nm. The results are calculated from three independent tests and indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. B. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunoblots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. C. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, PDI, pro-caspase3 vs that of individual β-actin. The results are calculated from three independent tests and presented as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. D. The relative densities of Bcl-2 and Bax specific bands normalized to β-actin are presented as ratio of Bax/Bcl-2. Statistical differences compared with controls are illustrated.
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pone-0038221-g003: Influences of expression of various PrP constructs on the cell viability and the relevant cellular factors.A. Cell viabilities assayed by CCK-8 kit. Each group is assigned to three repeating parallel units and measured with a spectrophotometer under 450 nm. The results are calculated from three independent tests and indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. B. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunoblots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. C. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, PDI, pro-caspase3 vs that of individual β-actin. The results are calculated from three independent tests and presented as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. D. The relative densities of Bcl-2 and Bax specific bands normalized to β-actin are presented as ratio of Bax/Bcl-2. Statistical differences compared with controls are illustrated.

Mentions: Previous studies have repeatedly confirmed that transient expressions of PrP-KEDL and PrPs with extra-octarepeat insertions cause cell apoptosis and pathological abnormality in animal models or cell lines [17], [18], [19]. Under these experimental conditions, expressions of PrP-KEDL and PrP-PG15 in 293-T cells significantly decreased cell viability (Fig. 3A). To see the changes in cellular agents related to ER stress and apoptosis, cells receiving different PrP constructs were harvested 48 h posttransfection and levels of individual molecules were evaluated by Western blots. In line with the observation in scrapie-infected hamsters, the levels of two ER-chaperone members, Grp78 and PDI, were markedly increased in cells expressing PrP-KEDL and PrP-PG15, whereas that in cells expressing wild-type PrP remained unchanged compared with the mock (Fig. 3B and C). Consistent with that, an executor of ER apoptosis, caspase-12, was activated by expressions of abnormal PrPs, which possessed a significant decrease in its precursor (Fig. 3C). Meanwhile, levels of pro-caspase-3 were downregulated, whereas that of Bax were upregulated in cells expressing those two PrP mutants, representing activation of the apoptotic executors caspase-3 and Bax (Fig. 3C and D). Those results indicate that ER stress and caspase activation contribute to PrP-KEDL and PrP-PG15-induced cytotoxicity.


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)

Influences of expression of various PrP constructs on the cell viability and the relevant cellular factors.A. Cell viabilities assayed by CCK-8 kit. Each group is assigned to three repeating parallel units and measured with a spectrophotometer under 450 nm. The results are calculated from three independent tests and indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. B. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunoblots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. C. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, PDI, pro-caspase3 vs that of individual β-actin. The results are calculated from three independent tests and presented as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. D. The relative densities of Bcl-2 and Bax specific bands normalized to β-actin are presented as ratio of Bax/Bcl-2. Statistical differences compared with controls are illustrated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369880&req=5

pone-0038221-g003: Influences of expression of various PrP constructs on the cell viability and the relevant cellular factors.A. Cell viabilities assayed by CCK-8 kit. Each group is assigned to three repeating parallel units and measured with a spectrophotometer under 450 nm. The results are calculated from three independent tests and indicated as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. B. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunoblots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. C. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, PDI, pro-caspase3 vs that of individual β-actin. The results are calculated from three independent tests and presented as mean ± SD. Statistical differences compared with controls are illustrated as *P<0.05. D. The relative densities of Bcl-2 and Bax specific bands normalized to β-actin are presented as ratio of Bax/Bcl-2. Statistical differences compared with controls are illustrated.
Mentions: Previous studies have repeatedly confirmed that transient expressions of PrP-KEDL and PrPs with extra-octarepeat insertions cause cell apoptosis and pathological abnormality in animal models or cell lines [17], [18], [19]. Under these experimental conditions, expressions of PrP-KEDL and PrP-PG15 in 293-T cells significantly decreased cell viability (Fig. 3A). To see the changes in cellular agents related to ER stress and apoptosis, cells receiving different PrP constructs were harvested 48 h posttransfection and levels of individual molecules were evaluated by Western blots. In line with the observation in scrapie-infected hamsters, the levels of two ER-chaperone members, Grp78 and PDI, were markedly increased in cells expressing PrP-KEDL and PrP-PG15, whereas that in cells expressing wild-type PrP remained unchanged compared with the mock (Fig. 3B and C). Consistent with that, an executor of ER apoptosis, caspase-12, was activated by expressions of abnormal PrPs, which possessed a significant decrease in its precursor (Fig. 3C). Meanwhile, levels of pro-caspase-3 were downregulated, whereas that of Bax were upregulated in cells expressing those two PrP mutants, representing activation of the apoptotic executors caspase-3 and Bax (Fig. 3C and D). Those results indicate that ER stress and caspase activation contribute to PrP-KEDL and PrP-PG15-induced cytotoxicity.

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