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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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Influences of knockdown of endogenous PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence down-regulating PDI on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for PDI and PrP were reacted with individual antibodies. B. 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<0.001. C. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. D. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, 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. E. 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-g005: Influences of knockdown of endogenous PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence down-regulating PDI on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for PDI and PrP were reacted with individual antibodies. B. 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<0.001. C. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. D. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, 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. E. 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: To determine the effectiveness of removal of PDI on the release of ER stress caused by expressions of PrP mutants, PDI-specific siRNAs were synthesized and introduced into 293-T cells. Evaluation of expression levels of cellular PDI by Western blot verified a clearly downregulated situation after transfected PDI-specific siRNA, caused roughly 40 to 60% reduction in expression of endogenous PDI, but did not affect PrP levels when co-transfected with different PrP-expressing plasmids (Fig 5A). CCK-8 assays did not reveal remarkable cytotoxicity on cultured cells after introduction of PDI-specific siRNA (Fig 5B). Co-expression of PrPKEDL with PDI-specific siRNA induced slightly lower cell viability than the expression of PrPKEDL alone, but without statistical difference (Fig 5B). However, co-expression of PrPPG15 with PDI-specific siRNA did not aggravate, but improved PrPPG15-induced cytotoxicity (Fig 5B). Western blots revealed downregulated pro-caspase 3 and Bcl-2, and upregulated Bax in cells receiving plasmid-expressing PrPKEDL and PDI-specific siRNA, and levels of molecules tested in cells receiving PrPPG15 and PDI-specific siRNA comparable to that of the control (Fig 5C, D and E). These data suggest that knockingdown endogenous PDI produces different effects on those two PrP mutants.


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 knockdown of endogenous PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence down-regulating PDI on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for PDI and PrP were reacted with individual antibodies. B. 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<0.001. C. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. D. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, 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. E. 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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pone-0038221-g005: Influences of knockdown of endogenous PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence down-regulating PDI on the expressions of various PrP constructs by Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for PDI and PrP were reacted with individual antibodies. B. 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<0.001. C. Western blots. Cell lysates were separated in 15% SDS-PAGE and the specific immunolots for Grp78, pro-caspase12, PDI, pro-caspase3, Bcl-2 and Bax were detected with individual antibodies. D. Quantitative analysis of each gray numerical value of Grp78, pro-caspase12, 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. E. 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: To determine the effectiveness of removal of PDI on the release of ER stress caused by expressions of PrP mutants, PDI-specific siRNAs were synthesized and introduced into 293-T cells. Evaluation of expression levels of cellular PDI by Western blot verified a clearly downregulated situation after transfected PDI-specific siRNA, caused roughly 40 to 60% reduction in expression of endogenous PDI, but did not affect PrP levels when co-transfected with different PrP-expressing plasmids (Fig 5A). CCK-8 assays did not reveal remarkable cytotoxicity on cultured cells after introduction of PDI-specific siRNA (Fig 5B). Co-expression of PrPKEDL with PDI-specific siRNA induced slightly lower cell viability than the expression of PrPKEDL alone, but without statistical difference (Fig 5B). However, co-expression of PrPPG15 with PDI-specific siRNA did not aggravate, but improved PrPPG15-induced cytotoxicity (Fig 5B). Western blots revealed downregulated pro-caspase 3 and Bcl-2, and upregulated Bax in cells receiving plasmid-expressing PrPKEDL and PDI-specific siRNA, and levels of molecules tested in cells receiving PrPPG15 and PDI-specific siRNA comparable to that of the control (Fig 5C, D and E). These data suggest that knockingdown endogenous PDI produces different effects on those two PrP mutants.

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