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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 overexpression of PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence overexpressing 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<0.05. 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-g004: Influences of overexpression of PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence overexpressing 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<0.05. 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 see the potential role of PDI in misfolded PrP-induced cytotoxicity, a PDI-expressing plasmid was introduced into the cell model to create a situation of overexpression of PDI. Western blots revealed that transfection of pcDNA-PDI increased cellular PDI levels but did not influence levels of expressed PrPs when co-transfected with different PrP-expressing plasmids (Fig 4A). CCK-8 assays showed that transient expression of PDI alone did not affect cell growth, which revealed cell viability similar to mock and the preparation of wild-type PrP (Fig 4B). Co-transfection of the plasmid-expressing PDI and PrP mutants reversed to a modest degree PrP mutants-induced cytotoxicity, but showed distinct effectiveness, in which overexpression of PDI rescued PrPKDEL- induced cytotoxicity with statistical significance but seemed to have less effect on PrPPG15-induced cytotoxicity (Fig. 4B). Western blots of cell lysates identified that, in the preparation co-expressing PDI and PrPKDEL, the levels of Grp78, pro-caspase-12, and pro-caspase-3 recovered to a degree comparable to the control, whereas levels of pro-caspase-3 in the preparation co-expressing PDI and PrPPG15 were obviously lower than the controls (Fig 4C and D). Additionally, levels of Bax in those two preparations were still higher than controls (Fig. 4E). It seems that overexpression of PDI in cultured cells sufficiently releases ER stress and antagonizes apoptosis induced by misfolded PrP mainly accumulated in the ER, but has a limited effect on the apoptosis of other organelles.


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 overexpression of PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence overexpressing 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<0.05. 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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getmorefigures.php?uid=PMC3369880&req=5

pone-0038221-g004: Influences of overexpression of PDI on PrP mutants-induced cytotoxicity and the alterations of relevant cellular factors.A. Evaluation of the influence overexpressing 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<0.05. 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 see the potential role of PDI in misfolded PrP-induced cytotoxicity, a PDI-expressing plasmid was introduced into the cell model to create a situation of overexpression of PDI. Western blots revealed that transfection of pcDNA-PDI increased cellular PDI levels but did not influence levels of expressed PrPs when co-transfected with different PrP-expressing plasmids (Fig 4A). CCK-8 assays showed that transient expression of PDI alone did not affect cell growth, which revealed cell viability similar to mock and the preparation of wild-type PrP (Fig 4B). Co-transfection of the plasmid-expressing PDI and PrP mutants reversed to a modest degree PrP mutants-induced cytotoxicity, but showed distinct effectiveness, in which overexpression of PDI rescued PrPKDEL- induced cytotoxicity with statistical significance but seemed to have less effect on PrPPG15-induced cytotoxicity (Fig. 4B). Western blots of cell lysates identified that, in the preparation co-expressing PDI and PrPKDEL, the levels of Grp78, pro-caspase-12, and pro-caspase-3 recovered to a degree comparable to the control, whereas levels of pro-caspase-3 in the preparation co-expressing PDI and PrPPG15 were obviously lower than the controls (Fig 4C and D). Additionally, levels of Bax in those two preparations were still higher than controls (Fig. 4E). It seems that overexpression of PDI in cultured cells sufficiently releases ER stress and antagonizes apoptosis induced by misfolded PrP mainly accumulated in the ER, but has a limited effect on the apoptosis of other organelles.

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