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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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Comparative analysis of the levels of PDIs in the brain tissues of normal and 263K-infected hamsters.A. Western blots. The infected hamsters were collected about 60 to 70 dpi. Same amounts of individual brain homogenate were loaded in 15% SDS-PAGE and immunoblotted with PrP specific mAb 3F4. PK+: with PK (50 µg/ml); PK-: without PK. B. Western blots of Grp78, Grp58, PDI and pro-caspase3. C. Quantitative analysis of each gray numerical value of Grp78, Grp58, PDI and pro-caspase3 vs that of individual β-actin. The average values are calculated from four infected hamsters or three normal hamsters and presented as mean ± SD. Statistical differences compared with controls are illustrated. D, Dynamic analyses of Grp78, Grp58, PDIs, pro-caspase3 and cleveage-caspase3 in the brain tissues of scrapie agent 263K-infected hamsters collected on 0, 20, 40 and 60 dpi during incubation period with Western blots. E. Log-fold changes of the gray values of Grp78, Grp58, PDIs, pro-caspase3 in the 263K-infected hamster’s brains collected on 20, 40 and 60 dpi vs that of 0 dpi.
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pone-0038221-g001: Comparative analysis of the levels of PDIs in the brain tissues of normal and 263K-infected hamsters.A. Western blots. The infected hamsters were collected about 60 to 70 dpi. Same amounts of individual brain homogenate were loaded in 15% SDS-PAGE and immunoblotted with PrP specific mAb 3F4. PK+: with PK (50 µg/ml); PK-: without PK. B. Western blots of Grp78, Grp58, PDI and pro-caspase3. C. Quantitative analysis of each gray numerical value of Grp78, Grp58, PDI and pro-caspase3 vs that of individual β-actin. The average values are calculated from four infected hamsters or three normal hamsters and presented as mean ± SD. Statistical differences compared with controls are illustrated. D, Dynamic analyses of Grp78, Grp58, PDIs, pro-caspase3 and cleveage-caspase3 in the brain tissues of scrapie agent 263K-infected hamsters collected on 0, 20, 40 and 60 dpi during incubation period with Western blots. E. Log-fold changes of the gray values of Grp78, Grp58, PDIs, pro-caspase3 in the 263K-infected hamster’s brains collected on 20, 40 and 60 dpi vs that of 0 dpi.

Mentions: To assess the possible change of PDI in the brains with prion disease, the levels of some PDI family members in hamsters brain tissues infected with scrapie strain 263K were evaluated with individual Western blots. Obvious PK-resistant PrP signals (PrPres) were detected in infected hamsters brains collected at the terminal stage, which were collected about 60 to 70 days post-inoculation (dpi) (Fig. 1A). Two candidates from the PDI family, Grp58 and PDI as well as another ER-anchored chaperone Grp78/Bip were individually detected with the dependable specimens. Significant upregulation in the expression levels of Grp78/Bip, Grp58, and PDI were detected in the samples of infected hamsters (Fig. 1B). Mean relative gray values of three ER makers in scrapie-infected animals showed statistical differences compared with that of normal controls, after they were normalized with individual data from β-actin (Fig 1C). Additionally, the amounts of pro-Caspase3 (pro-CASP3) in the brains of the infected hamsters were lower than that of controls (Fig 1, B and C), revealing signs of apoptosis.


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)

Comparative analysis of the levels of PDIs in the brain tissues of normal and 263K-infected hamsters.A. Western blots. The infected hamsters were collected about 60 to 70 dpi. Same amounts of individual brain homogenate were loaded in 15% SDS-PAGE and immunoblotted with PrP specific mAb 3F4. PK+: with PK (50 µg/ml); PK-: without PK. B. Western blots of Grp78, Grp58, PDI and pro-caspase3. C. Quantitative analysis of each gray numerical value of Grp78, Grp58, PDI and pro-caspase3 vs that of individual β-actin. The average values are calculated from four infected hamsters or three normal hamsters and presented as mean ± SD. Statistical differences compared with controls are illustrated. D, Dynamic analyses of Grp78, Grp58, PDIs, pro-caspase3 and cleveage-caspase3 in the brain tissues of scrapie agent 263K-infected hamsters collected on 0, 20, 40 and 60 dpi during incubation period with Western blots. E. Log-fold changes of the gray values of Grp78, Grp58, PDIs, pro-caspase3 in the 263K-infected hamster’s brains collected on 20, 40 and 60 dpi vs that of 0 dpi.
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Related In: Results  -  Collection

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pone-0038221-g001: Comparative analysis of the levels of PDIs in the brain tissues of normal and 263K-infected hamsters.A. Western blots. The infected hamsters were collected about 60 to 70 dpi. Same amounts of individual brain homogenate were loaded in 15% SDS-PAGE and immunoblotted with PrP specific mAb 3F4. PK+: with PK (50 µg/ml); PK-: without PK. B. Western blots of Grp78, Grp58, PDI and pro-caspase3. C. Quantitative analysis of each gray numerical value of Grp78, Grp58, PDI and pro-caspase3 vs that of individual β-actin. The average values are calculated from four infected hamsters or three normal hamsters and presented as mean ± SD. Statistical differences compared with controls are illustrated. D, Dynamic analyses of Grp78, Grp58, PDIs, pro-caspase3 and cleveage-caspase3 in the brain tissues of scrapie agent 263K-infected hamsters collected on 0, 20, 40 and 60 dpi during incubation period with Western blots. E. Log-fold changes of the gray values of Grp78, Grp58, PDIs, pro-caspase3 in the 263K-infected hamster’s brains collected on 20, 40 and 60 dpi vs that of 0 dpi.
Mentions: To assess the possible change of PDI in the brains with prion disease, the levels of some PDI family members in hamsters brain tissues infected with scrapie strain 263K were evaluated with individual Western blots. Obvious PK-resistant PrP signals (PrPres) were detected in infected hamsters brains collected at the terminal stage, which were collected about 60 to 70 days post-inoculation (dpi) (Fig. 1A). Two candidates from the PDI family, Grp58 and PDI as well as another ER-anchored chaperone Grp78/Bip were individually detected with the dependable specimens. Significant upregulation in the expression levels of Grp78/Bip, Grp58, and PDI were detected in the samples of infected hamsters (Fig. 1B). Mean relative gray values of three ER makers in scrapie-infected animals showed statistical differences compared with that of normal controls, after they were normalized with individual data from β-actin (Fig 1C). Additionally, the amounts of pro-Caspase3 (pro-CASP3) in the brains of the infected hamsters were lower than that of controls (Fig 1, B and C), revealing signs of apoptosis.

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