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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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Expressions of transiently transfected PrP constructs and co-localizations of the expressed PrPs with PDI in 293-T cells.A. Western blots of the expressions of wild-type PrP (PrPWT) and PrP mutants (PrPKDEL and PrPPG15). B. Immunocytochemial assays of the cells transfected with various PrP constructs 48 h after transfection. The images of PrP (green), PDI (red), DAPI (blue) and merge are monitored under a confocal microscopy and indicated above. (×1000).
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pone-0038221-g002: Expressions of transiently transfected PrP constructs and co-localizations of the expressed PrPs with PDI in 293-T cells.A. Western blots of the expressions of wild-type PrP (PrPWT) and PrP mutants (PrPKDEL and PrPPG15). B. Immunocytochemial assays of the cells transfected with various PrP constructs 48 h after transfection. The images of PrP (green), PDI (red), DAPI (blue) and merge are monitored under a confocal microscopy and indicated above. (×1000).

Mentions: Our previous studies demonstrated that transient expressions of the PrP construct retained in ER (PrP-KDEL) and PrP with extra-octarepeats insertions [15] on the cultured cells induced ER stress and cell apoptosis. To obtain more detailed insight into the involvement of PDI in the prion-associated abnormality, recombinant plasmids expressing wild-type PrP (pcDNA-PrP-WT), PrP with additional ER-anchored peptide (pcDNA-PrP-KDEL) and PrP with 10 extra-octarepeats (pcDNA-PrP-PG15) were individually introduced into 293-T cells that were confirmed without detectable endogenous PrPC in routine Western blot assays [15]. Clear PrP-specific signals were observed in the cells receiving the various PrP-expressing plasmids, in which wild-type PrP (PrPWT) and PrP with additional ER-anchored peptide (PrPKDEL) migrated at the same position (ranging from 25 to 35 kDa), while PrP with ten extra-octarepeats (PrPPG15) at the position from 35 to 45 kDa (Fig 2A).


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)

Expressions of transiently transfected PrP constructs and co-localizations of the expressed PrPs with PDI in 293-T cells.A. Western blots of the expressions of wild-type PrP (PrPWT) and PrP mutants (PrPKDEL and PrPPG15). B. Immunocytochemial assays of the cells transfected with various PrP constructs 48 h after transfection. The images of PrP (green), PDI (red), DAPI (blue) and merge are monitored under a confocal microscopy and indicated above. (×1000).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369880&req=5

pone-0038221-g002: Expressions of transiently transfected PrP constructs and co-localizations of the expressed PrPs with PDI in 293-T cells.A. Western blots of the expressions of wild-type PrP (PrPWT) and PrP mutants (PrPKDEL and PrPPG15). B. Immunocytochemial assays of the cells transfected with various PrP constructs 48 h after transfection. The images of PrP (green), PDI (red), DAPI (blue) and merge are monitored under a confocal microscopy and indicated above. (×1000).
Mentions: Our previous studies demonstrated that transient expressions of the PrP construct retained in ER (PrP-KDEL) and PrP with extra-octarepeats insertions [15] on the cultured cells induced ER stress and cell apoptosis. To obtain more detailed insight into the involvement of PDI in the prion-associated abnormality, recombinant plasmids expressing wild-type PrP (pcDNA-PrP-WT), PrP with additional ER-anchored peptide (pcDNA-PrP-KDEL) and PrP with 10 extra-octarepeats (pcDNA-PrP-PG15) were individually introduced into 293-T cells that were confirmed without detectable endogenous PrPC in routine Western blot assays [15]. Clear PrP-specific signals were observed in the cells receiving the various PrP-expressing plasmids, in which wild-type PrP (PrPWT) and PrP with additional ER-anchored peptide (PrPKDEL) migrated at the same position (ranging from 25 to 35 kDa), while PrP with ten extra-octarepeats (PrPPG15) at the position from 35 to 45 kDa (Fig 2A).

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