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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

Assays of mitochondrial dysfunction in the cells expressing various PrP constructs under the condition of overexpression of PDI or knockdown of endogenous PDI.A. Images of the cells with abnormal mitochondrial transmembrane potentials under a fluorescence microscopy. Cells under apoptotic process are colored as green and alive cells are red (×200). B. Quantificational analysis of the percentages of apoptotic cells among the total cells. Data are calculated from three independent tests and presented as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05. C. Qualitative evaluation of the prepared cell fractions with Western blots of VDAC1 (mitochondrial maker), PDI (ER maker) and actin (cytosol marker). D. Western blots for C-CytC outflux in the cytosol fractions and M-CtyC in the mitochondrial fraction of various preparations. The expressing levels of PDI and PrP in the cell lysates of various preparations were also evaluated with individual Western blots. E. Quantitative analysis of C-CytC with normalizing for mitochondrial membrane-associated Cyt C. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001 F. Cytoflowmetry analysis of the cells with mitochondrial dysfunction each preparation with a mitochondrial apoptosis detection kit. The percentages of apoptotic cells are shown in Y-axis. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001. G. Caspase-3 activity assays of various preparations with a commercial kit. 2 U/µl of recombinant human capsase-3 (marked as Caspase-3) supplied in the kit was used as the positive control. ABS indicates as absorbance at 405 nm. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001.
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pone-0038221-g006: Assays of mitochondrial dysfunction in the cells expressing various PrP constructs under the condition of overexpression of PDI or knockdown of endogenous PDI.A. Images of the cells with abnormal mitochondrial transmembrane potentials under a fluorescence microscopy. Cells under apoptotic process are colored as green and alive cells are red (×200). B. Quantificational analysis of the percentages of apoptotic cells among the total cells. Data are calculated from three independent tests and presented as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05. C. Qualitative evaluation of the prepared cell fractions with Western blots of VDAC1 (mitochondrial maker), PDI (ER maker) and actin (cytosol marker). D. Western blots for C-CytC outflux in the cytosol fractions and M-CtyC in the mitochondrial fraction of various preparations. The expressing levels of PDI and PrP in the cell lysates of various preparations were also evaluated with individual Western blots. E. Quantitative analysis of C-CytC with normalizing for mitochondrial membrane-associated Cyt C. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001 F. Cytoflowmetry analysis of the cells with mitochondrial dysfunction each preparation with a mitochondrial apoptosis detection kit. The percentages of apoptotic cells are shown in Y-axis. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001. G. Caspase-3 activity assays of various preparations with a commercial kit. 2 U/µl of recombinant human capsase-3 (marked as Caspase-3) supplied in the kit was used as the positive control. ABS indicates as absorbance at 405 nm. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001.

Mentions: Organelle-specific initiation of cell death can be induced by various misfolded proteins based on different situations [20], [21]. To identify the involvement of mitochondrial dysfunction in apoptosis induced by those two PrP mutants, the mitochondrial transmembrane potentials (MTP) of the cells challenged with PrP mutants under conditions of overexpressing or downregulating PDI were evaluated. With the help of a commercial mitocapture kit that could distinguish the apoptotic cells (green) with mitochondria dysfunction from the live cells (red), more green-stained cells were observed in preparations of PrPKDEL and PrPPG15, showing statistical significance after comparing percentages of apoptotic cells in preparations of PrP mutants with that of PrPWT (Fig. 6A and B). Furthermore, levels of cytochrome c in cytoplasm (C-CytC) were measured with Western blots, which were represented as sequential events of MTP disruption leading to release of cytochrome c from mitochondria to cytoplasm. In parallel, the levels of membrane cytochrome c (M-CytC) were evaluated with Western blots. The cytosolic fraction from crude lysates was carefully prepared, which was marked with actin and PDI, but without voltage-dependent anion channel 1 (VDAC1, a biomarker for the outer membrane of mitochondria, Fig. 6C). Western blots showed comparable levels of M-CytC in all preparations (Fig 6D). Interestingly, in the group of transfection of PrPs alone, cells expressing PrPKDEL or PrPPG15 showed stronger signals of C-CytC than that of PrPWT and control, with statistical significance after the digitized gray values of C-CytC were normalized with that of individual M-CytC (Fig 6 D and E). Expression of PDI or PDI-specific siRNA alone seemed to not alter the level of C-CytC (Fig 6D). However, co-expression of PDI in the cells of PrPKDEL partially reduced the level of C-CytC but not in that of PrPPG15, whereas co-expression of PDI-specific siRNA in cells of PrPPG15 remarkably decreased the level of C-CytC but not in PrPKDEL (Fig 6D and E). Flow cytometry analysis revealed that the proportions of apoptotic cells in cells transfected with plasmids expressing PrPKDEL and PrPPG15 alone, the cells co-expressing PrPKDEL and PDI-specific siRNA and cells co-expressing PrPPG15 and PDI were significantly higher than in the other preparations (Fig 6F), which were consistent with the results of assays of cell viability and levels of C-CytC. Assays of the activities of caspase-3 identified higher enzyme activities in cells expressing either PrPKDEL or PrPPG15, the cells co-challenged with PrPKDEL and PDI-specific siRNA, as well as the cells co-expressing PrPPG15 and PDI (Fig 6F). Furthermore, the activities of caspase-3 in cells expressing natural fCJD associated PrP mutants (PrP-G114V and PrP-PG14) were significantly higher than that of mock, whereas the activity of caspase 3 in cells expressing PS1-KDEL remained unchanged (Supplemental Fig S3). Those data imply that, in addition to ER stress and ER-related apoptosis, mitochondrial dysfunction contributes to apoptosis caused by expressions of PrP mutants. Changes in cellular PDI levels result in distinct effects on mitochondria function in cells expressing different misfolded 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)

Assays of mitochondrial dysfunction in the cells expressing various PrP constructs under the condition of overexpression of PDI or knockdown of endogenous PDI.A. Images of the cells with abnormal mitochondrial transmembrane potentials under a fluorescence microscopy. Cells under apoptotic process are colored as green and alive cells are red (×200). B. Quantificational analysis of the percentages of apoptotic cells among the total cells. Data are calculated from three independent tests and presented as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05. C. Qualitative evaluation of the prepared cell fractions with Western blots of VDAC1 (mitochondrial maker), PDI (ER maker) and actin (cytosol marker). D. Western blots for C-CytC outflux in the cytosol fractions and M-CtyC in the mitochondrial fraction of various preparations. The expressing levels of PDI and PrP in the cell lysates of various preparations were also evaluated with individual Western blots. E. Quantitative analysis of C-CytC with normalizing for mitochondrial membrane-associated Cyt C. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001 F. Cytoflowmetry analysis of the cells with mitochondrial dysfunction each preparation with a mitochondrial apoptosis detection kit. The percentages of apoptotic cells are shown in Y-axis. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001. G. Caspase-3 activity assays of various preparations with a commercial kit. 2 U/µl of recombinant human capsase-3 (marked as Caspase-3) supplied in the kit was used as the positive control. ABS indicates as absorbance at 405 nm. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369880&req=5

pone-0038221-g006: Assays of mitochondrial dysfunction in the cells expressing various PrP constructs under the condition of overexpression of PDI or knockdown of endogenous PDI.A. Images of the cells with abnormal mitochondrial transmembrane potentials under a fluorescence microscopy. Cells under apoptotic process are colored as green and alive cells are red (×200). B. Quantificational analysis of the percentages of apoptotic cells among the total cells. Data are calculated from three independent tests and presented as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05. C. Qualitative evaluation of the prepared cell fractions with Western blots of VDAC1 (mitochondrial maker), PDI (ER maker) and actin (cytosol marker). D. Western blots for C-CytC outflux in the cytosol fractions and M-CtyC in the mitochondrial fraction of various preparations. The expressing levels of PDI and PrP in the cell lysates of various preparations were also evaluated with individual Western blots. E. Quantitative analysis of C-CytC with normalizing for mitochondrial membrane-associated Cyt C. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001 F. Cytoflowmetry analysis of the cells with mitochondrial dysfunction each preparation with a mitochondrial apoptosis detection kit. The percentages of apoptotic cells are shown in Y-axis. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001. G. Caspase-3 activity assays of various preparations with a commercial kit. 2 U/µl of recombinant human capsase-3 (marked as Caspase-3) supplied in the kit was used as the positive control. ABS indicates as absorbance at 405 nm. Data are calculated from three independent tests and indicated as mean values ± SD. Statistical differences compared with controls are illustrated as *P<0.05 or **P<0.001.
Mentions: Organelle-specific initiation of cell death can be induced by various misfolded proteins based on different situations [20], [21]. To identify the involvement of mitochondrial dysfunction in apoptosis induced by those two PrP mutants, the mitochondrial transmembrane potentials (MTP) of the cells challenged with PrP mutants under conditions of overexpressing or downregulating PDI were evaluated. With the help of a commercial mitocapture kit that could distinguish the apoptotic cells (green) with mitochondria dysfunction from the live cells (red), more green-stained cells were observed in preparations of PrPKDEL and PrPPG15, showing statistical significance after comparing percentages of apoptotic cells in preparations of PrP mutants with that of PrPWT (Fig. 6A and B). Furthermore, levels of cytochrome c in cytoplasm (C-CytC) were measured with Western blots, which were represented as sequential events of MTP disruption leading to release of cytochrome c from mitochondria to cytoplasm. In parallel, the levels of membrane cytochrome c (M-CytC) were evaluated with Western blots. The cytosolic fraction from crude lysates was carefully prepared, which was marked with actin and PDI, but without voltage-dependent anion channel 1 (VDAC1, a biomarker for the outer membrane of mitochondria, Fig. 6C). Western blots showed comparable levels of M-CytC in all preparations (Fig 6D). Interestingly, in the group of transfection of PrPs alone, cells expressing PrPKDEL or PrPPG15 showed stronger signals of C-CytC than that of PrPWT and control, with statistical significance after the digitized gray values of C-CytC were normalized with that of individual M-CytC (Fig 6 D and E). Expression of PDI or PDI-specific siRNA alone seemed to not alter the level of C-CytC (Fig 6D). However, co-expression of PDI in the cells of PrPKDEL partially reduced the level of C-CytC but not in that of PrPPG15, whereas co-expression of PDI-specific siRNA in cells of PrPPG15 remarkably decreased the level of C-CytC but not in PrPKDEL (Fig 6D and E). Flow cytometry analysis revealed that the proportions of apoptotic cells in cells transfected with plasmids expressing PrPKDEL and PrPPG15 alone, the cells co-expressing PrPKDEL and PDI-specific siRNA and cells co-expressing PrPPG15 and PDI were significantly higher than in the other preparations (Fig 6F), which were consistent with the results of assays of cell viability and levels of C-CytC. Assays of the activities of caspase-3 identified higher enzyme activities in cells expressing either PrPKDEL or PrPPG15, the cells co-challenged with PrPKDEL and PDI-specific siRNA, as well as the cells co-expressing PrPPG15 and PDI (Fig 6F). Furthermore, the activities of caspase-3 in cells expressing natural fCJD associated PrP mutants (PrP-G114V and PrP-PG14) were significantly higher than that of mock, whereas the activity of caspase 3 in cells expressing PS1-KDEL remained unchanged (Supplemental Fig S3). Those data imply that, in addition to ER stress and ER-related apoptosis, mitochondrial dysfunction contributes to apoptosis caused by expressions of PrP mutants. Changes in cellular PDI levels result in distinct effects on mitochondria function in cells expressing different misfolded 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