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Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

Jodoin J, Misiewicz M, Makhijani P, Giannopoulos PN, Hammond J, Goodyer CG, LeBlanc AC - PLoS ONE (2009)

Bottom Line: However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation.These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death.Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

View Article: PubMed Central - PubMed

Affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Canada.

ABSTRACT
Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ((M)) or valine ((V)) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V), D202N(V), P102L(V) and Q217R(V) retained, whereas the P102L(M), P105L(V), Y145stop(M) and Q212P(M) PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

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GSS-associated PrP mutants F198SV, D202NV, P102LV and Q217RV, but not P102LM, P105LV, Y145stopM, and Q212PM, prevent Bax-mediated condensed chromatin in MCF-7 cells.A. Schematic diagram of human PrP showing the signal peptide, the octapeptide repeats, the transmembrane domain (TM), the β-sheets (β1 and β2), the α-helices (α1, α2, α3), the glycosyl phosphatidylinositol (GPI)-anchor signal peptide, the polymorphic codon at amino acid 129, and the GSS PrP mutations. B. Percentage of cell death assessed by condensed chromatin in MCF-7 cells transfected with pBud-EGFP or pBud-EGFP-Bax (control), pBud-EGFP/PrP or PrP mutants, and pBud-EGFP-Bax/PrP or PrP mutants carrying a methionine (M) or a valine (V) at codon 129. Data represent the mean±SEM of six independent experiments. At least 600 cells were counted for each condition. * and # indicate a statistically significant difference (p≤0.05) compared to the control and wild type PrP, respectively.
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pone-0006647-g001: GSS-associated PrP mutants F198SV, D202NV, P102LV and Q217RV, but not P102LM, P105LV, Y145stopM, and Q212PM, prevent Bax-mediated condensed chromatin in MCF-7 cells.A. Schematic diagram of human PrP showing the signal peptide, the octapeptide repeats, the transmembrane domain (TM), the β-sheets (β1 and β2), the α-helices (α1, α2, α3), the glycosyl phosphatidylinositol (GPI)-anchor signal peptide, the polymorphic codon at amino acid 129, and the GSS PrP mutations. B. Percentage of cell death assessed by condensed chromatin in MCF-7 cells transfected with pBud-EGFP or pBud-EGFP-Bax (control), pBud-EGFP/PrP or PrP mutants, and pBud-EGFP-Bax/PrP or PrP mutants carrying a methionine (M) or a valine (V) at codon 129. Data represent the mean±SEM of six independent experiments. At least 600 cells were counted for each condition. * and # indicate a statistically significant difference (p≤0.05) compared to the control and wild type PrP, respectively.

Mentions: GSS-associated PrP mutations P105L, A117V, F198S, D202N, and Q217R have been reported with a valine at codon 129, whereas P102L, Y145stop, and Q212P carry a methionine at codon 129 [1], [3], [34]–[36]. In some rare cases, P102L is associated with a valine at codon 129 [34]. To assess if the PrP anti-Bax function was altered in GSS mutants, specific single point mutations with their respective methionine or valine at codon 129 into the prion protein cDNA were produced and the mutant cDNA inserted into the bigenic pBudCE4.1 construct under the CMV promoter (Fig. 1A). This construct also expresses either EGFP or EGFP-Bax protein under the EF-1α promoter. Therefore, cell death is specifically induced by over-expression of Bax and the anti-Bax function of co-expressed wild type (WT) PrP or PrP mutants is directly assessed. Bax overexpression resulted in the condensation of chromatin in approximately 65% of the transfected MCF-7 cells within 20 hrs of transfection (Fig. 1B). Co-expression of WT PrP with either a methionine (PrPM) or valine (PrPV) at codon 129 prevented Bax-mediated condensed chromatin. Similar to our previous observations with CJD mutants, the P102LM, P105LV, Y145stopM and Q212PM PrP mutants completely lost their ability to prevent Bax-mediated condensed chromatin. However, F198SV, D202NV, and Q217RV completely while the P102LV partially retained the anti-Bax function. None of the GSS-associated PrP mutants co-expressed with EGFP induced chromatin condensation in MCF-7 cells, except for the Y145stopM which, consistent with previous observations [37], significantly increased the number of cells with condensed chromatin by 15% (Fig. 1B). Together, these results indicate that only a few of the GSS-associated mutants retain PrP's ability to prevent Bax-mediated condensed chromatin in MCF-7 cells.


Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

Jodoin J, Misiewicz M, Makhijani P, Giannopoulos PN, Hammond J, Goodyer CG, LeBlanc AC - PLoS ONE (2009)

GSS-associated PrP mutants F198SV, D202NV, P102LV and Q217RV, but not P102LM, P105LV, Y145stopM, and Q212PM, prevent Bax-mediated condensed chromatin in MCF-7 cells.A. Schematic diagram of human PrP showing the signal peptide, the octapeptide repeats, the transmembrane domain (TM), the β-sheets (β1 and β2), the α-helices (α1, α2, α3), the glycosyl phosphatidylinositol (GPI)-anchor signal peptide, the polymorphic codon at amino acid 129, and the GSS PrP mutations. B. Percentage of cell death assessed by condensed chromatin in MCF-7 cells transfected with pBud-EGFP or pBud-EGFP-Bax (control), pBud-EGFP/PrP or PrP mutants, and pBud-EGFP-Bax/PrP or PrP mutants carrying a methionine (M) or a valine (V) at codon 129. Data represent the mean±SEM of six independent experiments. At least 600 cells were counted for each condition. * and # indicate a statistically significant difference (p≤0.05) compared to the control and wild type PrP, respectively.
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getmorefigures.php?uid=PMC2722024&req=5

pone-0006647-g001: GSS-associated PrP mutants F198SV, D202NV, P102LV and Q217RV, but not P102LM, P105LV, Y145stopM, and Q212PM, prevent Bax-mediated condensed chromatin in MCF-7 cells.A. Schematic diagram of human PrP showing the signal peptide, the octapeptide repeats, the transmembrane domain (TM), the β-sheets (β1 and β2), the α-helices (α1, α2, α3), the glycosyl phosphatidylinositol (GPI)-anchor signal peptide, the polymorphic codon at amino acid 129, and the GSS PrP mutations. B. Percentage of cell death assessed by condensed chromatin in MCF-7 cells transfected with pBud-EGFP or pBud-EGFP-Bax (control), pBud-EGFP/PrP or PrP mutants, and pBud-EGFP-Bax/PrP or PrP mutants carrying a methionine (M) or a valine (V) at codon 129. Data represent the mean±SEM of six independent experiments. At least 600 cells were counted for each condition. * and # indicate a statistically significant difference (p≤0.05) compared to the control and wild type PrP, respectively.
Mentions: GSS-associated PrP mutations P105L, A117V, F198S, D202N, and Q217R have been reported with a valine at codon 129, whereas P102L, Y145stop, and Q212P carry a methionine at codon 129 [1], [3], [34]–[36]. In some rare cases, P102L is associated with a valine at codon 129 [34]. To assess if the PrP anti-Bax function was altered in GSS mutants, specific single point mutations with their respective methionine or valine at codon 129 into the prion protein cDNA were produced and the mutant cDNA inserted into the bigenic pBudCE4.1 construct under the CMV promoter (Fig. 1A). This construct also expresses either EGFP or EGFP-Bax protein under the EF-1α promoter. Therefore, cell death is specifically induced by over-expression of Bax and the anti-Bax function of co-expressed wild type (WT) PrP or PrP mutants is directly assessed. Bax overexpression resulted in the condensation of chromatin in approximately 65% of the transfected MCF-7 cells within 20 hrs of transfection (Fig. 1B). Co-expression of WT PrP with either a methionine (PrPM) or valine (PrPV) at codon 129 prevented Bax-mediated condensed chromatin. Similar to our previous observations with CJD mutants, the P102LM, P105LV, Y145stopM and Q212PM PrP mutants completely lost their ability to prevent Bax-mediated condensed chromatin. However, F198SV, D202NV, and Q217RV completely while the P102LV partially retained the anti-Bax function. None of the GSS-associated PrP mutants co-expressed with EGFP induced chromatin condensation in MCF-7 cells, except for the Y145stopM which, consistent with previous observations [37], significantly increased the number of cells with condensed chromatin by 15% (Fig. 1B). Together, these results indicate that only a few of the GSS-associated mutants retain PrP's ability to prevent Bax-mediated condensed chromatin in MCF-7 cells.

Bottom Line: However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation.These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death.Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

View Article: PubMed Central - PubMed

Affiliation: Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montréal, Canada.

ABSTRACT
Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ((M)) or valine ((V)) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V), D202N(V), P102L(V) and Q217R(V) retained, whereas the P102L(M), P105L(V), Y145stop(M) and Q212P(M) PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.

Show MeSH
Related in: MedlinePlus