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The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore.

Everett H, Barry M, Sun X, Lee SF, Frantz C, Berthiaume LG, McFadden G, Bleackley RC - J. Exp. Med. (2002)

Bottom Line: Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR.Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR.We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G2H7, Canada.

ABSTRACT
M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

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PBR expression augments the protective effects of myxoma virus infection. Jurkat or Jurkat-PBR cells were mock-infected (mock) or infected with myxoma virus (Myx) or the M11L knockout virus (M11L−). The percentage of cells that underwent a loss of TMRE fluorescence as a result of staurosporine treatment (A) or Fas stimulation (B) is shown. Results are the average of three independent experiments (±SD). In parallel, Jurkat or Jurkat-PBR cells that had been mock- or virus-infected were analyzed for the presence of M11L using immunoprecipitation and immunoblotting (C, top panel). Alternatively, virus infection was verified by the production of the early viral protein, M-T7 (C, bottom panel).
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fig6: PBR expression augments the protective effects of myxoma virus infection. Jurkat or Jurkat-PBR cells were mock-infected (mock) or infected with myxoma virus (Myx) or the M11L knockout virus (M11L−). The percentage of cells that underwent a loss of TMRE fluorescence as a result of staurosporine treatment (A) or Fas stimulation (B) is shown. Results are the average of three independent experiments (±SD). In parallel, Jurkat or Jurkat-PBR cells that had been mock- or virus-infected were analyzed for the presence of M11L using immunoprecipitation and immunoblotting (C, top panel). Alternatively, virus infection was verified by the production of the early viral protein, M-T7 (C, bottom panel).

Mentions: To study the biological importance of the physical association between M11L and the PBR, we investigated the ability of myxoma virus infection to protect cells from staurosporine-mediated apoptosis in the context of different PBR expression levels. PBR-deficient Jurkat T lymphocytes or PBR overexpressing Jurkat-PBR cells were infected with the myxoma virus, vMyxlac, to allow the expression of M11L. For control purposes, parallel samples from both cell lines were mock-infected or infected with a M11L-knockout myxoma virus (vMyxM11L−). Cells were treated with 2 μM staurosporine or DMSO solvent as a control and TMRE fluorescence was used to measure ΔΨm loss. As shown in Fig. 6 A, a marked loss of TMRE fluorescence occurred after staurosporine treatment of both Jurkat and Jurkat-PBR cells that had been mock infected, with the presence of PBR only providing a slight protective effect. Myxoma virus infection was found to protect both cell lines from staurosporine-induced ΔΨm loss. However, vMyxlac infection of Jurkat-PBR cells provided a level of protection against ΔΨm loss that was twofold greater than the protection afforded control Jurkat cells. Infection of both cell lines with the M11L-deficient vMyxM11L− did not provide protection against staurosporine-induced ΔΨm loss. Similarly, when apoptosis was induced by Fas stimulation the same trends could be observed (Fig. 6 B).


The myxoma poxvirus protein, M11L, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore.

Everett H, Barry M, Sun X, Lee SF, Frantz C, Berthiaume LG, McFadden G, Bleackley RC - J. Exp. Med. (2002)

PBR expression augments the protective effects of myxoma virus infection. Jurkat or Jurkat-PBR cells were mock-infected (mock) or infected with myxoma virus (Myx) or the M11L knockout virus (M11L−). The percentage of cells that underwent a loss of TMRE fluorescence as a result of staurosporine treatment (A) or Fas stimulation (B) is shown. Results are the average of three independent experiments (±SD). In parallel, Jurkat or Jurkat-PBR cells that had been mock- or virus-infected were analyzed for the presence of M11L using immunoprecipitation and immunoblotting (C, top panel). Alternatively, virus infection was verified by the production of the early viral protein, M-T7 (C, bottom panel).
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Related In: Results  -  Collection

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fig6: PBR expression augments the protective effects of myxoma virus infection. Jurkat or Jurkat-PBR cells were mock-infected (mock) or infected with myxoma virus (Myx) or the M11L knockout virus (M11L−). The percentage of cells that underwent a loss of TMRE fluorescence as a result of staurosporine treatment (A) or Fas stimulation (B) is shown. Results are the average of three independent experiments (±SD). In parallel, Jurkat or Jurkat-PBR cells that had been mock- or virus-infected were analyzed for the presence of M11L using immunoprecipitation and immunoblotting (C, top panel). Alternatively, virus infection was verified by the production of the early viral protein, M-T7 (C, bottom panel).
Mentions: To study the biological importance of the physical association between M11L and the PBR, we investigated the ability of myxoma virus infection to protect cells from staurosporine-mediated apoptosis in the context of different PBR expression levels. PBR-deficient Jurkat T lymphocytes or PBR overexpressing Jurkat-PBR cells were infected with the myxoma virus, vMyxlac, to allow the expression of M11L. For control purposes, parallel samples from both cell lines were mock-infected or infected with a M11L-knockout myxoma virus (vMyxM11L−). Cells were treated with 2 μM staurosporine or DMSO solvent as a control and TMRE fluorescence was used to measure ΔΨm loss. As shown in Fig. 6 A, a marked loss of TMRE fluorescence occurred after staurosporine treatment of both Jurkat and Jurkat-PBR cells that had been mock infected, with the presence of PBR only providing a slight protective effect. Myxoma virus infection was found to protect both cell lines from staurosporine-induced ΔΨm loss. However, vMyxlac infection of Jurkat-PBR cells provided a level of protection against ΔΨm loss that was twofold greater than the protection afforded control Jurkat cells. Infection of both cell lines with the M11L-deficient vMyxM11L− did not provide protection against staurosporine-induced ΔΨm loss. Similarly, when apoptosis was induced by Fas stimulation the same trends could be observed (Fig. 6 B).

Bottom Line: Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR.Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR.We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G2H7, Canada.

ABSTRACT
M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.

Show MeSH
Related in: MedlinePlus