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Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

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p20 sensitizes mitochondria to caspase-8–induced cyt.c release. H1299 cells were mock infected, or co-infected with AdRTA (control) and AdMFpk3FLICE or with Adp20 and AdMFpk3FLICE. 16 h post-infection, FK1012Z or vehicle alone (DMSO; Wang et al., 2003) were added for 45 or 90 min, and the amount of cyt.c in the post-mitochondrial supernatant and tBID in the mitochondrial fraction were assessed by SDS-PAGE and Western blot. The intensity of the cyt.c (A) and tBID (B) signals, relative to loading controls, was determined using ImageQuantTM software (Amersham Biosciences) and is expressed in arbitrary units. Shown is a representative of three independent experiments.
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fig3: p20 sensitizes mitochondria to caspase-8–induced cyt.c release. H1299 cells were mock infected, or co-infected with AdRTA (control) and AdMFpk3FLICE or with Adp20 and AdMFpk3FLICE. 16 h post-infection, FK1012Z or vehicle alone (DMSO; Wang et al., 2003) were added for 45 or 90 min, and the amount of cyt.c in the post-mitochondrial supernatant and tBID in the mitochondrial fraction were assessed by SDS-PAGE and Western blot. The intensity of the cyt.c (A) and tBID (B) signals, relative to loading controls, was determined using ImageQuantTM software (Amersham Biosciences) and is expressed in arbitrary units. Shown is a representative of three independent experiments.

Mentions: Given that BAP31 is a caspase-8 substrate, p20 might cooperate with other products generated by caspase-8, such as tBID, to enhance mitochondrial dysfunction. According to this model, immediately after its expression, p20 should activate a signal that is slow to induce cyt.c release on its own, but able to synergize with other apoptotic signals during Fas-mediated apoptosis. Therefore, we investigated whether p20 could enhance caspase-8–driven cyt.c release. Death receptor–dependent caspase-8 activation was mimicked by infecting H1299 cells with adenovector-expressing triplicate copies of Fpk (a mutant of FKBP) fused to the catalytic subunits of caspase-8 (AdMFpk3FLICE; Muzio et al., 1998). After its expression in cells, oligomerization and autoactivation of the caspase-8 fusion protein was induced with the Fpk-dimerizing compound, FK1012Z. This approach has the benefit of delivering synchronized caspase-8 signals to cells without stimulating caspase-8–independent pathways activated by death receptors (Schulze-Osthoff et al., 1998; Wang et al., 2001). In Fig. 3, H1299 cells were co-infected with AdRTA (control adenovector) and AdMFpk3FLICE, or with Adp20 and AdMFpk3FLICE. 16 h after infection, a time when Adp20 alone did not induce cyt.c release or caspase activation (Figs. 2 and 3), the cells were exposed to a short treatment (45 or 90 min) with FK1012Z or vehicle (DMSO) alone, and the mitochondrial and post-mitochondrial fractions were isolated. Compared with caspase-8 activation in the presence of the control protein RTA, caspase-8 activation in the presence of ectopic p20 strongly induced release of cyt.c to the cytosol (Fig. 3 A), but it did not affect the amount of caspase-8–generated tBID that was recovered in the mitochondrial fraction (Fig. 3 B). In all cases, equivalent amounts of MFpk3FLICE were produced (unpublished data). Therefore, these results suggest that p20-mediated signals from the ER might cooperate with other caspase-8–generated signals to increase cyt.c release from mitochondria.


Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol.

Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC - J. Cell Biol. (2003)

p20 sensitizes mitochondria to caspase-8–induced cyt.c release. H1299 cells were mock infected, or co-infected with AdRTA (control) and AdMFpk3FLICE or with Adp20 and AdMFpk3FLICE. 16 h post-infection, FK1012Z or vehicle alone (DMSO; Wang et al., 2003) were added for 45 or 90 min, and the amount of cyt.c in the post-mitochondrial supernatant and tBID in the mitochondrial fraction were assessed by SDS-PAGE and Western blot. The intensity of the cyt.c (A) and tBID (B) signals, relative to loading controls, was determined using ImageQuantTM software (Amersham Biosciences) and is expressed in arbitrary units. Shown is a representative of three independent experiments.
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Related In: Results  -  Collection

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fig3: p20 sensitizes mitochondria to caspase-8–induced cyt.c release. H1299 cells were mock infected, or co-infected with AdRTA (control) and AdMFpk3FLICE or with Adp20 and AdMFpk3FLICE. 16 h post-infection, FK1012Z or vehicle alone (DMSO; Wang et al., 2003) were added for 45 or 90 min, and the amount of cyt.c in the post-mitochondrial supernatant and tBID in the mitochondrial fraction were assessed by SDS-PAGE and Western blot. The intensity of the cyt.c (A) and tBID (B) signals, relative to loading controls, was determined using ImageQuantTM software (Amersham Biosciences) and is expressed in arbitrary units. Shown is a representative of three independent experiments.
Mentions: Given that BAP31 is a caspase-8 substrate, p20 might cooperate with other products generated by caspase-8, such as tBID, to enhance mitochondrial dysfunction. According to this model, immediately after its expression, p20 should activate a signal that is slow to induce cyt.c release on its own, but able to synergize with other apoptotic signals during Fas-mediated apoptosis. Therefore, we investigated whether p20 could enhance caspase-8–driven cyt.c release. Death receptor–dependent caspase-8 activation was mimicked by infecting H1299 cells with adenovector-expressing triplicate copies of Fpk (a mutant of FKBP) fused to the catalytic subunits of caspase-8 (AdMFpk3FLICE; Muzio et al., 1998). After its expression in cells, oligomerization and autoactivation of the caspase-8 fusion protein was induced with the Fpk-dimerizing compound, FK1012Z. This approach has the benefit of delivering synchronized caspase-8 signals to cells without stimulating caspase-8–independent pathways activated by death receptors (Schulze-Osthoff et al., 1998; Wang et al., 2001). In Fig. 3, H1299 cells were co-infected with AdRTA (control adenovector) and AdMFpk3FLICE, or with Adp20 and AdMFpk3FLICE. 16 h after infection, a time when Adp20 alone did not induce cyt.c release or caspase activation (Figs. 2 and 3), the cells were exposed to a short treatment (45 or 90 min) with FK1012Z or vehicle (DMSO) alone, and the mitochondrial and post-mitochondrial fractions were isolated. Compared with caspase-8 activation in the presence of the control protein RTA, caspase-8 activation in the presence of ectopic p20 strongly induced release of cyt.c to the cytosol (Fig. 3 A), but it did not affect the amount of caspase-8–generated tBID that was recovered in the mitochondrial fraction (Fig. 3 B). In all cases, equivalent amounts of MFpk3FLICE were produced (unpublished data). Therefore, these results suggest that p20-mediated signals from the ER might cooperate with other caspase-8–generated signals to increase cyt.c release from mitochondria.

Bottom Line: Cell.Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway.Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.

ABSTRACT
Stimulation of cell surface death receptors activates caspase-8, which targets a limited number of substrates including BAP31, an integral membrane protein of the endoplasmic reticulum (ER). Recently, we reported that a caspase-resistant BAP31 mutant inhibited several features of Fas-induced apoptosis, including the release of cytochrome c (cyt.c) from mitochondria (Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Mol. Cell. Biol. 20:6731-6740), implicating ER-mitochondria crosstalk in this pathway. Here, we report that the p20 caspase cleavage fragment of BAP31 can direct pro-apoptotic signals between the ER and mitochondria. Adenoviral expression of p20 caused an early release of Ca2+ from the ER, concomitant uptake of Ca2+ into mitochondria, and mitochondrial recruitment of Drp1, a dynamin-related protein that mediates scission of the outer mitochondrial membrane, resulting in dramatic fragmentation and fission of the mitochondrial network. Inhibition of Drp1 or ER-mitochondrial Ca2+ signaling prevented p20-induced fission of mitochondria. p20 strongly sensitized mitochondria to caspase-8-induced cyt.c release, whereas prolonged expression of p20 on its own ultimately induced caspase activation and apoptosis through the mitochondrial apoptosome stress pathway. Therefore, caspase-8 cleavage of BAP31 at the ER stimulates Ca2+-dependent mitochondrial fission, enhancing the release of cyt.c in response to this initiator caspase.

Show MeSH
Related in: MedlinePlus