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DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9.

Ekert PG, Silke J, Hawkins CJ, Verhagen AM, Vaux DL - J. Cell Biol. (2001)

Bottom Line: MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis.DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID.Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute, The Royal Melbourne Hospital, Victoria 3050, Australia.

ABSTRACT
MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

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MIHA acts downstream of mitochondrial cytochrome c and DIABLO release. NT2 cells stably transfected with LacZ, Bcl-2, or MIHA were exposed to UV radiation for the indicated times. The cells were harvested and membrane and cytosolic fractions were separated. The two fractions were then immunoblotted with anti-caspase 3, anti-caspase 9, anti-cytochrome c and anti-DIABLO antibodies. Control (LacZ) cells show translocation of cytochrome c and DIABLO to the cytosolic fraction and cleavage of cytosolic caspase 3 and caspase 9 (lanes 1–6). All these processes were inhibited in cells expressing Bcl-2 (lanes 7–12). MIHA inhibited processing of caspase 9 and caspase 3, but cytochrome c and DIABLO translocated from membrane to cytosolic fractions to the same degree as in control cells (lanes 13–18).
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Figure 4: MIHA acts downstream of mitochondrial cytochrome c and DIABLO release. NT2 cells stably transfected with LacZ, Bcl-2, or MIHA were exposed to UV radiation for the indicated times. The cells were harvested and membrane and cytosolic fractions were separated. The two fractions were then immunoblotted with anti-caspase 3, anti-caspase 9, anti-cytochrome c and anti-DIABLO antibodies. Control (LacZ) cells show translocation of cytochrome c and DIABLO to the cytosolic fraction and cleavage of cytosolic caspase 3 and caspase 9 (lanes 1–6). All these processes were inhibited in cells expressing Bcl-2 (lanes 7–12). MIHA inhibited processing of caspase 9 and caspase 3, but cytochrome c and DIABLO translocated from membrane to cytosolic fractions to the same degree as in control cells (lanes 13–18).

Mentions: The primary antibodies used were: anti-Flag M2 (Sigma-Aldrich), anti-caspase 3 (rabbit polyclonal; PharMingen), anti-caspase 9 (rabbit polyclonal; BD PharMingen) (designated A in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5), anti-caspase 9 (rabbit polyclonal; New England Biolabs, Inc.) (designated B in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5), anti-Smac (rabbit polyclonal; a kind gift from X. Wang, The Howard Hughes Medical Institute, University of Texas, Dallas, TX), anti-CARD caspase 9 (mouse monoclonal; a kind gift from Y. Lazebnik, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), anti-cytochrome c (mouse monoclonal; BD PharMingen), and anti-XIAP (mouse monoclonal; MBL [Abacus]). The secondary antibodies used were: goat anti–rabbit-HRP (Southern Biotechnology Associates, Inc.) and goat anti–mouse-HRP (Southern Biotechnology Associates, Inc.). ECL Western blotting reagents (Amersham Pharmacia Biotech) were used to detect antibody signal. All autoradiographs were scanned into Adobe Photoshop®, and the figures were made using Adobe Freehand v9.


DIABLO promotes apoptosis by removing MIHA/XIAP from processed caspase 9.

Ekert PG, Silke J, Hawkins CJ, Verhagen AM, Vaux DL - J. Cell Biol. (2001)

MIHA acts downstream of mitochondrial cytochrome c and DIABLO release. NT2 cells stably transfected with LacZ, Bcl-2, or MIHA were exposed to UV radiation for the indicated times. The cells were harvested and membrane and cytosolic fractions were separated. The two fractions were then immunoblotted with anti-caspase 3, anti-caspase 9, anti-cytochrome c and anti-DIABLO antibodies. Control (LacZ) cells show translocation of cytochrome c and DIABLO to the cytosolic fraction and cleavage of cytosolic caspase 3 and caspase 9 (lanes 1–6). All these processes were inhibited in cells expressing Bcl-2 (lanes 7–12). MIHA inhibited processing of caspase 9 and caspase 3, but cytochrome c and DIABLO translocated from membrane to cytosolic fractions to the same degree as in control cells (lanes 13–18).
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Figure 4: MIHA acts downstream of mitochondrial cytochrome c and DIABLO release. NT2 cells stably transfected with LacZ, Bcl-2, or MIHA were exposed to UV radiation for the indicated times. The cells were harvested and membrane and cytosolic fractions were separated. The two fractions were then immunoblotted with anti-caspase 3, anti-caspase 9, anti-cytochrome c and anti-DIABLO antibodies. Control (LacZ) cells show translocation of cytochrome c and DIABLO to the cytosolic fraction and cleavage of cytosolic caspase 3 and caspase 9 (lanes 1–6). All these processes were inhibited in cells expressing Bcl-2 (lanes 7–12). MIHA inhibited processing of caspase 9 and caspase 3, but cytochrome c and DIABLO translocated from membrane to cytosolic fractions to the same degree as in control cells (lanes 13–18).
Mentions: The primary antibodies used were: anti-Flag M2 (Sigma-Aldrich), anti-caspase 3 (rabbit polyclonal; PharMingen), anti-caspase 9 (rabbit polyclonal; BD PharMingen) (designated A in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5), anti-caspase 9 (rabbit polyclonal; New England Biolabs, Inc.) (designated B in Fig. 1Fig. 2Fig. 3Fig. 4Fig. 5), anti-Smac (rabbit polyclonal; a kind gift from X. Wang, The Howard Hughes Medical Institute, University of Texas, Dallas, TX), anti-CARD caspase 9 (mouse monoclonal; a kind gift from Y. Lazebnik, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), anti-cytochrome c (mouse monoclonal; BD PharMingen), and anti-XIAP (mouse monoclonal; MBL [Abacus]). The secondary antibodies used were: goat anti–rabbit-HRP (Southern Biotechnology Associates, Inc.) and goat anti–mouse-HRP (Southern Biotechnology Associates, Inc.). ECL Western blotting reagents (Amersham Pharmacia Biotech) were used to detect antibody signal. All autoradiographs were scanned into Adobe Photoshop®, and the figures were made using Adobe Freehand v9.

Bottom Line: MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis.DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID.Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

View Article: PubMed Central - PubMed

Affiliation: The Walter and Eliza Hall Institute, The Royal Melbourne Hospital, Victoria 3050, Australia.

ABSTRACT
MIHA is an inhibitor of apoptosis protein (IAP) that can inhibit cell death by direct interaction with caspases, the effector proteases of apoptosis. DIABLO is a mammalian protein that can bind to IAPs and antagonize their antiapoptotic effect, a function analogous to that of the proapoptotic Drosophila molecules, Grim, Reaper, and HID. Here, we show that after UV radiation, MIHA prevented apoptosis by inhibiting caspase 9 and caspase 3 activation. Unlike Bcl-2, MIHA functioned after release of cytochrome c and DIABLO from the mitochondria and was able to bind to both processed caspase 9 and processed caspase 3 to prevent feedback activation of their zymogen forms. Once released into the cytosol, DIABLO bound to MIHA and disrupted its association with processed caspase 9, thereby allowing caspase 9 to activate caspase 3, resulting in apoptosis.

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