Limits...
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.

Show MeSH
(A) MIHA and Bcl-2 inhibit caspase 9 and caspase 3 processing in response to UV radiation but not serum withdrawal, and MIHA binds to endogenous, processed caspase 3. NT 2 cells stably transfected with MIHA (M) or LacZ (C) were exposed to 100 J/m2 UV radiation and lysates were harvested at 6 h. Kinetics of caspase processing was followed using Western blot immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Full-length caspase 9 and the cleaved 37-kD fragment of caspase 9, full-length caspase 3, and the 20- and 17-kD large subunits are indicated. Almost complete processing of caspase 9 was observed in control cells, and this was inhibited by the stable expression of MIHA. Caspase 3 processing was also inhibited by MIHA, since most caspase 3 in the MIHA line remained in the unprocessed form (lane 7) compared with control cells (lane 8). (B) NT2 cells stably transfected with MIHA (M) or LacZ (C) were grown under serum-free conditions and lysates were harvested and immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Caspase 3 cleavage occurred in both MIHA- and LacZ-expressing cells. Over the time course, most caspase 9 remained in the full-length rather than cleaved form. (C) NT2 cells stably expressing MIHA, LacZ, or Bcl-2 were exposed to 100 J/m2 UV radiation and the lysates were harvested 24 h later and immunoblotted with anti-caspase 9 and anti-caspase 3 antibodies. MIHA lines inhibited caspase 9 and caspase 3 cleavage but not to the same degree as Bcl-2. In all the Western blots, anti-HSP70 immunoblotting was done as loading controls. (D) Flag-tagged MIHA was immunoprecipitated from the lysates of NT2 cells stably expressing MIHA or LacZ that were cultured in serum-containing media (C), or exposed to 100 J/m2 (UV), or were cultured in serum-free media for 24 h (−S), and the immunoprecipitate was probed with anti-caspase 3 and anti-caspase 9 antibodies. Processed endogenous caspase 3 coimmunoprecipitated with MIHA after serum withdrawal and UV radiation. No association with caspase 9 in either its zymogen or processed form was detected.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195997&req=5

Figure 1: (A) MIHA and Bcl-2 inhibit caspase 9 and caspase 3 processing in response to UV radiation but not serum withdrawal, and MIHA binds to endogenous, processed caspase 3. NT 2 cells stably transfected with MIHA (M) or LacZ (C) were exposed to 100 J/m2 UV radiation and lysates were harvested at 6 h. Kinetics of caspase processing was followed using Western blot immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Full-length caspase 9 and the cleaved 37-kD fragment of caspase 9, full-length caspase 3, and the 20- and 17-kD large subunits are indicated. Almost complete processing of caspase 9 was observed in control cells, and this was inhibited by the stable expression of MIHA. Caspase 3 processing was also inhibited by MIHA, since most caspase 3 in the MIHA line remained in the unprocessed form (lane 7) compared with control cells (lane 8). (B) NT2 cells stably transfected with MIHA (M) or LacZ (C) were grown under serum-free conditions and lysates were harvested and immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Caspase 3 cleavage occurred in both MIHA- and LacZ-expressing cells. Over the time course, most caspase 9 remained in the full-length rather than cleaved form. (C) NT2 cells stably expressing MIHA, LacZ, or Bcl-2 were exposed to 100 J/m2 UV radiation and the lysates were harvested 24 h later and immunoblotted with anti-caspase 9 and anti-caspase 3 antibodies. MIHA lines inhibited caspase 9 and caspase 3 cleavage but not to the same degree as Bcl-2. In all the Western blots, anti-HSP70 immunoblotting was done as loading controls. (D) Flag-tagged MIHA was immunoprecipitated from the lysates of NT2 cells stably expressing MIHA or LacZ that were cultured in serum-containing media (C), or exposed to 100 J/m2 (UV), or were cultured in serum-free media for 24 h (−S), and the immunoprecipitate was probed with anti-caspase 3 and anti-caspase 9 antibodies. Processed endogenous caspase 3 coimmunoprecipitated with MIHA after serum withdrawal and UV radiation. No association with caspase 9 in either its zymogen or processed form was detected.

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)

(A) MIHA and Bcl-2 inhibit caspase 9 and caspase 3 processing in response to UV radiation but not serum withdrawal, and MIHA binds to endogenous, processed caspase 3. NT 2 cells stably transfected with MIHA (M) or LacZ (C) were exposed to 100 J/m2 UV radiation and lysates were harvested at 6 h. Kinetics of caspase processing was followed using Western blot immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Full-length caspase 9 and the cleaved 37-kD fragment of caspase 9, full-length caspase 3, and the 20- and 17-kD large subunits are indicated. Almost complete processing of caspase 9 was observed in control cells, and this was inhibited by the stable expression of MIHA. Caspase 3 processing was also inhibited by MIHA, since most caspase 3 in the MIHA line remained in the unprocessed form (lane 7) compared with control cells (lane 8). (B) NT2 cells stably transfected with MIHA (M) or LacZ (C) were grown under serum-free conditions and lysates were harvested and immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Caspase 3 cleavage occurred in both MIHA- and LacZ-expressing cells. Over the time course, most caspase 9 remained in the full-length rather than cleaved form. (C) NT2 cells stably expressing MIHA, LacZ, or Bcl-2 were exposed to 100 J/m2 UV radiation and the lysates were harvested 24 h later and immunoblotted with anti-caspase 9 and anti-caspase 3 antibodies. MIHA lines inhibited caspase 9 and caspase 3 cleavage but not to the same degree as Bcl-2. In all the Western blots, anti-HSP70 immunoblotting was done as loading controls. (D) Flag-tagged MIHA was immunoprecipitated from the lysates of NT2 cells stably expressing MIHA or LacZ that were cultured in serum-containing media (C), or exposed to 100 J/m2 (UV), or were cultured in serum-free media for 24 h (−S), and the immunoprecipitate was probed with anti-caspase 3 and anti-caspase 9 antibodies. Processed endogenous caspase 3 coimmunoprecipitated with MIHA after serum withdrawal and UV radiation. No association with caspase 9 in either its zymogen or processed form was detected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195997&req=5

Figure 1: (A) MIHA and Bcl-2 inhibit caspase 9 and caspase 3 processing in response to UV radiation but not serum withdrawal, and MIHA binds to endogenous, processed caspase 3. NT 2 cells stably transfected with MIHA (M) or LacZ (C) were exposed to 100 J/m2 UV radiation and lysates were harvested at 6 h. Kinetics of caspase processing was followed using Western blot immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Full-length caspase 9 and the cleaved 37-kD fragment of caspase 9, full-length caspase 3, and the 20- and 17-kD large subunits are indicated. Almost complete processing of caspase 9 was observed in control cells, and this was inhibited by the stable expression of MIHA. Caspase 3 processing was also inhibited by MIHA, since most caspase 3 in the MIHA line remained in the unprocessed form (lane 7) compared with control cells (lane 8). (B) NT2 cells stably transfected with MIHA (M) or LacZ (C) were grown under serum-free conditions and lysates were harvested and immunoblotted with anti-caspase 3 and anti-caspase 9 antibodies. Caspase 3 cleavage occurred in both MIHA- and LacZ-expressing cells. Over the time course, most caspase 9 remained in the full-length rather than cleaved form. (C) NT2 cells stably expressing MIHA, LacZ, or Bcl-2 were exposed to 100 J/m2 UV radiation and the lysates were harvested 24 h later and immunoblotted with anti-caspase 9 and anti-caspase 3 antibodies. MIHA lines inhibited caspase 9 and caspase 3 cleavage but not to the same degree as Bcl-2. In all the Western blots, anti-HSP70 immunoblotting was done as loading controls. (D) Flag-tagged MIHA was immunoprecipitated from the lysates of NT2 cells stably expressing MIHA or LacZ that were cultured in serum-containing media (C), or exposed to 100 J/m2 (UV), or were cultured in serum-free media for 24 h (−S), and the immunoprecipitate was probed with anti-caspase 3 and anti-caspase 9 antibodies. Processed endogenous caspase 3 coimmunoprecipitated with MIHA after serum withdrawal and UV radiation. No association with caspase 9 in either its zymogen or processed form was detected.
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.

Show MeSH