Abstract
Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ∼50 kD to ∼38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or coelution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3343-3354 |
| Number of pages | 12 |
| Journal | Frontiers in Bioscience |
| Volume | 12 |
| Issue number | 9 |
| DOIs | |
| State | Published - May 1 2007 |
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Keywords
- Apaf-1
- Apoptosis
- Apoptosome
- Caspase-3
- Caspase-9
- Cytochrome c
- Inhibitor
- Research article
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Immunology and Microbiology(all)
- Medicine(all)
Cite this
Regulation of caspase-9 activity by differential binding to the apoptosome complex. / Saikumar, Pothana; Mikhailova, Margarita; Pandeswara, Srilakshmi.
In: Frontiers in Bioscience, Vol. 12, No. 9, 01.05.2007, p. 3343-3354.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Regulation of caspase-9 activity by differential binding to the apoptosome complex
AU - Saikumar, Pothana
AU - Mikhailova, Margarita
AU - Pandeswara, Srilakshmi
PY - 2007/5/1
Y1 - 2007/5/1
N2 - Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ∼50 kD to ∼38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or coelution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.
AB - Proteolytic processing of procaspase-9 is required for its activation, but processing in itself appears to be insufficient for its activity. We studied caspase activation in a cell-free system and found that incubation of cytosol from rat kidney proximal tubule cells with Cytochrome c (Cyt c) and dATP results in rapid autocatalytic processing of procaspase-9 from ∼50 kD to ∼38 kD size fragment. Moreover, Cyt c concentration influences the production of alternatively processed forms of caspase-9. At lower Cyt c concentration (0.01-0.05 mg/ml), two fragments of caspase-9 of the size 38 and 40 kD are produced. In contrast, at higher concentrations of Cyt c (>0.1 mg/ml) only 38 kD fragment will prevail. However, our failure to capture processed caspase-9 by affinity labeling or coelution with Apaf-1 suggested that caspase-9 undergoes a conformational change during its enzymatic action on effector caspases, resulting in its release from the apoptosome complex and inactivation. In support of this hypothesis, catalytic inhibitors of caspase-9 prevented its release from the apoptosome complex without affecting its auto-processing and allowed successful capture of active caspase-9 (38 kD) and its complex by affinity labeling. These observations suggest that complex allosteric interactions with the apoptosome complex influence caspase-9 activity and function by controlling not only the induction of its enzymatic activity, but also its rapid termination.
KW - Apaf-1
KW - Apoptosis
KW - Apoptosome
KW - Caspase-3
KW - Caspase-9
KW - Cytochrome c
KW - Inhibitor
KW - Research article
UR - http://www.scopus.com/inward/record.url?scp=34347272654&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34347272654&partnerID=8YFLogxK
U2 - 10.2741/2317
DO - 10.2741/2317
M3 - Article
C2 - 17485304
AN - SCOPUS:34347272654
VL - 12
SP - 3343
EP - 3354
JO - Frontiers in Bioscience - Landmark
JF - Frontiers in Bioscience - Landmark
SN - 1093-9946
IS - 9
ER -