Related Experiment Videos
A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis
1Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. seold+@pitt.edu
Abstract:
It is likely that endogenous inhibitors of the apical caspases such as caspase-9 exist to prevent undesirable activation of caspase cascades. A naturally occurring variant of caspase-9 named caspase-9S was cloned from human liver. Caspase-9S is missing most of the large subunit of caspase-9, including the catalytic site, but has the intact prodomain and small subunit. Caspase-9S did not show apoptotic activity in transfection analysis. Overexpression of caspase-9S inhibited apoptosis induced by caspase-9, indicating that caspase-9S is an endogenous dominant-negative of caspase-9. Moreover, caspase-9S inhibited apoptosis induced by tumor necrosis factor(TNF)-alpha, TNF factor-related apoptosis-inducing ligand (TRAIL), Bax, or Fas-associated death domain-containing protein (FADD) as well as the combination of Apaf-1 and caspase-9. In vitro binding assays demonstrated that caspase-9S binds to Apaf-1 and blocks the binding of caspase-9 to Apaf-1. Coexpression of caspase-9 and caspase-9S mRNA was identified in various cell lines. Thus, caspase-9S acts as a dominant-negative inhibitor of caspase-9 activation, at least in part, by blocking Apaf-1-caspase-9 interaction.
Insights
A newly identified caspase-9S protein acts as a natural inhibitor of apoptosis. This caspase-9S variant blocks the activation of caspase-9, preventing programmed cell death pathways in human cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Endogenous inhibitors of apical caspases, like caspase-9, are crucial for preventing uncontrolled apoptosis.
- Understanding these regulatory mechanisms is key to controlling cell death pathways.
Purpose of the Study:
- To identify and characterize endogenous inhibitors of caspase-9.
- To elucidate the mechanism by which caspase-9S inhibits apoptosis.
Main Methods:
- Cloning of caspase-9S from human liver.
- Transfection analysis to assess apoptotic activity.
- In vitro binding assays to study protein interactions.
- Analysis of mRNA coexpression in cell lines.
Main Results:
- Caspase-9S, a naturally occurring variant, lacks catalytic activity but inhibits caspase-9-induced apoptosis.
- Caspase-9S acts as a dominant-negative regulator, blocking apoptosis induced by various stimuli (TNF-alpha, TRAIL, Bax, FADD).
- Caspase-9S binds to Apaf-1, preventing caspase-9 interaction and subsequent activation.
Conclusions:
- Caspase-9S is an endogenous dominant-negative inhibitor of caspase-9.
- It functions by disrupting the Apaf-1-caspase-9 complex formation.
- This finding provides new insights into the regulation of apoptosis.