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Pre-processed caspase-9 contained in mitochondria participates in apoptosis
P Costantini1, J-M Bruey, M Castedo
1Centre National de la Recherche Scientifique, UMR1599, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, France.
Abstract:
As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.
Insights
Mitochondria contain pre-processed caspase-9 in non-apoptotic cells, a finding observed across various organs. This mitochondrial caspase-9 is released upon cell permeability changes, impacting apoptosis regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitochondria play a crucial role in apoptosis.
- Caspase-9 is a key initiator caspase in the intrinsic apoptosis pathway.
- The localization and processing of caspase-9 are critical for its function.
Purpose of the Study:
- To investigate the presence and processing state of caspase-9 within mitochondria.
- To determine the cellular localization of caspase-9 in different cell types and under various conditions.
- To elucidate the role of Bcl-2 in regulating mitochondrial caspase-9 activity.
Main Methods:
- Mitochondrial purification from various organs (liver, brain, kidney, spleen, heart).
- Induction of mitochondrial permeability transition in vitro.
- Subcellular fractionation and immunofluorescence assays.
- Western blotting using antibodies specific for pro-caspase-9 and processed caspase-9.
Main Results:
- Mitochondria from diverse organs contain both pro-caspase-9 and mature caspase-9.
- Liver mitochondria release mature caspase-9 upon induction of permeability transition.
- SHEP neuroblastoma cells constitutively harbor pre-processed caspase-9 in mitochondria, unlike HeLa cells.
- Apoptosis induction causes mitochondrial pro-caspase-9 translocation to cytosol/nucleus, an event inhibited by Bcl-2.
Conclusions:
- Mitochondria can store pre-processed caspase-9 in non-apoptotic cells.
- Bcl-2 regulates apoptosis by preventing mitochondrial caspase-9 interaction with cytosolic activators via membrane permeabilization control.
- This finding reveals a novel mechanism for apoptosis regulation involving mitochondrial caspase localization and processing.