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Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha
L P Deiss1, H Galinka, H Berissi
1Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot 76100, Israel.
Abstract:
A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon-gamma- and Fas/APO-1-induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF-alpha-induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady-state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well-known endoprotease plays an active role in cytokine-induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.
Insights
Cathepsin D, an aspartic protease, acts as a mediator in programmed cell death. Its inhibition or reduced expression protects cells from apoptosis, while its overexpression induces cell death.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Programmed cell death (apoptosis) is a crucial biological process.
- Identifying mediators of apoptosis is essential for understanding cellular regulation and disease.
- Previous research has implicated various proteases in apoptotic pathways.
Purpose of the Study:
- To identify positive mediators of programmed cell death using a functional gene cloning approach.
- To investigate the role of cathepsin D in cytokine-induced apoptosis.
Main Methods:
- Functional gene cloning via antisense cDNA expression libraries in HeLa cells.
- Selection of surviving cells under apoptotic stimuli.
- Analysis of cathepsin D expression and activity.
- Inhibition studies using Pepstatin A.
- Ectopic expression of cathepsin D.
Main Results:
- A cathepsin D antisense cDNA fragment was identified as a mediator protecting cells from apoptosis.
- High cathepsin D antisense RNA levels conferred resistance to interferon-gamma and Fas/APO-1 induced death.
- Pepstatin A inhibited cathepsin D and suppressed cell death, including TNF-alpha-induced apoptosis in U937 cells.
- Cathepsin D expression and processing were altered during cell death, leading to an active single-chain form.
- Overexpression of cathepsin D induced cell death independently of external stimuli.
Conclusions:
- Cathepsin D functions as a positive mediator in cytokine-induced programmed cell death.
- This study adds cathepsin D to the list of proteases involved in apoptosis.
- The findings highlight cathepsin D's active role in regulating cell death pathways.