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Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes

View Article: PubMed Central - PubMed

ABSTRACT

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B’s proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

No MeSH data available.


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Cleavage of Bcl-xl, Bax and Bid upon lysosomal lysis in the presence of the cystein cathepsin inhibitor E64d. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells (gray traces) upon NDI treatment in the presence of the cystein cathepsin inhibitor E64d. The average cellular traces are color coded to indicate different behavioral classes: blue traces do not show cleavage, black traces show delayed cleavage. (b) Representative FRET ratio–time series of cells in the dominant, inhibited cleavage (blue trace) class. FRET ratios are shown in false color. Lower ratios (cooler colors) indicate cleavage. Scale bar, 10 μm.
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fig3: Cleavage of Bcl-xl, Bax and Bid upon lysosomal lysis in the presence of the cystein cathepsin inhibitor E64d. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells (gray traces) upon NDI treatment in the presence of the cystein cathepsin inhibitor E64d. The average cellular traces are color coded to indicate different behavioral classes: blue traces do not show cleavage, black traces show delayed cleavage. (b) Representative FRET ratio–time series of cells in the dominant, inhibited cleavage (blue trace) class. FRET ratios are shown in false color. Lower ratios (cooler colors) indicate cleavage. Scale bar, 10 μm.

Mentions: The previous experiments suggest that cells undergoing lysosomal rupture initially attempt to mount an apoptotic response. Nevertheless, in the advancing course of lysosomal disruption to completion, this more favorable response could become overwhelmed by the action of ceaselessly released lysosomal proteases, instead resulting in uncontrolled necrotic cell death. Incubation of the cells expressing the different Bcl-2 family member FRET constructs with E64d either prevented their degradation or expanded the survival time during NDI treatment (Figure 3). The Bcl-xl and Bid sensor show a complete inhibition. In the case of Bid, both the first and the second delayed response were affected. For the Bax construct, the majority of cells (70%) still showed residual degradation, but the onset of their cleavage was delayed and only occurs after about 80 min. The degradation responses observed for Bid, Bax and Bcl-xl are thus likely attributable to lysosomal thiol cathepsins, thereby excluding a significant role of the abundant aspartyl cathepsin D.


Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes
Cleavage of Bcl-xl, Bax and Bid upon lysosomal lysis in the presence of the cystein cathepsin inhibitor E64d. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells (gray traces) upon NDI treatment in the presence of the cystein cathepsin inhibitor E64d. The average cellular traces are color coded to indicate different behavioral classes: blue traces do not show cleavage, black traces show delayed cleavage. (b) Representative FRET ratio–time series of cells in the dominant, inhibited cleavage (blue trace) class. FRET ratios are shown in false color. Lower ratios (cooler colors) indicate cleavage. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979493&req=5

fig3: Cleavage of Bcl-xl, Bax and Bid upon lysosomal lysis in the presence of the cystein cathepsin inhibitor E64d. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells (gray traces) upon NDI treatment in the presence of the cystein cathepsin inhibitor E64d. The average cellular traces are color coded to indicate different behavioral classes: blue traces do not show cleavage, black traces show delayed cleavage. (b) Representative FRET ratio–time series of cells in the dominant, inhibited cleavage (blue trace) class. FRET ratios are shown in false color. Lower ratios (cooler colors) indicate cleavage. Scale bar, 10 μm.
Mentions: The previous experiments suggest that cells undergoing lysosomal rupture initially attempt to mount an apoptotic response. Nevertheless, in the advancing course of lysosomal disruption to completion, this more favorable response could become overwhelmed by the action of ceaselessly released lysosomal proteases, instead resulting in uncontrolled necrotic cell death. Incubation of the cells expressing the different Bcl-2 family member FRET constructs with E64d either prevented their degradation or expanded the survival time during NDI treatment (Figure 3). The Bcl-xl and Bid sensor show a complete inhibition. In the case of Bid, both the first and the second delayed response were affected. For the Bax construct, the majority of cells (70%) still showed residual degradation, but the onset of their cleavage was delayed and only occurs after about 80 min. The degradation responses observed for Bid, Bax and Bcl-xl are thus likely attributable to lysosomal thiol cathepsins, thereby excluding a significant role of the abundant aspartyl cathepsin D.

View Article: PubMed Central - PubMed

ABSTRACT

The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B’s proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis.

No MeSH data available.


Related in: MedlinePlus