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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 the apoptosis-regulating proteins Bcl-xl, Bax and Bid upon lysosomal lysis. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells upon NDI treatment (at time point 0 min). The thick black trace shows the average behavior with the 95% confidence interval shown in the gray area. (b) Representative FRET ratio images of the constructs during NDI treatment. Lower ratios (cooler colors) indicate the cleavage of the sensors. Scale bar, 10 μm.
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fig1: Cleavage of the apoptosis-regulating proteins Bcl-xl, Bax and Bid upon lysosomal lysis. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells upon NDI treatment (at time point 0 min). The thick black trace shows the average behavior with the 95% confidence interval shown in the gray area. (b) Representative FRET ratio images of the constructs during NDI treatment. Lower ratios (cooler colors) indicate the cleavage of the sensors. Scale bar, 10 μm.

Mentions: All three biosensor constructs showed inverse sigmoidal FRET time profiles upon addition of NDI (Figure 1). Within each biosensor class, individual cells exhibit variable delays to the onset of cleavage, which then progresses rapidly. Cleavage of the Bcl-xl sensor in the different cells occurs in a relatively narrow time window between ~30 and 60 min after addition of NDI. Bax cleavage behavior is more heterogeneous, with a broadened time window, between ~30 and 90 min. The Bid sensor shows a strikingly different behavior, in which cells show a pronounced and very rapid cleavage in the first minutes upon addition of NDI, followed by a second cleavage response that, on average, is similar to that observed for Bax. For the comparison of the global behaviors of the three biosensors, we plotted the duration of cleavage against the time of maximal activity for each cell. These values are obtained by fitting the cell FRET responses with a Gauss error function (Supplementary Figure S2A), in which these parameters are represented by the standard deviation and average, respectively (Supplementary Figure S2B). This analysis shows that Bax degradation takes place at the latest times but its cleavage lasts shorter than for the other constructs. Bid cleavage (in the second wave) shows the largest variability in duration. Bcl-xl cleavage, though on average starting before the other constructs, is slower in comparison.


Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes
Cleavage of the apoptosis-regulating proteins Bcl-xl, Bax and Bid upon lysosomal lysis. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells upon NDI treatment (at time point 0 min). The thick black trace shows the average behavior with the 95% confidence interval shown in the gray area. (b) Representative FRET ratio images of the constructs during NDI treatment. Lower ratios (cooler colors) indicate the cleavage of the sensors. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Cleavage of the apoptosis-regulating proteins Bcl-xl, Bax and Bid upon lysosomal lysis. (a) Temporal response of the degradation of Bcl-xl (left), Bax (middle) and Bid (right) monitored by FRET in individual cells upon NDI treatment (at time point 0 min). The thick black trace shows the average behavior with the 95% confidence interval shown in the gray area. (b) Representative FRET ratio images of the constructs during NDI treatment. Lower ratios (cooler colors) indicate the cleavage of the sensors. Scale bar, 10 μm.
Mentions: All three biosensor constructs showed inverse sigmoidal FRET time profiles upon addition of NDI (Figure 1). Within each biosensor class, individual cells exhibit variable delays to the onset of cleavage, which then progresses rapidly. Cleavage of the Bcl-xl sensor in the different cells occurs in a relatively narrow time window between ~30 and 60 min after addition of NDI. Bax cleavage behavior is more heterogeneous, with a broadened time window, between ~30 and 90 min. The Bid sensor shows a strikingly different behavior, in which cells show a pronounced and very rapid cleavage in the first minutes upon addition of NDI, followed by a second cleavage response that, on average, is similar to that observed for Bax. For the comparison of the global behaviors of the three biosensors, we plotted the duration of cleavage against the time of maximal activity for each cell. These values are obtained by fitting the cell FRET responses with a Gauss error function (Supplementary Figure S2A), in which these parameters are represented by the standard deviation and average, respectively (Supplementary Figure S2B). This analysis shows that Bax degradation takes place at the latest times but its cleavage lasts shorter than for the other constructs. Bid cleavage (in the second wave) shows the largest variability in duration. Bcl-xl cleavage, though on average starting before the other constructs, is slower in comparison.

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