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

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.

Figure 1

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.

Figure 2

Early, short-lasting cystein cathepsin activation induces apoptosis. The occurrence of apoptosis is monitored by a FRET sensor for active caspase 3/7. (a) Treatment with NDI does not induce apoptosis. (b) Treatment with NDI, followed by the addition of cystein cathepsin inhibitor E64d after 5 min induces caspase 3/7 activation. (c) Apoptosis induced by incubation with trimeric Fas ligand. Lower ratios (cooler colors) indicate sensor cleavage. Scale bar, 10 μm.

Figure 3

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.

Figure 4

Cleavage of Bcl-xl, Bax and Bid upon lysosomal lysis in the presence of the CTSB-specific inhibitor CA-074Me. (a) Temporal degradation response 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 CTSB-specific inhibitor CA-074Me. Color coding of average cellular traces are as in Figure 3 for inhibited (blue) and delay-cleavage (black) behaviors. The new, red traces indicate a population that exhibit cleavage without prior delay. Notably, for Bax, this is the dominant behavior. (b) representative FRET ratio-time series of cells in the new, no delay-cleavage (red) class. FRET ratios are shown in false color. Lower ratios (cooler colors) indicate cleavage. Scale bar, 10 μm.

Figure 5

Model of the programmatic regulation of cell death during lysosomal lysis. The observed results with the degradation of the three studied bcl-2-family apoptosis-regulating proteins are summarized in their relation and in time. Numbers refer to the three actions in the rescue program as used in the discussion. CTSth refers to the Bax-degrading thiol cathepsin that is degraded by CTSB.