Cell death can take many shapes. Programmed ‘apoptotic' and uncontrolled ‘necrotic'
cell death mark the extremes of the spectrum of possibilities.
1
Whereas the induction of apoptosis follows distinct signaling steps and cleanly removes
a cell from its tissue, necrosis represents the cell's wholesale disintegration and
is in general harmful to the organism. Massive cellular insults lead to necrosis.
It appears, however, that cells have some choice in the mode of their demise depending
on the severity of the insult. Hypoxic cores in solid tumors
2
or perfusion-impaired brain tissue in stroke,
3
for instance are typically necrotic, but possess an apoptotic shell.
The same can be seen for lysosomal damage.
4, 5, 6
Lysosomes can rupture during ischemic or traumatic cell injury,
7
giving rise to both apoptotic and necrotic outcomes.
Lysosomes are cellular organelles with a proteolytic function and possess an impressive
set of protease enzymes
8
dedicated to the degradation of both intracellular material, such as damaged or old
organelles, and extracellular components such as matrix proteins. The release of the
lysosomal proteases into the cytosol sentences the cell to death by autodigestion.
In a recent Cell Death Discovery article,
9
we looked at the fate of the major regulators of apoptosis, the bcl-2 protein family,
in order to understand how cell fate upon lysosomal rupture is directed toward necrosis
or apoptosis. This protein family contains both pro- and anti-apoptotic members and
has been shown to be part of the proteolytic spectrum of cathepsin lysosomal proteases.
10
We reasoned that a regulated degradation of these proteins, by affecting the equilibrium
of pro- and anti-apoptotic signaling, might offer a means for switching between cell
death forms.
To this end, we imaged the proteolytic degradation of the major bcl-2 family representatives
bcl-xl, bid and bax upon induced lysosomal disruption in real time using förster resonance
energy transfer (FRET) microscopy of single cells. We created proteolytic FRET sensors
by sandwiching full-length bcl-2 proteins between cyan and yellow fluorescent proteins.
A strong FRET signal is detected in the intact sensor, which is lost upon cleavage
(Figure 1, left).
The bcl-2 sensors show a common sigmoidal ‘delay-snap' cleavage behavior: after a
delay of tens of minutes, cleavage occurs rapidly and is completed within minutes.
Among the observed bcl-2 members, anti-apoptotic bcl-xl is degraded before pro-apoptotic
bid and bax. Cells thus appear to accumulate pro-apoptotic reactants in the early
stages of lysosomal rupture.
The pro-apoptotic bid showed a conspicuous behavior. Before the overall sigmoidal
degradation, a pronounced and very rapid cleavage was observed in the first minutes
of lysosomal lysis. Fast proteolytic processing of bid is used in apoptosis and might
serve the same role in lysosomal lysis. In apoptosis, the caspase 8 protease activates
bid by its truncation, directly connecting the extrinsic and intrinsic apoptotic signaling
pathways. This mechanism is called the ‘bid shunt' and processing of bid by cathepsin
B has been suggested to operate similarly.
11
Our work places the bid shunt very early in lysosomal cell death signaling and this
is the first time this event is caught ‘on film'.
In order to gain a better understanding of the regulation of bcl-2 protein levels
by cathepsins and the involvement in programmatic steps in necrosis and apoptosis,
we investigated the interplay of both systems in more detail.
Given the relatively low amount of lysosomes that were disrupted in the early stages
after lysosomal disruption, we selected the neutral-active cathepsin B as likely candidate
for most of the early proteolytic actions. Hence, we repeated the experiments in the
presence of a selective cathepsin B inhibitor and an inhibitor for the thiol-cathepsin
class, to which cathepsin B belongs. Inhibition of the thiol-cathepsins confirmed
their predominant role in the degradation of bcl-2 family proteins, as the proteolysis
of all three bcl-2 constructs was inhibited. Selective cathepsin B inhibition helped
uncover its role in steering cell death. We found that early bid truncation was abolished,
confirming cathepsin B as the responsible protease. Furthermore, when we limited lysosomal
protease action to the time window of the early bid truncation, we detected late-apoptotic
caspase 3/7 activation, showing that cells had chosen an apoptotic exit.
Unexpectedly and paradoxically, bax and also bid exhibited a new accelerated proteolytic
behavior upon selective inhibition of cathepsin B. The delay phase before the onset
of cleavage was completely abolished, in particular for bax. As a general inhibition
of the thiol-cathepsin classes resulted in the complete inhibition of the proteolysis
of these same sensors, we concluded that their accelerated degradation upon cathepsin
B inhibition is most likely caused by another, unidentified, thiol-cathepsin. This
proteolytic cascade prevents the degradation of apoptosis-supporting proteins, shifting
the balance toward apotosis. The identity of this cathepsin requires further research.
Cells thus appear to be able to pull an ‘emergency brake' upon lethal injury with
damaging of lysosomes. Starting with cathepsin B, an early programmatic protease cascade
is initiated that appears to avoid necrosis in favor of apoptosis. In this apoptotic
exit effort, cathepsin B preferentially degrades anti-apoptotic bcl-xl, activates
bid by its rapid and selective truncation, and degrades a thiol-cathepsin to avoid
the removal of apoptotic bid and bax (Figure 1, right). Up to a certain level of lysosomal
damage, this three-pronged rescue program steers dying cells away from necrosis. When
massive damage overwhelms the cell with thiol-cathepsins that eat away at essential
cellular proteins, necrosis becomes unavoidable. Our research contributes to the understanding
of cell death on a molecular mechanistic level by uncovering programmatic steps at
the interface between necrosis and apoptosis. Knowledge on cell death decision signaling
is therapeutically relevant, for example, in the formulation of more effective chemotherapy
and in combatting toxic drug side effects.