Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Abstract

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.

Endoplasmic reticulum stress-induced cell death mediated by the proteasome

“…Phase-contrast microscopy revealed that inhibition of the proteasome with either lactacystin, MG262, or epoxomycin, protected cells from ER stress (BFA/CHX, for 24 h) to the same extent as Pefabloc ( Figure 1a). As was previously shown, we saw that caspase inhibition with either Z-VAD.fmk or Q-Val-Asp-(non-O-methylated)-OPh (Q-VD-Oph) ( Figure 1a and data not shown) did not prevent cells from undergoing ER stressinduced cell death, 11 and CHX treatment alone did not induce cell death (Figure 1a). …”

“…* Po0.001 treated versus untreated Z-VAD.fmk and Q-VD-OPh did not impose a complete block in ER stress-induced cell death (Figures 1 and 2, Ref. 11 and data not shown). This suggests that some fraction of the caspase-3 processing associated with BFA/CHX treatment might only be an epiphenomenon, and is not a key player in this pathway.…”

“…We previously showed in our model of ER stress-induced cell death using the BFA/CHX cocktail that over expression of Bcl-2 was protective, indicating mitochondrial involvement in this pathway. Furthermore, BFA/CHX treatment resulted in cytochrome c release from mitochondria ( Figure 4b, and Ref 11 ). Thus, we asked the question whether the deathinducing activity of the proteasome could affect the outermembrane permeability of the mitochondria.…”

“…Previously, it was shown that the serine protease inhibitor Pefabloc (AEBSF) could block ER stress-induced cell death, whereas the broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), could not, thus implicating the involvement of a serine protease (or other AEBSF-inhibitable activity) in caspase-independent ER stress-induced cell death. 11 In this report, we show that the activity of the proteasome is necessary for ER stress-induced cell death. Proteasomal inhibition blocked ER stress-induced degradation of antiapoptotic Bcl-2 family proteins, cytochrome c release, caspase maturation, phosphatidylserine exposure, and cell death.…”

“…8 We reported that ER stress-induced apoptosis was not prevented by caspase inhibition with Z-VAD.fmk but could be blocked by Pefabloc, a general inhibitor of serine protease activity. 11 We therefore sought to identify the serine protease(s) responsible for cell death observed in our system. We used a mass spectrometry (MS) approach to identify proteins in cytosolic extracts bound to serine protease active site-directed inhibitors.…”