Bax cleavage is mediated by calpain during drug-induced apoptosis

Abstract

The anti-apoptotic molecule Bcl-2 is located in the mitochondrial and endoplasmic reticulum membranes as well as the nuclear envelope. Although its location has not been as rigorously de®ned, the pro-apoptotic molecule Bax appears to be mainly a cytosolic protein which translocates to the mitochondria upon induction of apoptosis. Here we identify a protease activity in mitochondria-enriched membrane fractions from HL-60 cells capable of cleaving Bax which is absent from the cytosolic fraction. Bax protease activity is blocked in vitro by cysteine protease inhibitors including E-64 which distinguishes it from all known caspases and granzyme B, both of which are involved in apoptosis. Protease activity is also blocked by inhibitors against the calciumactivated neutral cysteine endopeptidase calpain. Partial puri®cation of the Bax protease activity from HL-60 cell membrane fractions by column chromatography revealed that a calpain-like activity was the protease responsible for Bax cleavage. In addition, puri®ed calpain enzymes cleaved Bax in a calcium-dependent manner. Pretreatment of HL-60 cells with the speci®c calpain inhibitor calpeptin eectively blocked both drug-induced Bax cleavage and calpain activation, but not PARP cleavage or cell death. These results suggest that calpains and caspases are activated during drug-induced apoptosis and that calpains, along with caspases, may be involved in modulating cell death by acting selectively on cellular substrates.

Early mitochondrial alterations in ATRA-induced cell death

“…It has been shown that Bax can be cleaved either by caspases or calpain. 30,31 However, under the conditions of our experiments, neither the pancaspase inhibitor z-VAD-fmk nor different calpain inhibitors such as E64d, PD150606 and calpeptin were able to prevent cleavage of Bax (Figure 3b). …”

BCL-2 in the crosshairs: tipping the balance of life and death

“…43 The amino terminus of BAX has been implicated in BH3 ligand binding, 59 intracellular localization, 89,90 and negative regulation. 91,92 Calpain-mediated cleavage of BAX just prior to its relatively short unstructured loop generates a truncated form with enhanced apoptogenic activity, [93][94][95] which may reflect its functional conversion to a BH3-only-type protein. 96 Multidomain pro-and antiapoptotic proteins contain Cterminal transmembrane domains that insert into the mitochondrial outer membrane.…”

Multiple death pathways in retina-derived 661W cells following growth factor deprivation: crosstalk between caspases and calpains

“…18,19 At the same time, calpains directly proteolyse executioner caspases, mediating their activation, 20,21 as well as proapoptotic Bcl-2 family members that eventually cause the release of apoptogenic factors from the mitochondria involved in the triggering of the caspase cascade. [22][23][24] While the time course of developmental apoptosis in the mouse retina has been extensively described in several studies, 10,[25][26][27] the biochemical death pathways involved are not fully understood. In particular, relatively little is known about the molecular mechanisms of developmental apoptosis in cone photoreceptors, due to the paucity of cone-specific markers at early stages of development.…”