Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the “executioner” caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochromec/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis.

TACE Trafficking: The cytokine tumor necrosis factor (TNF) is a major driver of inflammation and contributes to the immune pathology seen in a variety of diseases, including inflammatory bowel disease, rheumatoid arthritis, and sepsis. Soluble TNF is produced by cleavage of its ectodomain by the ADAM family metalloprotease, TNFα-converting enzyme (TACE). However, the molecular regulation of TACE is not understood (see the Perspective by Lichtenthaler ). Adrain et al. (p. 225 ) and McIlwain et al. (p. 229 ) now show that the rhomboid family member iRhom2 interacts with TACE in macrophages and is required for its proper intracellular trafficking and activation. In the absence of iRhom2, TACE was not released from the endoplasmic reticulum, and active protease did not reach the cell surface. Because of an inability to produce TNF, iRhom2-deficient mice were more resistant to lipopolysaccharide-induced septic shock but could not adequately control a Listeria monocytogenes infection.

Smac/DIABLO is a mitochondrial protein that potentiates some forms of apoptosis, possibly by neutralizing one or more members of the IAP family of apoptosis inhibitory proteins. Smac has been shown to exit mitochondria and enter the cytosol during apoptosis triggered by UV‐ or γ‐irradiation. Here, we report that Smac/DIABLO export from mitochondria into the cytosol is provoked by cytotoxic drugs and DNA damage, as well as by ligation of the CD95 death receptor. Mitochondrial efflux of Smac/DIABLO, in response to a variety of pro‐apoptotic agents, was profoundly inhibited in Bcl‐2‐overexpressing cells. Thus, in addition to modulating apoptosis‐associated mitochondrial cytochrome c release, Bcl‐2 also regulates Smac release, suggesting that both molecules may escape via the same route. However, whereas cell stress‐associated mitochondrial cytochrome c release was largely caspase independent, release of Smac/DIABLO in response to the same stimuli was blocked by a broad‐spectrum caspase inhibitor. This suggests that apoptosis‐associated cytochrome c and Smac/DIABLO release from mitochondria do not occur via the same mechanism. Rather, Smac/DIABLO efflux from mitochondria is a caspase‐catalysed event that occurs downstream of cytochrome c release.

The Apaf‐1 apoptosome is a multi‐subunit caspase‐activating scaffold that is assembled in response to diverse forms of cellular stress that culminate in apoptosis. To date, most studies on apoptosome composition and function have used apoptosomes reassembled from recombinant or purified proteins. Thus, the precise composition of native apoptosomes remains unresolved. Here, we have used a one‐step immunopurification approach to isolate catalytically active Apaf‐1/caspase‐9 apoptosomes, and have identified the major constituents of these complexes using mass spectrometry methods. Using this approach, we have also assessed the ability of putative apoptosome regulatory proteins, such as Smac/DIABLO and PHAPI, to regulate the activity of native apoptosomes. We show that Apaf‐1, caspase‐9, caspase‐3 and XIAP are the major constituents of native apoptosomes and that cytochrome c is not stably associated with the active complex. We also demonstrate that the IAP‐neutralizing protein Smac/DIABLO and the tumor‐suppressor protein PHAPI can enhance the catalytic activity of apoptosome complexes in distinct ways. Surprisingly, PHAPI also enhanced the activity of purified caspase‐3, suggesting that it may act as a co‐factor for this protease.