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 cytochrome c/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.

The cytokine tumor necrosis factor (TNF) is the primary trigger of inflammation. Like many extracellular signaling proteins, TNF is synthesized as a transmembrane protein; the active signal is its ectodomain, which is shed from cells after cleavage by an ADAM family metalloprotease, TACE/ADAM17. We report that iRhom2/RHBDF2, a proteolytically inactive member of the rhomboid family, is required for TNF release in mice. iRhom2 binds TACE and promotes exit from the endoplasmic reticulum. The failure of TACE ER exit in the absence of iRhom2 prevents furin-mediated maturation and its trafficking to the cell surface, the site of TNF cleavage. Given the role of TNF in autoimmune and inflammatory diseases, iRhom2 may represent an attractive therapeutic target.Proteolytic release of the extracellular domain of transmembrane proteins is an important mechanism for generating signals that regulate major aspects of animal development, physiology, immunity and pathology (1, 2). An important example of regulated ectodomain shedding is the cytokine TNF, the primary trigger of inflammatory responses. TNF is associated with many human diseases including rheumatoid arthritis, Crohn's disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. Its blockade is licensed as a therapy for a number of conditions, and is being assessed for others (3). Soluble, active TNF is shed from the plasma membrane by the ADAM family metalloprotease TACE (TNFα converting enzyme; also known as ADAM17) (4). Despite the medical importance of TNF and other transmembrane signaling proteins, the regulation of ectodomain shedding remains poorly understood. Both the transmembrane forms of the signaling proteins themselves, and the shedding proteases, are subject to control by posttranslational modification, interaction with specific partners, and regulated intracellular trafficking and compartmentalization (5-9). The relative physiological importance, however, of these different modes of regulation is unclear.We have investigated the regulation of ectodomain shedding by genetic and cellular approaches, both in Drosophila and mammalian cells. This has led to the recent discovery of a new class of polytopic endoplasmic reticulum (ER) proteins, the iRhoms, which are noncatalytic relatives of rhomboid intramembrane proteases (Fig. 1A) (10). Drosophila iRhom regulates epidermal growth factor (EGF) receptor signaling by interacting with EGF family ligands in the ER, shunting them into the ER-associated degradation (ERAD) pathway (11). iRhoms are conserved in all metazoans, and in cell culture assays their mammalian Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts counterparts, iRhom1 and iRhom2, can also promote ERAD of EGF. In mammals, however, their physiological role is unknown. We therefore generated a null mutation in the gene that encodes iRhom2/RHBDF2 in mice (Fig. S1A). iRhom2 −/− mice appeared normal: they were viable and fertile, with no morphological defects. Unlike iRhom1, which is widely expressed, iRhom2 is...

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.

No abstract

Caspases participate in the molecular control of apoptosis in several guises; as triggers of the death machinery, as regulatory elements within it, and ultimately as a subset of the effector elements of the machinery itself. The mammalian caspase family is steadily growing and currently contains 14 members. At present, it is unclear whether all of these proteases participate in apoptosis. Thus, current research in this area is focused upon establishing the repertoire and order of caspase activation events that occur during the signalling and demolition phases of cell death. Evidence is accumulating to suggest that proximal caspase activation events are typically initiated by molecules that promote caspase aggregation. As expected, distal caspase activation events are likely to be controlled by caspases activated earlier in the cascade. However, recent data has cast doubt upon the functional demarcation of caspases into signalling (upstream) and effector (downstream) roles based upon their prodomain lengths. In particular, caspase-3 may perform an important role in propagating the caspase cascade, in addition to its role as an effector caspase within the death programme. Here, we discuss the apoptosis-associated caspase cascade and the hierarchy of caspase activation events within it.

Bcl-2 family proteins play central roles in apoptosis by regulating the release of mitochondrial intermembrane space proteins such as cytochrome c. Deathpromoting Bcl-2 family members, such as Bax, can promote cytochrome c release and fragmentation of the mitochondrial network, whereas apoptosis-inhibitory members, such as Bcl-2 and Bcl-xL, can antagonize these events. It remains unclear whether CED-9, the worm Bcl-2 relative, can regulate mitochondrial fission/fusion dynamics or the release of proteins from the mitochondrial intermembrane space. Here, we show that CED-9 interacts with Mitofusin-2/fuzzy onions and can promote mitochondrial clustering and dramatic reorganization of mitochondrial networks. Consistent with its ability to neutralize CED-9 function, EGL-1 antagonized CED-9-dependent remodeling of the mitochondrial network. However, CED-9 failed to inhibit mitochondrial cytochrome c release or apoptosis induced by diverse triggers in mammalian cells. These data suggest that the ability to regulate mitochondrial fission/fusion dynamics is an evolutionarily conserved property of the Bcl-2 family.

Loss of iRhom2, a catalytically inactive rhomboid-like protein, blocks maturation of TACE/ADAM17 in macrophages, resulting in defective shedding of the cytokine tumor necrosis factor. Apart from the resulting inflammatory defects, iRhom2-null mice appear normal: they do not show the several defects seen in TACE knockouts, suggesting that TACE maturation is independent of iRhom2 in cells other than macrophages. Here we show that the physiological role of iRhoms is much broader. iRhom1 knockout mice die within 6 weeks of birth. They show a severe phenotype, with defects in several tissues including highly penetrant brain haemorrhages. The non-overlapping phenotypes imply that iRhom 1 and 2 have distinct physiological roles, although at a cellular level both promote the maturation of TACE (but not other ADAM proteases). Both iRhoms are co-expressed in many contexts where TACE acts. We conclude that all TACE activity, constitutive and regulated, requires iRhom function. iRhoms are therefore essential and specific regulators of TACE activity, but our evidence also implies that they must have additional physiologically important clients.

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.