G van Loo

The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet

Mitochondria are ‘life-essential’ organelles for the production of metabolic energy in the form of ATP. Paradoxically mitochondria also play a key role in controlling the pathways that lead to cell death. This latter role of mitochondria is more than just a ‘loss of function’ resulting in an energy deficit but is an active process involving different mitochondrial proteins. Cytochrome c was the first characterised mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be actively implicated in apoptotic cell death. Since then, other mitochondrial proteins, such as AIF, Smac/DIABLO, endonuclease G and Omi/HtrA2, were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process. Members of the Bcl-2 protein family control the integrity and response of mitochondria to apoptotic signals. The molecular mechanism by which mitochondrial intermembrane space proteins are released and the regulation of mitochondrial homeostasis by Bcl-2 proteins is still elusive. This review summarises and evaluates the current knowledge concerning the complex role of released mitochondrial proteins in the apoptotic process.

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated. Cell Death and Differentiation (2001) 8, 1136–1142

The serine protease Omi/HtrA2 is released from mitochondria during apoptosis. Omi interacts with caspase-inhibitor XIAP and induces enhanced caspase activity.

Proteome analysis of supernatant of isolated mitochondria exposed to recombinant tBid, a proapoptotic Bcl-2 member, revealed the presence of the serine protease Omi, also called HtrA2. This release was prevented in mitochondria derived from Bcl-2-transgenic mice. Release of Omi under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Omi release also occurs in apoptotic dying but not in necrotic dying fibrosarcoma L929 cells, treated with anti-Fas antibodies and TNF, respectively. The amino acid sequence reveals the presence of an XIAP interaction motif at the N-terminus of mature Omi. We demonstrate an interaction between endogeneous Omi and recombinant XIAP. Furthermore we show that endogenous Omi is involved in enhanced activation of caspases in cytosolic extracts.

Death receptor-induced apoptotic and necrotic cell death: differential role of caspases and mitochondria

In L929sAhFas cells, tumor necrosis factor (TNF) leads to necrotic cell death, whereas agonistic anti-Fas antibodies elicit apoptotic cell death. Apoptosis, but not necrosis, is correlated with a rapid externalization of phosphatidylserine and the appearance of a hypoploid population. During necrosis no cytosolic and organelle-associated active caspase-3 and -7 fragments are detectable. The necrotic process does not involve proteolytic generation of truncated Bid; moreover, no mitochondrial release of cytochrome c is observed. Bcl-2 overexpression slows down the onset of necrotic cell death. In the case of apoptosis, active caspases are released to the culture supernatant, coinciding with the release of lactate dehydrogenase. Following necrosis, mainly unprocessed forms of caspases are released. Both TNF-induced necrosis and necrosis induced by anti-Fas in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone are prevented by the serine protease inhibitor N-tosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, while Fas-induced apoptosis is not affected. Cell Death and Differentiation (2001) 8, 829–840

Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA.

Interferons enhance the cellular antiviral response by inducing expression of protective proteins. Many of these proteins are activated by dsRNA, a typical by-product of viral infection. Here we show that type-I and type-II interferons can sensitize cells to dsRNA-induced cytotoxicity. In caspase-8- or FADD-deficient Jurkat cells dsRNA induces necrosis, instead of apoptosis. In L929sA cells dsRNA-induced necrosis involves high reactive oxygen species production. The antioxidant butylated hydroxyanisole protects cells from necrosis, but shifts the response to apoptosis. Treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethylketone or overexpression of Bcl-2 prevent this shift and promote necrosis. Our results suggest that a single stimulus can initiate different death-signaling pathways, leading to either necrotic or apoptotic cell death. Inhibition of key events in these signaling pathways, such as caspase activation, cytochrome c release or mitochondrial reactive oxygen species production, tips the balance between necrosis and apoptosis, leading to dominance of one of these death programs.

A matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) analysis of proteins released from isolated liver mitochondria treated with recombinant truncated Bid.

A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), inducing release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins using a proteomic matrix-assisted laser desorption ionization post-source decay (MALDI-PSD) approach. In order to meet physiological conditions, the concentration of tBid was chosen such that it was unable to induce cytochrome c release in mitochondria derived from liver-specific Bcl-2-transgenic mice. Several mitochondrial proteins were identified to be released in a tBid-dependent way, among which cytochrome c, DIABLO/Smac, adenylate kinase 2, acyl-CoA-binding protein, endonuclease G, polypyrimidine tract-binding protein, a type-I RNA helicase, a WD-40 repeat-containing protein and the serine protease Omi. Western blotting confirmed the absence of adenylate kinase 3, a matrix mitochondrial protein. These results demonstrate that a physiologically relevant concentration of tBid is sufficient to induce release of particular intermembrane mitochondrial proteins belonging to a broad molecular-mass range.

Caspases are not localized in mitochondria during life or death

Caspases are crucial for the initiation, propagation and execution of apoptosis. They normally exist as proenzymes, which can be activated through recruitment into activating complexes and by proteolytic cleavage by other caspases or proteases. Perturbation of organelles such as nuclei, endoplasmatic reticulum and mitochondria results in the activation of caspases. A number of caspases (-2, -3, -8 and -9) were published as being localized in the intermembrane space of mitochondria. However, in three different models of apoptosis (anti-Fas-induced cell death in murine hepatocytes, Fas ligand-induced apoptosis in Jurkat cells and apoptosis induced by growth factor withdrawal in Ba/F3 cells) we could not identify a mitochondrial location of caspases, neither under control nor under apoptotic conditions. In all three apoptotic models caspases were found in the cytosolic (caspases-2, -3, -6, -7, -8, -9) and nuclear subcellular fractions (caspases-2, -3). In another approach we treated isolated liver mitochondria with truncated Bid. Although tBid-dependent release of Cytochrome c, AIF, adenylate kinase, Smac/DIABLO and Omi/HtrA2 could be demonstrated, none of the caspases were detectable both in the supernatant and the mitochondrial fraction after treatment. Our results demonstrate that, in contrast to previous studies, no caspases-2, -3, -8 and -9 are associated with the mitochondrial fraction. These findings support the concept of a separate compartmentalization between proapoptotic cofactors in the mitochondria and silent precursor caspases in the cytosol.

Keratinocyte-specific ablation of the NF-κB regulatory protein A20 (TNFAIP3) reveals a role in the control of epidermal homeostasis.

The ubiquitin-editing enzyme A20 (tumor necrosis factor-α-induced protein 3) serves as a critical brake on nuclear factor κB (NF-κB) signaling. In humans, polymorphisms in or near the A20 gene are associated with several inflammatory disorders, including psoriasis. We show here that epidermis-specific A20-knockout mice (A20EKO) develop keratinocyte hyperproliferation, but no signs of skin inflammation, such as immune cell infiltration. However, A20EKO mice clearly developed ectodermal organ abnormalities, including disheveled hair, longer nails and sebocyte hyperplasia. This phenotype resembles that of mice overexpressing ectodysplasin-A1 (EDA-A1) or the ectodysplasin receptor (EDAR), suggesting that A20 negatively controls EDAR signaling. We found that A20 inhibited EDAR-induced NF-κB signaling independent from its de-ubiquitinating activity. In addition, A20 expression was induced by EDA-A1 in embryonic skin explants, in which its expression was confined to the hair placodes, known to be the site of EDAR expression. In summary, our data indicate that EDAR-induced NF-κB levels are controlled by A20, which functions as a negative feedback regulator, to assure proper skin homeostasis and epidermal appendage development.

A20 prevents chronic liver inflammation and cancer by protecting hepatocytes from death.

An important regulator of inflammatory signalling is the ubiquitin-editing protein A20 that acts as a break on nuclear factor-κB (NF-κB) activation, but also exerts important cytoprotective functions. A20 knockout mice are cachectic and die prematurely due to excessive multi-organ inflammation. To establish the importance of A20 in liver homeostasis and pathology, we developed a novel mouse line lacking A20 specifically in liver parenchymal cells. These mice spontaneously develop chronic liver inflammation but no fibrosis or hepatocellular carcinomas, illustrating an important role for A20 in normal liver tissue homeostasis. Hepatocyte-specific A20 knockout mice show sustained NF-κB-dependent gene expression in the liver upon tumor necrosis factor (TNF) or lipopolysaccharide injection, as well as hepatocyte apoptosis and lethality upon challenge with sublethal doses of TNF, demonstrating an essential role for A20 in the protection of mice against acute liver failure. Finally, chronic liver inflammation and enhanced hepatocyte apoptosis in hepatocyte-specific A20 knockout mice was associated with increased susceptibility to chemically or high fat-diet-induced hepatocellular carcinoma development. Together, these studies establish A20 as a crucial hepatoprotective factor.

The ubiquitin editing enzyme A20 (TNFAIP3) is upregulated during permanent middle cerebral artery occlusion but does not influence disease outcome

The ubiquitin editing enzyme A20 (TNFAIP3) is upregulated during permanent middle cerebral artery occlusion but does not influence disease outcome