Life's smile, death's grin: vital functions of apoptosis-executing proteins

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Apoptosis is executed by caspases as well as caspase-independent death effectors. Caspases are expressed as inactive zymogens in virtually all animal cells and are activated in cells destined to undergo apoptosis. However, there are many examples where caspase activation is actually required for cellular processes not related to cell death, namely terminal differentiation, activation, proliferation, and cytoprotection. Several caspase-independent death effectors including apoptosis-inducing factor, endonuclease G and a serine protease (Omi/HtrA2) are released from the mitochondrial intermembrane space upon permeabilization of the outer membrane. Such proteins also have important roles in cellular redox metabolism and/or mitochondrial biogenesis. As a general rule, it thus appears that cell-death-relevant proteins, especially those involved in the core of the executing machinery, have a dual function in life and death. This has important implications for pathophysiology. The fact that the building blocks of the apoptotic machinery have normal functions not related to cell death may mean that essential parts of the apoptotic executioner cannot be lost and thus reduces the possibility of oncogenic mutations that block the apoptotic program. Moreover, therapeutic suppression of unwarranted cell death must be designed to target only the lethal (and not the vital) role of death effectors.

Introduction

Apoptosis is a regulated, energy-dependent form of cell death with a characteristic morphological appearance that involves cellular shrinkage and chromatin condensation. Two main pathways can lead to apoptosis: the intrinsic pathway and the extrinsic pathway (Figure 1). The extrinsic pathway involves a death receptor protein (prototype: Fas/CD95) and an adaptor protein (e.g. FADD, Fas-associated death domain protein), which in turn interacts with the cysteine aspartate protease pro-caspase-8 (and often pro-caspase-10). Activation of caspase-8 culminates in the explosive activation of other caspases (including the so-called executioner caspases-3, -6 and -7) or alternatively triggers the activation of caspase-3 in an indirect fashion, through a pathway that involves the mitochondrial release of cytochrome c. The intrinsic pathway directly releases soluble proteins contained in the mitochondrial intermembrane space [1••]. These molecules include cytochrome c, apoptosis inducing factor (AIF), endonuclease G (EndoG), Omi/HtrA2 and Smac/DIABLO. Cytochrome c, once in the cytosol, interacts with Apaf-1 (apoptotic protease activation factor 1) and pro-caspase-9 leading to the formation of the caspase-9 activation complex. Activated caspase-9 triggers the maturation of pro-caspase-3. Smac/DIABLO and Omi/HtrA2 activate apoptosis by neutralizing the inhibitory activity of IAPs (inhibitory apoptosis proteins) that associate with and inhibit caspases. Caspases are therefore implicated in different aspects of cell death. They initiate (in the case of caspase-8 and -9) the propagation of apoptotic signals and execute (in the case of caspase-3, -6 and -7) the apoptotic program through cleavage of an array of vital proteins. In addition, apoptosis can be induced by caspase-independent death effectors such as AIF, EndoG and Omi/HtrA2.

Apoptosis is essential for development and adult tissue homeostasis. An abnormal increase in apoptosis leading to the unwarranted demise of cells is involved in many pathological processes such as myocardial infarction, stroke, neurodegenerative disease and AIDS [2]. As a result, targeting of essential elements of the apoptotic machinery has been considered a promising therapeutic option, especially for the treatment of massive, life-threatening states of acute apoptotic death, for instance in septic shock or after ischemia of the heart or the brain. It would only be possible to target these components at an acceptable level of toxicity, however, if they had no important function for normal cellular metabolism or vital signal transduction processes. This review will focus on the function of these proteins in processes other than cell death (Figure 1).

Section snippets

Implication of caspases in differentiation processes

The involvement of caspases in differentiation (Table 1) was initially thought to be associated with the enucleation process, which is regarded as a caspase-dependent incomplete apoptotic process. Erythroblasts, keratinocytes and lens epithelial cells lose their nucleus, as well as other organelles, during terminal differentiation, yet continue to be metabolically active. For instance, erythropoiesis involves the sequential formation in the bone marrow of a series of erythrocyte precursors

Caspases in the immune system

Beyond the well-known function of the inflammatory caspases-1 and -11 in interleukin-1 production, loss-of-function mutations in caspase-8 are linked to defects in the activation of T, B and NK cells that culminate in an immunodeficiency syndrome [16••]. These data, which have been obtained in the human system, have been corroborated in the mouse model. Genetic elimination of caspase-8 (and knockout of its death-domain-containing cytosolic adaptor FADD) in mice results in impaired heart muscle

Caspases as cytoprotective agents?

It has been widely assumed that apoptotic death of mammalian cells would be closely associated with the activation of caspases. In consequence, it would be expected that pharmacological inhibition of caspases would abrogate apoptotic cell death. However, when mammalian cells are treated with caspase inhibitors such as N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk), they are actually sensitized to cell death induction in vitro. That caspase inhibition can be deleterious has been

Cytochrome c: essential in life and death

Cytochrome c may be considered as the quintessential example of a protein whose role in normal life — as an obligate electron shuttling protein between complexes III and IV of the respiratory chain — was well characterized before its role in apoptotic demise was discovered. Only holo-cytochrome c (which contains the prosthetic heme group) has a proapoptotic function. Substitution of the iron atom within heme (which abolishes electron transfer by cytochrome c) does not affect caspase activation

Apoptosis-inducing factor — a Janus protein

AIF is a phylogenetically conserved protein essential for embryonic development [27]. It is synthesized from a nuclear gene as an immature precursor with a ∼100-amino-acid-long N-terminal mitochondrial localization sequence (MLS). Upon import into the mitochondrial intermembrane space, the MLS is removed from the protein, which refolds and incorporates flavine adenine nucleotide (FAD) [28]. The resulting ∼57 kDa AIF flavoprotein is redox-active and behaves as an NADH oxidase. Upon apoptosis

Other caspase-independent mitochondrial death effectors: EndoG and Omi/HtrA2

EndoG is a mitochondrial nuclease encoded by a nuclear gene. Once liberated into the cytosol, EndoG translocates to the nucleus where it causes oligonucleosomal DNA fragmentation even in the presence of caspase inhibitors [36]. In mammalian cells, EndoG cooperates with exonuclease and DNase I to facilitate DNA processing [37], while it is thought to cooperate with AIF in C. elegans 38.•, 39.. EndoG homozygous mutant embryos die between embryonic days 2.5 and 3.5. This essential function of

Concluding remarks

In evolutionary terms, it appears unlikely that any protein has been generated with the exclusive function of killing cells. Indeed, it appears that most pro-apoptotic proteins, perhaps with the exception of the pro-apoptotic proteins from the Bcl-2 family, have additional functions in normal metabolism (as opposed to apoptotic catabolism) or signal transduction that are not related to apoptosis. One example is AIF, which has a pro-apoptotic function, yet causes the death of vulnerable cells

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

The authors’ own work has been sponsored by a special grant from League against Cancer, as well as by the French Ministry of Science and the EU Trans-Death consortium (to G.K.), ‘Fondation contre la Leucemie’, ‘Ligue National contre le Cancer’ and ‘Comité de la Nièvre de la Ligue contre le Cancer’ (to C.G). We thank S Gurbuxani, E Schmitt and A Parcellier for their help.

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