Apoptosis: checkpoint at the mitochondrial frontier

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Abstract

Apoptosis, an evolutionarily conserved form of cell death, requires a regulated program. Central to the apoptotic program is a family of cysteine proteases, known as caspases, that cleave a subset of cellular proteins, resulting in the stereotypic morphological changes of apoptotic cell death. In living cells caspases are present as inactive zymogens and become activated in response to pro-apoptotic stimuli. Mitochondria participate in the activation of caspases by releasing cytochrome c into the cytosol where it binds to the adaptor molecule Apaf-1 (apoptotic protease activating factor 1) and causes its oligomerization. This renders Apaf-1 competent to recruit and activate the cell death initiator caspase, pro-caspase-9. Once caspase-9 is activated, it cleaves and activates downstream cell death effector caspases. Bcl-2, an apoptosis inhibitor localized to mitochondrial outer membranes, prevents cytochrome c release, caspase activation and cell death. This review discusses recent advances on the role of mitochondria and cytochrome c in the central pathway leading to apoptotic cell death.

Section snippets

Mitochondria reawaken in the control of apoptosis

Mitochondria were thought not to be involved in the cell death program since cells without mitochondrial genome (ρo cells) died by apoptosis and Bcl-2, a cell death inhibitor residing in the mitochondrial outer membrane, still prevented this cell death [1].

However, recent studies with cell-free systems of apoptosis indicated that mitochondria are part of the central apoptotic pathway. In a cell-free system of apoptosis with Xenopus egg extracts, apoptosis was dependent on a heavy membrane

Cytochrome c, a double agent for life and death

Recent discoveries indicated that cytochrome c regulates not just bio-energetics but also apoptosis [19]. Cytochrome c is encoded by a nuclear gene as apo-cytochrome c, which undergoes selective import across the mitochondrial outer membrane. Once in the mitochondrial intermembrane space apo-cytochrome c acquires a heme group, resulting in a conformational change that locks mature holo-cytochrome c in the mitochondria. In living cells, holo-cytochrome c shuttles electrons from respiratory

The cytochrome c/Apaf-1 pathway for caspase activation

Apoptosis is evolutionarily conserved. In Caenorhabditis elegans (C. elegans), three main cell death regulators have been isolated including ced-3, ced-4 and ced-9. Ced-3 encodes a C. elegans caspase and is activated by the adaptor molecule Ced-4. Ced-9 shares homology with the mammalian Bcl-2 protein and binds Ced-4, inhibiting its ability to activate Ced-3.

In mammals, 14 members of the caspase family have been identified which cleave their substrates after aspartic acid (Asp) [23]. Activation

Mechanism of cytochrome c translocation

Heme attachment induces a conformational change in cytochrome c which traps the molecule in the mitochondrial intermembrane space. How then does cytochrome c cross the outer membrane of mitochondria in apoptotic cells? At least three mechanism have been proposed that may account for the release of cytochrome c in apoptosis [36].

First, cytochrome c release may be mediated by opening of the PTP. The PTP is a multiprotein complex formed at the contact sites between the mitochondrial outer and

Bcl-2 family proteins–gatekeepers of mitochondrial function

The Bcl-2 family of proteins plays a pivotal role in the regulation of cell death and cell survival. Some proteins of the family such as Bax, Bad, Bid, Bak, Bcl-xL, Bik, Bim, Hrk, C. elegans Egl-1 promote cell death while others inhibit cell death including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1 [50]. Bcl-2 family members form homo- and hetero-dimers and the balance between anti- and pro-apoptotic Bcl-2 proteins may dictate the response to an apoptotic signal. They can function either independently or

Surviving cytochrome c release

Is cytochrome c release from mitochondria sufficient to induce apoptosis? Data on sympathetic neurons suggest that cytochrome c release is not always enough to trigger cell death and additional events are required, which render the cell sensitive to enter the apoptotic program [65]. Sympathetic neurons undergoing apoptosis by NGF withdrawal exhibit loss of cytochrome c from mitochondria. Micro-injection of cytochrome c was insufficient to induce cell death in NGF-maintained sympathetic neurons.

Cytochrome c-dependent and -independent cell death pathways

Do all forms of apoptosis require a mitochondrial pathway? When Fas ligand binds the Fas receptor, the adaptor molecule FADD becomes recruited to the receptor, allowing binding and auto-processing of pro-caspase-8 25, 26, 72, 73. Once caspase-8 is activated, it can directly process downstream effector caspases and therefore could by-pass a mitochondrial requirement [74]. However, mitochondria substantially amplify the caspase-8-induced caspase cleavage activity in vitro 75, 76, 77. Caspase-8 is

Caspase-independent cell death

Cytochrome c release in Fas-mediated cell death is blocked by caspase inhibitors and the cells maintain full clonogenic potential 41, 77, 85, 86. However, caspase inhibition does not prevent cytochrome c release in apoptosis induced by growth factor withdrawal, etoposide, actinomycin D, UV, staurosporine and overexpression of Myc, Bax or Bak and the cells die a slow necrotic death 46, 47, 77, 87, 88. Thus, cytochrome c release may have two effects. On one hand, cytochrome c activates caspases

Concluding remarks

A model of apoptosis implicating mitochondria in the basic cell-death machinery is shown in Fig. 1. Understanding the role of mitochondria in apoptosis may build the foundation for the development of new cytotoxic and cytoprotective drugs for the treatment of cancer and degenerative disorders, in which the apoptotic program is deregulated.

Acknowledgements

E.B.-W. is supported by a fellowship of the Swiss National Science Foundation (823A-046638). We thank Douglas Ethell, Michael Pinkoski and Nigel Waterhouse for the critical reading of the manuscript.

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