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Programmed cell death, or apoptosis, is currently one of the hottest areas of modern biology. It describes the orchestrated collapse of a cell, staging membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and DNA degradation followed by rapid engulfment of corpses by neighbouring cells.
The excitement ensued when it became clear that apoptosis is an essential part of life for any multicellular organism and that the way in which most cells die is conserved from worm to mammal. Optimum body maintenance means that about 10 billion of our cells will die on a normal day just to counter the numbers of new cells that arise through mitosis. During development apoptosis helps to sculpture the body, shape the organs, and carve out fingers and toes. Both the nervous system and the immune system arise through overproduction of cells followed by the death of those that fail to establish functional synaptic connections or productive antigen specificities, respectively. Apoptosis is necessary to purge the body of pathogen-invaded cells, but is also needed to eliminate activated or auto-aggressive immune cells.
Such massacre has to be tightly regulated as too little or too much cell death may lead to pathology, including developmental defects, autoimmune diseases, neurodegeneration or cancer. Not surprisingly then that the hunt is on to understand which cells die when, why and how precisely, and to find drugs that interfere with specific steps along the pathway. Naturally, with over 50,000 publications on the subject to date (source: ISI–Web of Science), it is impossible to be comprehensive, but we hope that this Nature Insight provides our readers with a taster of the latest developments in this rapidly moving field.
Michael Hengartner sets the stage on page 770 and introduces the assassins and victims in this molecular 'murder mystery'. On page 777 Andrew Wyllie and co-workers discuss why and how DNA damage results in apoptosis in some cells but not in others and what the consequences are if cells with damaged genomes fail to die. Once a cell is committed to die, its corpse must be removed and destroyed by phagocytic cells, as discussed by John Savill and Valerie Fadok on page 784 . Peter Krammer outlines the importance of apoptosis for the immune system on page 789, focusing on the role of the infamous CD95 death receptor. On page 796 Gerard Evan and colleagues outline the molecular mechanisms that bring about apoptosis during development of various organisms, and highlight the conservation of cell death mechanisms during evolution. The role of programmed cell death in the construction and pathological deconstruction of the brain is discussed on page 802by Junying Yuan and Bruce Yankner. It is evident from reading these reviews that death or serious illness may result if cells that should die survive, or cells that should live die. There is clearly huge therapeutic potential, but will apoptosis deliver its promise to medicine? On page 810 Donald Nicholson discusses the opportunities and limitations of taking apoptosis from the bench to the clinic.
We are indebted to all of the contributors to this Insight for their considerable efforts, despite space and time constraints, in producing an enlightening and thought-provoking collection of reviews.
Marie-Thérèse Heemels Senior Editor
Ritu Dhand Insight Programme Editor
Liz Allen Publisher
The biochemistry of apoptosis
MICHAEL O. HENGARTNER
Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724 , USA
Apoptosis — the regulated destruction of a cell — is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.
Multicellular animals often need to get rid of cells that are in excess, in the way, or potentially dangerous. To this end, they use an active dedicated molecular programme. As important as cell division and cell migration, regulated (or programmed) cell death allows the organism to tightly control cell numbers and tissue size, and to protect itself from rogue cells that threaten homeostasis.
Discovered and rediscovered several times by various developmental biologists and cytologists, programmed cell death acquired a number of names over the past two centuries1. The term finally adopted is apoptosis, coined by Currie and colleagues in 1972 to describe a common type of programmed cell death that the authors repeatedly observed in various tissues and cell types2. The authors noticed that these dying cells shared many morphological features, which were distinct from the features observed in cells undergoing pathological, necrotic cell death, and they suggested that these shared morphological features might be the result of an underlying common, conserved, endogenous cell death programme3.
Caspases: the central executioners
Most of the morphological changes that were observed by Kerr et al . are caused by a set of cysteine proteases that are activated specifically in apoptotic cells. These death proteases are homologous to each other, and are part of a large protein family known as the caspases4. Caspases are highly conserved through evolution, and can be found from humans all the way down to insects, nematodes and hydra5-7. Over a dozen caspases have been identified in humans; about two-thirds of these have been suggested to function in apoptosis7, 8.
All known caspases possess an active-site cysteine, and cleave substrates at Asp-Xxx bonds (that is, after aspartic acid residues); a caspase's distinct substrate specificity is determined by the four residues amino-terminal to the cleavage site9. Caspases have been subdivided into subfamilies based on their substrate preference, extent of sequence identity and structural similarities.
Because they bring about most of the visible changes that characterize apoptotic cell death, caspases can be thought of as the central executioners of the apoptotic pathway. Indeed, eliminating caspase activity, either through mutation or the use small pharmacological inhibitors, will slow down or even prevent apoptosis7. Thus, blocking caspases can rescue condemned cells from their apoptotic fate — a fact that has not escaped the notice of the pharmaceutical industry (see review in this issue by Nicholson, pages 810–816).
It slices, it dices, and that's not all!
What exactly do the caspases do that is so important for apoptosis? Activation of caspases does not result in the wholesale degradation of cellular proteins. Rather, caspases selectively cleave a restricted set of target proteins, usually at one, or at most a few positions in the primary sequence (always after an aspartate residue). In most cases, caspase-mediated 'protein surgery' results in inactivation of the target protein (Box 1). But caspases can also activate proteins, either directly, by cleaving off a negative regulatory domain, or indirectly, by inactivating a regulatory subunit ( Box 1).
Several important caspase substrates have been identified in recent years. One of the more exciting discoveries has been the elucidation of the mechanism of activation of the nuclease responsible for the famous nucleosomal ladder. First described by Wyllie10, this nuclease cuts the genomic DNA between nucleosomes, to generate DNA fragments with lengths corresponding to multiple integers of approximately 180 base pairs. The presence of this DNA ladder has been used (and abused) extensively as a marker for apoptotic cell death.
In an elegant series of experiments, the groups of Wang and Nagata showed that the DNA ladder nuclease (now known as caspase-activated DNase, or CAD) pre-exists in living cells as an inactive complex with an inhibitory subunit, dubbed ICAD (ref. 11). Activation of CAD occurs by means of caspase-3-mediated cleavage of the inhibitory subunit, resulting in the release and activation of the catalytic subunit12-14.
Caspase-mediated cleavage of specific substrates also explains several of the other characteristic features of apoptosis. For example, cleavage of the nuclear lamins is required for nuclear shrinking and budding15, 16. Loss of overall cell shape is probably caused by the cleavage of cytoskeletal proteins such as fodrin and gelsolin17. Finally, caspase-mediated cleavage of PAK2, a member of the p21-activated kinase family, seems to mediate the active blebbing observed in apoptotic cells. Interestingly, in this last case, caspase cleavage occurs between the negative regulatory subunit and the catalytic subunit, and results in a constitutive activation of PAK2 (ref. 18).
Close to 100 additional caspase substrates have been reported over the years, and there will certainly be many more7, 19. Why are there so many substrates? Perhaps apoptosis is just much more complicated that we currently believe. Indeed, several of the key apoptotic subprogrammes, such as cell shrinking and the emission of pro-engulfment signals (see review in this issue by Savill and Fadok, pages 784–788 ), are still poorly understood. Alternatively, it is possible that many of the described caspase substrates are not relevant substrates, but simply 'innocent bystanders' that get caught in the action. According to this line of reasoning, there might be little selection against the presence of fortuitous caspase cleavage sites on irrelevant proteins, as the cell is about to stop functioning anyway. Further experimentation might allow this issue to be resolved.
How to activate a caspase
Given the great importance of caspases in the apoptotic process, it is reasonable to propose that a proper understanding of apoptosis will require us to understand how caspases are activated.
As is true of most proteases, caspases are synthesized as enzymatically inert zymogens. These zymogens are composed of three domains: an N-terminal prodomain, and the p20 and p10 domains, which are found in the mature enzyme. In all cases examined so far, the mature enzyme is a heterotetramer containing two p20/p10 heterodimers and two active sites7. Although much has been made about the fact that active caspases are dimers containing two active sites, there is no obvious structural reason why this should be so, and it seems quite possible that caspases could exist as active monomers under the right conditions.
Three general mechanisms of caspase activation have been described so far. Each of them is described briefly below (see also Box 2).
Processing by an upstream caspase Most caspases are activated by proteolytic cleavage of the zymogen between the p20 and p10 domains, and usually also between the prodomain and the p20 domain. Interestingly, all these cleavage sites occur at Asp-X sites — candidate caspase substrate sites — suggesting the possibility of autocatalytic activation9. Indeed, the simplest way to activate a procaspase is to expose it to another, previously activated caspase molecule (Box 2 ). This 'caspase cascade' strategy of caspase activation is used extensively by cells for the activation of the three short prodomain caspases, caspase-3, -6 and -7. These three downstream effector caspases are considered the workhorses of the caspase family, and are usually more abundant and active than their long prodomain cousins.
The caspase cascade is a useful method to amplify and integrate pro-apoptotic signals, but it cannot explain how the first, most upstream caspase gets activated. At least two other approaches are used to get the ball rolling.
Induced proximity Caspase-8 is the key initiator caspase in the death-receptor pathway (see review in this issue by Krammer, pages 789–795). Upon ligand binding, death receptors such as CD95 (Apo-1/Fas) aggregate and form membrane-bound signalling complexes (Box 3). These complexes then recruit, through adapter proteins, several molecules of procaspase-8, resulting in a high local concentration of zymogen. The induced proximity model posits that under these crowded conditions, the low intrinsic protease activity of procaspase-8 (ref. 20) is sufficient to allow the various proenzyme molecules to mutually cleave and activate each other (Box 2). A similar mechanism of action has been proposed to mediate the activation of several other caspases, including caspase-2 and the nematode caspase CED-3 (ref. 21). Although forced crowding of zymogens clearly is sufficient in many cases to activate caspases22, it is a rather crude a way to control the fate of a cell. Whereas the basic concept is probably correct, additional levels of regulation surely must exist in vivo to modulate the process.
|Biochemistry (Moscow), Series A: Membrane and Cell Biology. 2004. V. 69. P. 441-450|
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