Discovery of Apoptosis
Apoptosis is an evolutionary conserved programmed cell death process that is of fundamental importance in tissue homeostasis. The word Apoptosis comes from the Greek word meaning the dropping of leaves from a tree. The term apoptosis was first introduced in 1972 by John Kerr and colleagues to describe the morphological changes they observed within a dying cell under a variety of conditions.
Apoptosis & Physiology
Apoptosis is a vital component of several physiological process such as embryogenesis, normal tissue development and the immune-response (Vaux and Korsmeyer, 1999). Examples of the requirement of programmed cell death (Apoptosis) under such physiological conditions include, the removal of the tissue between digits in the developing foetus, the elimination of surplus cells in order to form correct connections between neurons in the developing brain, the deletion of the tadpole tail and the elimination of unwanted cells by the immune system on a daily basis (Oppenheim, 1991; Tata, 1966).
Apoptosis & Disease
Apoptosis also plays a major role in the pathogenesis of disease. Enhanced apoptosis can lead to neurodegenerative diseases, stroke and AIDS whereas its inhibition can result in carcinogenesis and chemoresistance (Brown and Attardi, 2005; Gougeon et al., 2000; Green and Evan, 2002; Hanahan and Weinberg, 2000; Plati et al., 2008; Stennicke et al., 2002; Thompson, 1995). Apoptotic cell death is dependent on the activation of a specific group of cysteine proteases known as caspases (Cysteine aspartate specific proteases) which are the principal effectors of apoptosis in cells destined for programmed cell death (Alnemri et al., 1996).
Caspases are the main proteases involved in apoptosis. Caspases, are also regulated by the proteasome through interaction with members of the Inhibitor of Apoptosis Protein (IAP) family. Some IAPs contain a C-terminal RING zinc-finger domain that has ubiquitin E3 ligase activity (Lorick et al., 1999).
The RING motif is a common motif found in E3 ligases. The IAP RING targets not only IAPs themselves, but also other proteins such as caspases and Smac/DIABLO,which antagonises the inhibitory function of IAPS (Creagh et al., 2004; Hu and Yang, 2003; Huang et al., 2000; MacFarlane et al., 2002; Suzuki et al., 2001; Weissman, 2001; Yang et al., 2000).
The formation of apoptotic bodies is characterized by a distinct pattern of changes such as cytoplasmic shrinkage, active membrane blebbing, chromatin condensation and typically the fragmentation into membrane enclosed vesicles (apoptotic bodies) (Kerr et al., 1972; Wyllie et al., 1980). These transformations are also accompanied by certain biochemical changes in the cell which include the mitochondrial membrane permeabilisation, the creation of reactive oxygen species, externalisation of phosphatidylserine, and the degradation of nuclear DNA (Lazebnik et al., 1994; Martin et al., 1995; Wyllie et al., 1984; Zamzami et al., 1995).
The apoptosome is a large quarternary protein structure formed during Apoptosis. The formation of the apoptosome is triggered following the release of cyctochrome C in response to intrinsic and extrinsic death stimuli.
It is important to note the crucial function that cytochrome c plays in the mitochondrial pathway of caspase activation. Such requirement seems to be exclusive to mammalian systems as cytochrome c is not required for the formation of the apoptosome in C. elegans or Drosophila (Dorstyn et al., 2002; Zimmermann et al., 2002). Waterhouse et al illustrated that mitochondrial outer membrane permeabilisation coincided with cytochrome c release and was entirely released within five minutes. Outer mitochondrial membrane permeabilisation is regulated by members of the Bcl-2 family (Adams and Cory, 1998; Waterhouse et al., 2002).
Upon permeablisation of the outer mitochondrial membrane additional proteins are released from the mitochondria, these include apoptosis inducing factor (AIF), a flavoprotein thought to be involved in inducing DNA fragmentation, (Daugas et al., 2000; Landshamer et al., 2008) Smac/DIABLO (second mitochondrial-derived activator of apoptosis), a protein that functions by binding to and inhibiting a family of endogenous regulators of apoptosis, the IAPs (Adrain et al., 2001; Du et al., 2000; Verhagen et al., 2000; Zhou et al., 2005), and Omi/Atr2, a serine protease involved in the induction of apoptosis at multiple levels (Faccio et al., 2000; Gray et al., 2000; Suzuki et al., 2001a).
The structure of the apoptosome is comprised of seven Apaf-1 monomers arranged in a symmetric wheel like structure (Acehan et al., 2002; Shi, 2006). Pro-caspase 9 is recruited and activated by the apoptosome. Binding of cytochrome c to the C-terminal of Apaf-1 results in the inhibitory action of the WD40 repeat region on Apaf-1 self-association being abolished (Hu et al., 1998). This binding induces a conformational change that allows Apaf-1 to bind to ATP/dATP to form the apoptosome (Li et al., 1997; Riedl et al., 2005). This in turn mediates the recruitment of the initiator caspase, pro-caspase-9 to the CARD motif at the Apaf-1 N-terminus.
Once activated the mature caspase-9 remains part of the apoptosome complex. Apaf-1 functions as an allosteric regulator of its activity. It allows it to cleave and activate downstream effector caspases such as pro-caspase-3 and pro-caspase-7 (Adrain et al., 1999; Rodriguez and Lazebnik, 1999; Slee et al., 1999; Srinivasula et al., 1998).
Caspase-9 crystal structure
The crystal structure of caspase-9 revealed that in its active form it is a dimer and it does not require cleavage for its activation. Rather it involves dimerisation of caspase-9 monomers within the apoptosome, with the dimer interface providing surfaces compatible with catalytic organization of the active site (Riedl et al., 2005; Shi, 2006).
The executioner caspases-3 and -7 exist within the cytosol as inactive dimers (Boatright et al., 2003; Donepudi and Grutter, 2002). Cleavage by other capsases within the linker L2 segment is required for activation (Donepudi and Grutter, 2002). When activated these caspases cleave and activate further downstream caspases such as caspases-2 and-6. Caspase-6 is then involved in the processing of caspases-8 and -10 (Slee et al., 1999). Caspase-3 is also involved in a feed-back amplification loop to further activate caspase-9 (Slee et al., 1999; Srinivasula et al., 1998).
The proteasome is not only involved in apoptosis regulation but also other important signalling pathways, including cell cycle progression (Hershko, 1997; King et al., 1996) and gene expression (Ciechanover, 1994; Karin and Ben-Neriah, 2000). Disruption of proteasomal activity can be detrimental to cellular processes. Recently there has been great interest in the development of drugs to regulate the function of the proteasome, such as proteasomal inhibitors, e.g. bortezomib, to more effectively treat cancers and stroke (Kisselev and Goldberg, 2001; Pratt et al., 2007; Wojcik and Di Napoli, 2004).