I

MOMP

MOMP

proCED-3

CED-4

apoptosis caspase-independent cell death apoptosis caspase-dependent cell death proCED-3

CED-4

apoptosis

CED-3

caspase-independent cell death apoptosis caspase-dependent cell death

Fig. 2.1 Cell death by mitotic catastrophe. The induction of catastrophic breakdown of the mitochondrion derived from experimental results of mammalian cells (black) is shown. Various insults lead to the activation of BAX and BAK, which eventually trigger MOMP, resulting in the release of various enzymes. Depending on different factors, e.g., concentration, these enzymes cause cell death either in a caspase-dependent or -independent manner. C. elegans cell death pathways are shown in red and Drosophila mechanisms in green. Pathways for both animal models have also been shown to be dependent on caspases: The classical apoptotic pathway in C. elegans is based on the release of CED-4 from CED-9 leading to caspase-dependent cell death. The caspases CED-3 and CED-4 are also involved in the release of WAH-1. In Drosophila, the process of MOMP and the release of the HtrA2/Omi homologues, result in the downregulation of the caspase inhibitor DIAP1 and eventually the elevated activity of the caspase DRONC. It is not clear yet in which cell death pathway WWOX is involved. DRONC Drosophila Nedd2-like caspase; MOMP mitochondrial outer membrane permeabilization; CED cell death abnormality; EGL egg-laying deficiency; WAH worm AIF homolog; CPS CED-3 protease suppressor; CRN cell-death-related nuclease; AIF apoptosis inducing factor; EndoG endonuclease G; DIABLO direct inhibitor of apoptosis-binding protein with low pI; WWOX WW domain-containing oxidoreductase

In summary, although Bcl-2 family members have been shown to be involved in apoptosis and CED-9 is localized to the mitochondrial membrane, it is not known whether it is associated with MOMP (Estaquier and Arnoult 2006). In the fruit fly Drosophila, the involvement of mitochondria in cell death is less clear and they probably do not undergo MOMP (Varkey et al. 1999), although recent research findings may overturn this notion (see below; Challa et al. 2007; Igaki et al. 2007).

In mammalian cells the consequence of MOMP is the release of several factors (cytochrome c, Smac/Diablo, Omi/HtrA2, AIF and EndoG in mammals) from the intermembrane space into the cytosol, where they function as either caspase-dependent or -independent death executors (Kim et al. 2006). Cytochrome c release causes activation of Apaf-1 and leads to the classical apoptotic pathway, in which Smac/Diablo also plays a role as counteractor of IAPs (inhibitor of apoptosis proteins; Hengartner 2000). The release of apoptosis inducing factor (AIF) HtrA2/ Omi and EndoG have been shown to initiate caspase-independent mechanisms of cell death (Lorenzo and Susin 2004).

In mouse cells the endonuclease EndoG was identified as an apoptotic DNase that is released from mitochondria, subsequently localizing to the nucleus and fragmenting DNA independently of the activity of caspases. UV irradiation-induced DNA fragmentation mediated by EndoG still occurs in the presence of caspase inhibitors (Li et al. 2001). The function of the protein in mitochondria is the generation of RNA primers initiating DNA synthesis, a process important during mito-chondrial replication (Cote and Ruiz-Carrillo 1993). The C. elegans homolog of mammalian EndoG, CPS-6 represents the first mitochondrial protein that has been identified to be involved in developmental programmed cell death in the nematode, indicating that an evolutionarily conserved family of nucleases plays an important role in apoptotic DNA degradation (Parrish et al. 2001). The activity of CPS-6 appears to be caspase-dependent, since down regulated CPS-6 function enhances cell survival in developing nematodes baring mutations in the caspases CED-3 and CED-4 (Parrish et al. 2001). Some interactors of CPS-6 have been identified: WAH-1, the C. elegans homolog of AIF (Wang et al. 2002), which is discussed below, and CRN-1, the homolog of human flap endonuclease-1 (FEN-1) (Parrish et al. 2003). CRN-1 possesses a 5-3' exonuclease and a structure-specific endonuclease activity. It acts as a co-factor of CPS-6, which is an endonuclease generating single-stranded nicks in DNA. Together they mediate stepwise DNA degradation (Parrish et al. 2003). Several more CRN nucleases might be involved in this process (Parrish and Xue 2003).

AIF was first identified in mammals as an effector of apoptotic cell death causing chromatin condensation and large-scale DNA fragmentation after localizing to the nucleus (Susin et al. 1999). Although it has been connected to the release of caspase-9 and therefore acting in the caspase-dependent pathway of cell death (Susin et al. 1999), AIF is also thought to be involved in a caspase-independent mechanism called "apoptosis-like" cell death (Leist and Jaattela 2001). The mechanism of releasing AIF to the cytosol is still under debate: The protein is embedded in the inner membrane of mitochondria and needs to be cleaved by proteases in order to be released. Cleavage occurs after the permeabilization of the outer membrane of mitochondria and is processed by the cysteine proteases cathepsins and calpains (Yuste et al. 2005). Such a scenario is supported by the fact that AIF is released to the cytosol through the same pore but much slower than cytochrome c, Smac/Diablo and Omi/HtrA2 (Munoz-Pinedo et al. 2006). However this notion contradicts earlier findings, where blocking caspase activity through zVAD-fmk prevents the release of AIF from mitochondria (Arnoult et al. 2003). Given that zVAD-fmk also blocks the activity of cysteine proteases these data need to be re-evaluated (Modjtahedi et al. 2006; Krantic et al. 2007).

The precise mechanism by which AIF promotes apoptosis-like cell death is not fully understood. Human AIF likely interacts with DNA since it shows a strong positive electrostatic potential (Ye et al. 2002) and most likely recruits potential partners such as nucleases to degrade DNA, triggering cell death (Lorenzo and Susin 2004). Indirectly, AIF may activate cell death via generation of free radicals after being released to the cytosol. AIF exhibits NADH oxidase activity, reducing O2 (Miramar et al. 2001). However, AIF also plays the role of a free radical scavenger, as shown in the Harlequin mouse (Klein et al. 2002). Thus, AIF might fulfill a dual role depending on its actual localization either to the cytosol (oxidase and cell death executor) or to the inner membrane of mitochondria (free radical scavenger involved in the mitochondrial respiratory chain; Porter and Urbano 2006).

Insight into the mode of AIF action has been obtained by studies of the C. elegans AIF homolog wah-1 in developmental cell death. Wang and colleagues demonstrated that WAH-1 and the C. elegans EndoG (CPS-6) can be released from mitochondria by EGL-1 in a way similar to the release of cytochrome c and EndoG from mammalian mitochondria. Both proteins cooperate and act in the same pathway to promote apoptotic DNA degradation (Wang et al. 2002). Surprisingly, speed of WAH-1 release observed in a time-course study is at least partially dependent on caspase CED-3 activity, suggesting that C. elegans AIF and EndoG define a single, mitochondria-initiated apoptotic DNA degradation pathway that is conserved between C. elegans and mammals (Wang et al. 2002; Wang unpublished results). This assumption was recently confirmed by the discovery that WAH-1 promotes plasma membrane phosphatidylserine externalization and initiates cell engulfment typical for classical apoptosis in the nematode through activation of phospholipid scramblase 1 (SCRM-1; Wang et al. 2007).

The death effector Omi/HtrA2 was first identified in mammals as inhibitor of the X-chromosome linked inhibitor of apoptosis (XIAP) similar to Smac/Diablo. The same investigation showed the induction of a second mechanism of mediating cell death independent of caspases, probably due to its serine protease function (Suzuki et al. 2001). In mammals the protein is processed after import to the mitochondria and 133 of 458 residues are removed, leaving an active form of 36 kDa. The amino-terminus shares high homology with Drosophila pro-death proteins Grim, Hid, Reaper and mammalian Smac/Diablo proteins (Lorenzo and Susin 2004). Some evidence about the mechanism of Omi/HtrA2 action comes from studies in Drosophila: The mitochondrial proteins dOmi and dmHtrA2 were independently identified as highly homologous to the human HtrA2/Omi, particularly within the serine protease domain. During UV-irradiation-induced cell death, labeled dmHtrA2 or dOmi proteins and also cytochrome c, were observed outside mitochondria (Challa et al. 2007; Igaki et al. 2007). Release is both caspase-dependent and -independent (Challa et al. 2007). In the cytosol dOmi induces cell death in S2 cells and in the developing fly eye by proteolytically degrading DIAP1 (an IAP family caspase inhibitor), which finally displaces DRONC and acts in the classical apoptosis pathway (Challa et al. 2007; Igaki et al. 2007).

Another recently investigated gene involved in caspase-independent cell death is hspinl, a homolog of the Drosophila spin gene (Yanagisawa et al. 2003). Mutations in spin interfere with programmed cell death during the development of Drosophila nurse cells and neurons. Persistence of surviving cells leads to neurodegeneration and death of oocytes in the ovary (Nakano et al. 2001). In human cells HSpin1, which contains membrane spanning domains, causes necrotic cell death when overexpressed. HSpin1 binds to the antiapoptotic proteins Bcl-2 and Bcl-xL and its activity can be blocked by the necrosis inhibitor pyrrolidine dithiocarbamate (PDTC) but not by the caspase-inhibitors zVAD-fmk and p35. This indicates that HSpin1 titrates Bcl-2 and/or Bcl-xL by localizing to the mitochondria and thereby promoting cell death in a caspase-independent way (Yanagisawa et al. 2003). Three homologs of the spin gene are encoded in the C. elegans genome and have not been characterized in detail (Nakano et al. 2001).

Additional proteins that are involved in mitochondrial caspase-independent cell death have been identified in mammalian cells: WWOX or FOR, the AIF homologue mitochondrion-associated inducer of death (AMID) and the p53 regulated gene 3 (PRG3). All these show sequence similarity to AIF (Lorenzo and Susin 2004). WWOX has a homolog in Drosophila, which has been shown to protect from ionizing radiation when overexpressed (O'Keefe et al. 2005).

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