including the regulation of pre-existing membrane pores of the mitochondrium by
association with proapoptotic family members (Shimizu et al., 1999; 2001).
Combining all available information, a three-step regulatory model appears likely,
at least for vertebrates. In the most downstream step, the all-BH proapoptotic
members, such as Bax, trigger the mitochondrial permeability by an as yet unknown
mechanism. This apoptosis-inducing action can be prevented by association with the
antiapoptotic all-BH proteins such as Bcl-2 and Bcl-xL. The inhibitory effect of these
proteins can, in turn, be abrogated by interaction with proapoptotic BH3-only
proteins. The variety of BH3-only proteins can be regulated in different fashions,
reflecting the fact that the mitochondrium is really at the intersection of many
apoptosis pathways (Kroemer, 1999). Bid, a BH3-only protein with an important
role in receptor-induced apoptosis, is activated by caspase-8 cleavage (Li, H. et al.,
1998; Luo et al., 1998). Other BH3-only proteins, such as Noxa and Puma, are
transcriptionally induced and play a likely role in p53-induced apoptosis (Oda et al.,
2000; Nakano and Vousden, 2001; Yu et al., 2001). In nematodes, the ced-9 protein
seems to work by a totally different mechanism, although it is related to bcl-2.
Cytochrome c release does not play a role in nematode apoptosis, where, instead,
ced-9 keeps ced-4 inactive by a direct interaction. There is no evidence that vertebrate
bcl-2 members can work in a similar fashion by directly regulating APAF1 function.
The ced-9 protein itself is regulated by binding of the egl-1 gene product, which is
transcriptionally regulated, and bears some resemblance to BH3 regions (Liu and
Hengartner, 1999a). So it seems that at least this regulatory aspect is conserved
between animal lineages.
5.2
The NB-ARC and NACHT NTPase domainsThe NB-ARC domain, also known as AP-ATPase domain, is a homology domain
found in a number of apoptosis regulators (van der Biezen and Jones, 1998; Aravind
et al., 1999). It was first observed in the central region of APAF1, where it is located
between the N-terminal CARD and the C-terminal WD-40 repeat domain, and the
nematode Ced-4 protein, which is related to APAFl but lacks the WD-40 repeats.
Interestingly, this domain is also found in a number of plant-derived ‘R’ gene
products, which are involved in the hypersensitive response, an apoptosis-related,
host defense mechanism of plants (Heath, 2000). As will be discussed in Section 6,
these plant R-proteins have an overall architecture similar to animal apoptosis
regulators. Additional copies of the NB-ARC domain are found in a number of
bacterial proteins of unknown function, which frequently also contain putative DNA
binding motifs. A typical NB-ARC domain spans 300–350 residues and contains all
the hallmarks of an ATPase, including a conserved P-loop (Walker A motif) and a
moderately well-conserved Walker B motif. Since these conserved elements are typical
of both ATPases and GTPases, and a specific nucleotide hydrolysis preference for
these proteins has not been described, the assignment as ATPases is speculative.
FUNCTIONAL DOMAINS IN APOPTOSIS PROTEINS 93