It can be noted that caspase-2 knockout mouse embryonic fibroblasts (MEFs) were only partially resistant to PIDD overexpression, whereas RAIDD knockout cells were fully protected, suggesting that PIDD can also induce cell death independent of caspase-2. A member of the mammalian Ced-4 protein family named DEFCAP (death effector filament-forming Ced-4-like apoptosis protein), also known as NALP1 (NACHT, LRR and PYD domains-containing protein 1) or CARD7, identified in a CARD-domain homology database search, is able to interact with capsase-2.34Similar to Apaf-1, DEFCAP has a nucleotide-binding domain and a CARD, and both isoforms of the protein identified, DEFCAP-L and DEFCAP-S, co-immunoprecipitated with overexpressed caspase-2 or -9, but did not bind to caspases-3, -8 and -10 or the adaptors FADD or RAIDD. or the caspase activation and recruitment domain (CARD) found in caspases-2 and -9, as well as short prodomain containing effector caspases-3, -6 and -7. Initiator caspases are activated by autocatalytic processing, which is initiated upon prodomain-dependent dimerization of zymogens at specific activation platforms such as the Sennidin B apoptosome or the death-inducing signaling complex (DISC).1The number of substrates cleaved by initiator caspases is thought to be limited to themselves and downstream effector caspases. The BH3- only Bcl-2 family member Bid appears to be the only exception, as it can also be cleaved by initiator caspases, to connect the extrinsic apoptotic pathway with the mitochondrial apoptosis signaling pathway.2,3 A molecule at odds with current dogma is caspase-2 Sennidin B (earlier synonyms Ich-1: ICE/CED-3 homolog 1 or Nedd-2, forneural precursor cells-expressed,developmentallydownregulated), the most conserved caspase across species, sharing 55% similarity withCaenorhabditis eleganscaspase Ced-3.46On the one hand, the predicted cleavage specificity of caspase-2 appears to place it more closely to effector caspases-3 and -7.7On the other, caspase-2 contains a long CARD prodomain, through which it can interact with adaptor proteins, which is typical for initiator caspases. Overall, biochemical analysis supports a role of caspase-2 as an initiator caspase, but many contradictory findings render the exact function of this enzyme unresolved. In this review, we aim to give an overview on the steadily growing body of literature about this enigmatic enzyme, aiming to provide a synopsis on the present knowledge and to point out important aspects that still need to be tackled in future research. == Activation and Processing of Caspase-2 == During apoptosis induction, caspase-2 is thought to be activated by CARD-mediated, dimerization-induced intrasubunit cleavage (@D333) and subsequent removal of the prodomain (@D169) as well as of the linker region (@D347), connecting its large p19 subunit with the small p12 fragment. In addition to these cleavage site residues, it was found that cystein C320 in the catalytic site is critical for autoprocessing of the protein.8,9Consistent with the CREB4 initiator status of caspase-2, a D333G mutant that remains a monomer, according to the examination by gel filtration, has no enzymatic activity in fluorescence substrate assays.9In contrast, other mutations that prohibit processing, but not dimerization, retain catalytic and apoptosis-inducing activity, including a C436G mutant,9which prevents the formation of the unique disulfide bond between two caspase-2 monomers.10 Although caspase-2 can form a stable dimer in solution, this may not be sufficient for full activation that may depend on specific activation platforms, which is analogous to the activation of initiator caspases-9 or -8.11,12The fact that caspase-2 is highly active and appears to autoprocess, even when expressed without prodomain, is believed to be due to self-association at high concentrations,9and this has also been documented for other caspases when bacterially expressed.13When present at endogenous levels, however, it is believed that the prodomain is essential for autoprocessing, which is supported by the observation that caspase-3 may be converted into an autoprocessing caspase with hugely enhanced killing activity when fused to the prodomain of caspase-2.14In addition to activation by autoprocessing, several studies showed that caspase-2 is readily cleaved by caspases-3, and -8, and with less efficiency also by others.1517Taken together, these experiments show that for activating caspase-2, and initiator caspases in general, dimerization is the crucial activation step and that, different from effector caspases, the appearance of cleavage products in western blotting analysis is not Sennidin B synonymous with their activation. == Interaction Partners of Caspase-2 == The number of molecules reported to interact with caspase-2 through its prodomain has steadily increased in recent years, suggesting the involvement of caspase-2 in different signaling pathways (Figure 1). The first among these proteins was the death adaptor molecule RAIDD/CRADD (receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain; CASP2 and RIPK1 domain containing adaptor with death domain),20,21which has an N-terminal CARD and a C-terminal death domain (DD). Through its CARD, RAIDD/CRADD can bind to the CARD of caspase-2 by homophilic interaction (as well as to that of theC. eleganscaspase Ced-3) that connects caspase-2 to different protein complexes. One such DD-containing protein was found to be RIP-1 kinase, which was in turn associated with TRADD (tumor necrosis factor receptor-1 (TNF-R1)-associated DD protein) and implicated a role for caspase-2 in TNF-R signaling.20However, although the association of this complex could be documented upon overexpression, its physiological relevance remains doubtful considering that neither catalytically inactive caspase-2 nor dominant-negative RAIDD constructs were able to block TNF-R1-induced cell death,20nor are cells from the relevant knock-out mice resistant to killing by TNF.22Nonetheless, some studies have in.