Oncogene (2001) 20, 6473 ± 6481 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc The NOD: a signaling module that regulates apoptosis and host defense against pathogens Naohiro Inohara1 and Gabriel NunÄ ez*,1 1Department of Pathology and Comprehensive Cancer Center, The University of Michigan Medical School, Ann Arbor, Michigan, MI 48109, USA Nods, a growing family of proteins containing a molecule that induces systemic acquired resistance nucleotide-binding oligomerization domain (NOD), are (SAR) (Feys and Parker, 2000). In higher multicellular involved in the regulation of programmed cell death organisms, appropriate development and tissue home- (PCD) and immune responses. Members of the family ostasis require highly regulated activation of PCD. include Apaf-1, Ced-4, Nod1, Nod2, and the cytosolic Whereas PCD induced during the defense against products of plant disease resistance genes. The NOD pathogens in both animal and plants is accompanied module is homologous to the ATP-binding cassette by the production of pro-in¯ammatory molecules, that (ABC) found in a large number of proteins with diverse associated with tissue development and homeostasis is biological function. The centrally located NOD promotes devoid of an in¯ammatory response (Jacobson et al., activation of eector molecules through self-association 1997; Feys and Parker, 2000). Despite these dierences, and induced proximity of binding partners. The C- both types of PCD are associated with the induction of terminal domain of Nods serves as a sensor for similar morphological features including membrane intracellular ligands, whereas the N-terminal domain blebbing, cytosolic fragmentation, nuclear condensa- mediates binding to dowstream eector molecules and tion and fragmentation as well as biochemical events activation of diverse signaling pathways. Thus, Nods such as degradation of genomic DNA, proteolysis and activate, through the NOD module, diverse signaling redistribution of membrane lipids (Kerr et al., 1972; pathways involved in the elimination of cells via PCD Wyllie, 1980). These changes associated with PCD, and the host defense against pathogens. Oncogene generally referred as apoptosis, are largely due to the (2001) 20, 6473 ± 6481. activation of caspases, a family of cysteine proteases (NuÂnÄ ez et al., 1998; Kumar, 1999). Mammalian cells Keywords: apoptosis; Apaf-1/Nod1/CED4 family; pro- have more than 14 caspases and synthesize them as grammed cell death; ATPase/GTPases; NOD domain enzymatically inactive forms. Once an apical caspase such as caspase-8 or caspase-9 is activated, the protease activates downstream (eector) caspases and non- caspase proteases causing massive proteolytic activity in the cytosol and the orderly demise of the cell Introduction (Kumar, 1999). Therefore, activation of apical caspases is a critical step in that it determines cell fate and as Programmed cell death (PCD) or apoptosis plays a such is strictly regulated by speci®c regulatory critical role not only during embryonic development molecules. and cellular homeostasis in the adult, but also in the clearance of invading pathogens by host tissues (Weinrauch and Zychlinsky, 1999). Multicellular Ced-4 and Apaf-1 contain a NOD domain that is critical organisms have several host defense systems to prevent for functional activity proliferation and propagation of invading pathogens. One of the most ancient types of immune reactions is Early genetic studies in the nematode Caenorhabditis the elimination of pathogen infected cells by PCD elegans revealed molecules which play critical roles in (Weinrauch and Zychlinsky, 1999). In addition, the induction, regulation and execution of PCD during infected host cells produce defensins and pro-in¯am- worm development. Ced-3 and Ced-4 are required for matory cytokines that play an important role in the the execution step of PCD, whereas Ced-9 protects clearance of the invading pathogen. In plants, patho- cells from PCD during normal development (Ellis and gens trigger a specialized form of PCD known as the Horvitz, 1986; Metzstein et al., 1998). Homology hypersensitive response (HR) and the production of analysis and functional assays demonstrated that defensins and salicylic acid, a phenolic signaling Ced-3 is a caspase, Ced-4 is the activator of Ced-3 and Ced-9 is a pro-survival member of the Bcl-2 family (Yuan and Horvitz, 1992; Yuan et al., 1993; *Correspondence: G NunÄ ez; E-mail: [email protected] Hengartner and Horvitz, 1994; Chinnaiyan et al., Proteins containing nucleotide-binding oligomerization domains N Inohara and G NunÄez 6474 1997a). Both Ced-3 and Ced-4 have N-terminal Ced-4 and Apaf-1 activate target caspases through caspase-recruitment domains (CARDs) that associate induced proximity with each other through homophilic interaction between their CARDs (Hofmann et al., 1997; Wu et The structural similarity between Ced-4 and Apaf-1 al., 1997a; Irmler et al., 1997; Seshagiri et al., 1998). re¯ects the fact that both molecules use a common Ced-4 has an additional module, the nucleotide-binding mechanism to activate their target caspases. The N- domain (NBD), which is essential for Ced-3 activation terminal CARD of each protein mediates interaction (Chinnaiyan et al., 1997b; Chaudhary et al., 1998). with that of the target caspase, whereas the NOD self- Biochemical approaches to identify cytosolic factors associates and oligomerizes (Figure 1a). Through their that activate human caspase-3, an eector caspase, NODs and CARDs, Apaf-1 and presumably Ced-4 revealed a cell death machinery in mammals similar form a large protein complex containing oligomerized to that identi®ed by genetic studies in C. elegans. Apaf-1/Ced-4 bound to caspase-9 or Ced-3, respec- Puri®cation of caspase-3 activators and functional tively, which allows proximity and enzymatic self- reconstitution assays revealed caspase-9 as a direct activation of the bound caspases (Yang et al., 1998; Hu activator of caspase-3 (Zou et al., 1997). Apaf-1 was et al., 1998a; Srinivasula et al., 1998; Salvesen and identi®ed as the activator of caspase-9, a step that Dixit, 1999). The mechanism by which these caspases requires dATP/ATP and cytochrome c, a ligand for are activated by Ced-4 and Apaf-1 appears to be Apaf-1 released from damaged mitochondria (Li et similar to that proposed for activation of caspase-8 in al., 1997). Sequence analysis revealed that Apaf-1 is the death-inducing receptor complex (DISC) (Muzio et structurally and functionally related to the nematode al., 1998; Salvesen and Dixit, 1999). Caspase-8 is Ced-4 (Zou et al., 1997). The N-terminal CARD of another mammalian apical caspase linked to death Apaf-1 is required for interaction with the CARD of receptor signaling pathways (Boldin et al., 1996; Muzio caspase-9 and is fused to a centrally located NBD et al., 1996). Caspase-8 binds to its upstream activator (Zou et al., 1997; Li et al., 1997). The NBD of Apaf- FADD (also called MORT1) through an homophilic 1 and Ced-4 is homologous to the ABC-ATP/GTPase interaction via their N-terminal CARD-related a helix- module that is present in a large number of proteins rich domains, namely the death eector domains with diverse biological function (Walker et al., 1982). (DEDs) (Boldin et al., 1995; Chinnaiyan et al., 1995; Both the ABC domain present in these ATP/GTPases Bertin et al., 1997). FADD also has a C-terminal death and the NBD of Apaf-1/Ced-4 contain conserved domain (DD) which binds to DD-containing receptors binding sites for nucleotide (P-loop or Walker's A or adapter molecules and mediates recruitment of box) and Mg2+ (Walker's B box) (Walker et al., caspase-8 to the membrane receptor complex triggered 1982; Seshagiri and Miller, 1997; Chinnaiyan et al., upon binding of the trimeric ligand (Boldin et al., 1995, 1997b; Chaudhary et al., 1998; Jaroszewski et al., 1996; Chinnaiyan et al., 1995; Muzio et al., 1996). 2000). In addition, the NBD of both Apaf-1 and Trimerization of death receptors (DRs) induces the CED-4 mediates protein homooligomerization, a proximity and activation of caspase-8 (Muzio et al., function that is critical for Apaf-1/Ced-4 activity 1998). A model for caspase-8 activation in the tumor (Chaudhary et al., 1998; Yang et al., 1998; Hu et al., necrosis factor receptor-1 (TNFR1) signaling pathway 1998a; Srinivasula et al., 1998). Therefore, we refer is shown in Figure 1b. Although the caspase-8/DISC here to the NBD present in Apaf-1 and homologues and caspase-9/Apaf-1 machineries have signi®cant as nucleotide-binding oligomerization domain (NOD) dierences, enzymatic activation of caspases in both and to structurally related NOD-containing proteins systems appear to require binding to their upstream such as Apaf-1 and Nod1 as Nods. Although the regulators and the induced proximity of apical caspases NOD mediates both nucleotide binding and oligo- though oligomerization (or trimerization) (Inohara et merization, studies with Apaf-1 and Ced-4 have not al., 2000). revealed dierent subdomains within the NOD that mediate these activities (Hu et al., 1998a; Yang et al., 1998). Regulation of Ced-4/Apaf-1 oligomerization and caspase Apaf-1 also has a C-terminal regulatory domain recruitment containing WD40 repeats (WDR), which is essential for the ability of Apaf-1 to respond to cytochrome c Because caspase activation plays a critical role in (Zou et al., 1997; Li et al., 1997; Hu et al., 1999). determining cell fate, oligomerization of caspase Analyses of mice lacking Apaf-1 and caspase-9 activators such as Apaf-1 and the subsequent recruit- revealed that Apaf-1 and caspase-9 play important ment of target caspases to their activators are highly roles in apoptosis during development of multiple regulated events. For example, activation of Ced-3 by tissues including brain, limb and eye, comparable to Ced-4 is regulated by Ced-9.