ICEBERG A Novel Inhibitor of Interleukin-1β Generation

(Thornberry et al., 1992). The zymogen has low but detectable enzymatic activity. Upon receipt of a proinflammatory signal, caspase-1 is thought to oligomerize and Eric W. Humke,*† Stephanie K. Shriver,‡ Melissa A. Starovasnik,‡ Wayne J. Fairbrother,‡§ and Vishva M. Dixit†§ autoprocess to generate the active p10/p20 heterodi*Department of Cellular and Molecular Biology meric protease (Walker et al., 1994; Wilson et al., 1994; University of Michigan Medical School Ghayur et al., 1997). The N-terminal prodomain appears Ann Arbor, Michigan 48109 to play a critical role in this oligomerization-based acti†Department of Molecular Oncology vation of caspase-1 since its removal prevents pro‡Department of Protein Engineering cessing (Van Criekinge et al., 1996). Genentech, Inc. At least one potential mechanism by which casSouth San Francisco, California 94080 pase-1 is regulated became evident with the identification of a serine/threonine kinase RIP2/CARDIAK/RICK (Inohara et al., 1998; McCarthy et al., 1998; Thome et al., Summary 1998) that binds caspase-1 and promotes its processing (Thome et al., 1998). RIP2 engages caspase-1 through ProIL-1b is a proinflammatory cytokine that is proteoa direct protein–protein interaction involving correlytically processed to its active form by caspase-1. sponding caspase recruitment domains (CARDs) presUpon receipt of a proinflammatory stimulus, an ent at the C terminus of RIP2 and within the prodomain upstream adaptor, RIP2, binds and oligomerizes of caspase-1 (Hofmann et al., 1997; Thome et al., 1998). caspase-1 zymogen, promoting its autoactivation. The CARD module mediates the interaction between a ICEBERG is a novel protein that inhibits generation of number of large prodomain caspases and their correIL-1b by interacting with caspase-1 and preventing its sponding upstream activator adaptors, the prototypical association with RIP2. ICEBERG is induced by proinexamples being caspase-9 and Apaf-1 (Zou et al., 1997, flammatory stimuli, suggesting that it may be part of a 1999; Day et al., 1999; Qin et al., 1999). Structurally, the negative feedback loop. Consistent with this, enforced CARD motif resembles the death domain (DD) and the retroviral expression of ICEBERG inhibits lipopolysacdeath effector domain (DED). All possess six helices and charide-induced IL-1b generation. The structure of have a propensity to self associate. These homotypic ICEBERG reveals it to be a member of the deathinteraction motifs form the molecular glue that bind the domain-fold superfamily. The distribution of surface signaling machinery responsible for caspase activation charge is complementary to the homologous prodo(for a review, see Hofmann, 1999). main of caspase-1, suggesting that charge–charge inOne powerful way to modulate assembly of such sigteractions mediate binding of ICEBERG to the prodonaling complexes is through the presence of “decoy” main of caspase-1. molecules that attenuate the assembly process. The most dramatic examples of this are virally encoded Introduction DEDs from a number of g-herpesviruses including two human oncogenic viruses (Kaposi sarcoma associatedMembers of the caspase family of aspartate-specific herpesvirus and molluscum contagiosum virus) that cysteine proteases play a crucial role in both apoptosis bind the DEDs of FADD and/or caspase-8 (FLICE) and and inflammation. The first mammalian member of this disrupt assembly of the Fas-receptor associated death family cloned, interleukin-1b converting enzyme (ICE), inducing signaling complex (Hu et al., 1997; Thome et now termed caspase-1 (for cysteinyl aspartate-specific al., 1997). These viral proteins, termed v-FLIPs (for viral protease), was originally discovered as the cytoplasmic FLICE inhibitory proteins), function to effectively switch protease responsible for the conversion of the 34 kDa off proapoptotic signaling from the Fas death receptor inactive precursor IL-1b to the mature 17 kDa proinflam(for reviews see Tschopp et al., 1998a, 1998b). The matory cytokine (Cerretti et al., 1992; Thornberry et al., v-FLIPs are closely related to c-FLIP, an endogenous 1992; Miller et al. 1993). Caspase-1 has since been cellular protein present as various splice forms, the lonshown to process the cytokine precursor of IL-18 (intergest of which (c-FLIPL) is a potent inhibitor of FADDferon-g-inducing factor) (Ghayur et al., 1997; Gu et al., mediated caspase-8 activation (Hu et al., 1997; Irmler 1997). Underscoring the importance of this protease in et al., 1997). Therefore, both viral and cellular decoy cytokine processing is the finding that caspase-1-defimolecules are capable of regulating assembly of signalcient mice are remarkably resistant to LPS-induced ening complexes held together by homotypic interactions dotoxic shock. Thus, caspase-1 is an attractive target for involving six-helix-bundle interaction domains. In a simitherapeutic intervention in inflammatory diseases (Kuida lar vein, we find that activation of caspase-1 and subseet al., 1995; Li et al., 1995). quent generation of IL-1b is regulated by a small CARDCaspase-1 is synthesized as a single-chain polypepcontaining decoy molecule termed ICEBERG. This decoy tide zymogen consisting of an N-terminal prodomain, protein binds the corresponding CARD motif present and a large (p20) and a small (p10) catalytic domain in the prodomain of caspase-1, inhibiting and/or displacing the upstream activator RIP2. ICEBERG works through this mechanism to attenuate inflammation. The § To whom correspondence should be addressed (e-mail: fairbro@