Pattern-Recognition Receptors in Human Disease

A phylogenetic and functional overview of inflammatory caspases and caspase-1-related CARD-only proteins

K. Kersse, T. Vanden Berghe, M. Lamkanfi, P. Vandenabeele


Caspase 1 is a cysteinyl aspartate-specific proteinase involved in the maturation of inflammatory cytokines such as pro-IL-1β (interleukin-1β) and pro-IL-18. Caspase 1 clusters phylogenetically together with human caspases 4, 5 and 12 and murine caspases 11 and 12, and forms the group of the so-called inflammatory caspases. Caspase 1 consists of an N-terminal CARD (caspase recruitment domain) and a proteolytic domain containing the catalytic residues. The CARD-containing prodomain is involved in the formation of the protease-activating inflammasome complex. We have also found that the prodomain is necessary and sufficient for the activation of NF-κB (nuclear factor κB). The human genome also contains three caspase-1-related CARD-only decoy proteins [COP (CARD-only protein), INCA (inhibitory CARD) and ICEBERG], which are located near the caspase 1 locus. In this mini-review, we focus on the evolutionary aspects of the inflammatory caspase locus in the human, chimpanzee, Rhesus monkey, mouse and rat. Furthermore, we discuss the functional characteristics of the caspase-1-related CARD-only proteins in relation to caspase-1-mediated IL-1β maturation and NF-κB activation.

  • apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)
  • caspase 1
  • caspase recruitment domain (CARD)
  • inflammation
  • interleukin-1β (IL-1β)
  • nuclear factor κB (NF-κB)

Phylogenetic analysis of the caspase 1 locus

Caspase 1 was the first member of an evolutionarily conserved family of cysteine-dependent aspartate-specific proteases, better known as caspases, to be reported. Originally, caspase 1 was identified as a protease that proteolytically activates the proforms of IL-1β (interleukin-1β) and IL-18 in monocytes and macrophages. Together with caspases 4, 5, 11 and 12, caspase 1 constitutes the subset of inflammatory caspases [1]. These are termed ‘inflammatory’ because the main caspase 1 substrates identified to date are cytokines that are crucial mediators of innate immunity and inflammation [2]. Moreover, human, chimpanzee, Rhesus monkey, mouse and rat inflammatory caspases share a significant degree of similarity and are organized in a single locus. The locus in primates encodes four proteins, caspases 12, 4, 5 and 1, while the locus in rodents encodes three inflammatory caspases, caspases 1, 11 and 12 (Figure 1A). Sequence analysis of the inflammatory caspases suggests that caspases 4 and 5 probably arose following the duplication of a caspase 11 ancestor gene [3]. A common denominator for all mammalian inflammatory caspases is the presence of an N-terminal CARD (caspase recruitment domain). This CARD motif is a member of the DD (death domain) superfamily, which further encompasses the DD, the DED (death effector domain) and the Pyrin domain [4]. These homotypic interaction motifs are generally characterized by the presence of approx. 90 amino acids that are organized in six antiparallel amphipathic α-helices, the so-called DD fold [4].

Figure 1 An overview of inflammatory caspases and caspase-1-regulating complexes

(A) Chromosomal organization of the inflammatory caspase locus in the human, chimpanzee, Rhesus monkey, mouse and rat. (B) Phylogenetic analysis of inflammatory caspases based on the conserved CARD domain. Sequences were aligned by using ClustalW (gap weight=10.00; gap length weight=0.05). Trees were visualized in Mega 4.0. Homo sapiens (h), Pan troglodytes (p), Macaca mulatta (mac), Mus musculus (m) and Rattus norvegicus (r). Asterisks indicate inactive caspase. (C) Schematic representation of the three inflammasome components, including illustration of the interference points of the CARD-only and pyrin-only proteins. (D) The ratio of ASC/Pycard to RIP2 determines the functional outcome for caspase 1, either IL-1β maturation or NF-κB activation.

Besides encoding full-length biologically active inflammatory caspases, the human genome also encodes three CARD-only proteins, COP (CARD-only protein), INCA (inhibitory CARD) and ICEBERG, sharing a high degree of homology with the CARD prodomain of caspase 1 (Figure 1B) [5,6]. Interestingly, no orthologues of these proteins can be retrieved in the mouse or rat. However, putative orthologues of COP and INCA encoded on chromosome 11 of the chimpanzee and of COP, INCA and ICEBERG encoded on chromosome 14 of the Rhesus monkey can be identified (Figure 1B). This observation suggests that these CARD-only proteins must have emerged by gene duplication in an ancestral mammal during the divergence of rodents from primates. Paradoxically, an orthologue of ICEBERG, the most divergent of the caspase-1-related COPs, could only be retrieved in the Rhesus monkey genome, but not in the chimpanzee genome, although the Rhesus monkey, as a member of the old world monkeys, diverged earlier from the human–chimpanzee lineage. This may be due to the fact that the chimpanzee genome still contains some gaps at this locus on chromosome 9. Another fact demonstrating the plasticity and rapid evolution of the inflammatory caspase locus is the incorporation of SNPs (single nucleotide polymorphisms) into the human caspase 12 gene. These SNPs either result in the production of a COP or a full-length caspase 12, although lacking enzymatic activity since the conserved catalytic SHG (Ser-His-Gly) box remains mutated to SHS (Ser-His-Ser) [7]. On the other hand, both the chimpanzee and the Rhesus monkey genome encode full-length enzymatically active caspase 12 (Figure 1A). This indicates that the human-specific nature of the caspase 12 SNPs is a recent evolutionary event.

Caspase 1 activation and regulation

Like all other caspases, caspase 1 is synthesized as an inactive zymogen that needs to be processed in order to become enzymatically active. In the case of caspase 1, the activation occurs in large, multimeric protein platforms commonly referred to as inflammasomes [1]. In general, these complexes are composed of three distinct building blocks: a sensor, an adaptor and an effector (Figure 1C). Formation of the inflammasome starts with a sensor-platform protein belonging to the NLR [NACHT–LRR (leucine-rich repeat)] family that senses the presence of an instigating factor through its LRR, an evolutionarily conserved ligand-sensing domain. The best studied NLR-family members to date are NALP1 (NACHT-, LRR- and pyrin domain-containing protein 1), sensing Bacillus anthracis toxin, NALP3, sensing gout-associated uric acid crystals, bacterial RNA and bacterial pathogens, such as Staphylococcus aureus and Listeria monocytogenes, and Ipaf (IL-1β-converting enzyme protease-activating factor) that specifically detects the presence of flagellin from intracellular bacteria, such as Salmonella Typhimurium and Legionella pneumophila [1]. Adjacent to the LRR domain, these sensor-platform proteins also contain a nucleotide-binding oligomerization (NACHT) domain and a homotypic interaction motif belonging to the DD fold superfamily, a CARD or a pyrin. These DD motifs mediate, either directly or indirectly through the adaptor ASC (apoptosis-associated speck-like protein containing a CARD), the recruitment of caspase 1 effector molecules to the inflammasome. ASC, containing an N-terminal pyrin and a C-terminal CARD, ensures the link between the pyrin-containing NLR-family members and the CARD-containing caspase 1 and is therefore an essential component for inflammasome formation [1]. Once some caspase 1 molecules are recruited to the inflammasome, they process and thus activate one another before they get released into the cytosol as homodimers. Besides the maturation of IL-1β and IL-18, caspase 1 is also responsible for pyroptosis, an inflammatory form of cell death induced by Salmonella Typhimurium and other intracellular bacterial and viral pathogens [8]. Although mature IL-1β has many beneficial effects, excessive production is harmful and needs to be kept in check. One group of proteins safeguarding IL-1β levels by preventing activation of caspase 1 are the earlier described caspase-1-related COPs. They all interact with caspase 1 and thereby prevent recruitment and activation of caspase 1 to the inflammasome complexes (Figure 1C) [5,6]. Studies in caspase-12-deficient mice suggest that caspase 12 has a dominant-negative effect on caspase 1 independent of its catalytic activity. Therefore the human CARD-only variant of caspase 12 might have evolved as an additional CARD-only protein [9]. Another group of proteins with a similar action mechanism are the pyrin-only proteins, POP1 (pyrin-only protein 1) and POP2, which disturb inflammasome assembly by interfering with pyrin–pyrin interactions (Figure 1C) [10,11]. Apparently, poxviruses have evolved to encode a pyrin-only protein with similar inflammasome-interfering properties [12].

Caspase-1-mediated NF-κB (nuclear factor κB) activation

In addition to the well-established role of caspase 1 in the maturation of IL-1β and IL-18, caspase 1 is also capable of activating the nuclear factor of the κ-enhancer in B-cells, better known as NF-κB [13]. The activation of this pro-inflammatory transcription factor is entirely dependent on the CARD domain of caspase 1, while the catalytic domain is dispensable. The signalling pathway propagates through a homotypic interaction with the C-terminal CARD of RIP2 (receptor-interacting protein 2), a serine/threonine kinase involved in NF-κB activation induced by NOD (nucleotide-binding and oligomerization domain) 1 and 2, two members of the NLR family [13,14]. Apparently, there is some competition between ASC and RIP2 for the interaction with caspase 1. Depending on the ratio of ASC to RIP2, caspase 1 will either be activated in inflammasome complexes or give rise to NF-κB activation (Figure 1D) [15]. Remarkably, of the three caspase-1-related CARD-only proteins, it is only COP, the most homologous with caspase 1 CARD, which binds RIP2 and induces NF-κB activity. Neither INCA nor ICEBERG displays these characteristics [6,13].


This work was supported in part by grants from the Research Fund of Ghent University (Geconcerteerde Onderzoekstacties no. 12.0514.03 and 12.0505.02), the Interuniversity Poles of Attraction Programme–Belgian Science Policy (P6/18), the European Union (DeathTrain, MRTN-CT-035624) and the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (2G.0218.06 and G.0133.05).


  • Pattern-Recognition Receptors in Human Disease: A Biochemical Society Focused Meeting held at Queens' College, University of Cambridge, Cambridge, U.K., 8–10 August 2007. Organized and Edited by C. Bryant (Cambridge, U.K.), K. Fitzgerald (University of Massachusetts Medical School, U.S.A.), N. Gay (Cambridge, U.K.), P. Morley (GlaxoSmithKline, U.K.) and L. O'Neill (Trinity College Dublin, Ireland).

Abbreviations: CARD, caspase recruitment domain; ASC, apoptosis-associated speck-like protein containing a CARD; COP, CARD-only protein; DD, death domain; IL-1β, interleukin-1β; INCA, inhibitory CARD; LRR, leucine-rich repeat; NALP, NACHT-, LRR- and pyrin domain-containing protein; NF-κB, nuclear factor κB; NLR, NACHT–LRR; POP1, pyrin-only protein 1; RIP2, receptor-interacting protein 2; SNP, single nucleotide polymorphism


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