Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappaB

Apaf-1 and Nod1 are members of a protein family, each of which contains a caspase recruitment domain (CARD) linked to a nucleotide-binding domain, which regulate apoptosis and/or NF-kappaB activation. Nod2, a third member of the family, was identified. Nod2 is composed of two N-terminal CARDs, a nucleotide-binding domain, and multiple C-terminal leucine-rich repeats. Although Nod1 and Apaf-1 were broadly expressed in tissues, the expression of Nod2 was highly restricted to monocytes. Nod2 induced nuclear factor kappaB (NF-kappaB) activation, which required IKKgamma and was inhibited by dominant negative mutants of IkappaBalpha, IKKalpha, IKKbeta, and IKKgamma. Nod2 interacted with the serine-threonine kinase RICK via a homophilic CARD-CARD interaction. Furthermore, NF-kappaB activity induced by Nod2 correlated with its ability to interact with RICK and was specifically inhibited by a truncated mutant form of RICK containing its CARD. The identification of Nod2 defines a subfamily of Apaf-1-like proteins that function through RICK to activate a NF-kappaB signaling pathway.     

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD–NACHT linker and one in the NACHT itself.     

Polypeptide modulators of caspase recruitment domain (CARD)-CARD-mediated protein-protein interactions

The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display.     

Human CARD4 protein is a novel CED-4/Apaf-1 cell death family member that activates NF-kappaB

The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.     

Rapid folding and unfolding of Apaf-1 CARD

Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an alpha-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.     

Structural insights into the MDP binding and CARD–CARD interaction in zebrafish (Danio rerio) NOD2: a molecular dynamics approach

Nucleotide binding and oligomerization domain (NOD2) is a key component of innate immunity that is highly specific for muramyl dipeptide (MDP)—a peptidoglycan component of bacterial cell wall. MDP recognition by NOD2–leucine rich repeat (LRR) domain activates NF‐κB signaling through a protein–protein interaction between caspase activating and recruitment domains (CARDs) of NOD2 and downstream receptor interacting and activating protein kinase 2 (RIP2). Due to the lack of crystal/NMR structures, MDP recognition and CARD–CARD interaction are poorly understood. Herein, we have predicted the probable MDP and CARD–CARD binding surfaces in zebrafish NOD2 (zNOD2) using various in silico methodologies. The results show that the conserved residues Phe819, Phe871, Trp875, Trp929, Trp899, and Arg845 located at the concave face of zNOD2–LRR confer MDP recognition by hydrophobic and hydrogen bond (H‐bond) interactions. Molecular dynamics simulations reveal a stable association between the electropositive surface on zNOD2–CARDa and the electronegative surface on zRIP2–CARD reinforced mostly by H‐bonds and electrostatic interactions. Importantly, a 3.5 Å salt bridge is observed between Arg60 of zNOD2–CARDa and Asp494 of zRIP2–CARD. Arg11 and Lys53 of zNOD2–CARDa and Ser498 and Glu508 of zRIP2–CARD are critical residues for CARD–CARD interaction and NOD2 signaling. The 2.7 Å H‐bond between Lys104 of the linker and Glu508 of zRIP2–CARD suggests a possible role of the linker for shaping CARD–CARD interaction. These findings are consistent with existing mutagenesis data. We provide first insight into MDP recognition and CARD–CARD interaction in the zebrafish that will be useful to understand the molecular basis of NOD signaling in a broader perspective. Copyright © 2014 John Wiley & Sons, Ltd.     

CIPER, a novel NF kappaB-activating protein containing a caspase recruitment domain with homology to Herpesvirus-2 protein E10

We have identified and characterized CIPER, a novel protein containing a caspase recruitment domain (CARD) in its N terminus and a C-terminal region rich in serine and threonine residues. The CARD of CIPER showed striking similarity to E10, a product of the equine herpesvirus-2. CIPER formed homodimers via its CARD and interacted with viral E10 but not with several apoptosis regulators containing CARDs including ARC, RAIDD, RICK, caspase-2, caspase-9, or Apaf-1. Expression of CIPER induced NF-kappaB activation, which was inhibited by dominant-negative NIK and a nonphosphorylable IkappaB-alpha mutant but not by dominant-negative RIP. Mutational analysis revealed that the N-terminal region of CIPER containing the CARD was sufficient and necessary for NF-kappaB-inducing activity. Point mutations in highly conserved residues in the CARD of CIPER disrupted the ability of CIPER to activate NF-kappaB and to form homodimers, indicating that the CARD is essential for NF-kappaB activation and dimerization. We propose that CIPER acts in a NIK-dependent pathway of NF-kappaB activation.     

CARD games in apoptosis and immunity

A bewildering array of proteins containing the caspase recruitment domain (CARD) have now been identified. Previously, CARD-CARD interactions have been shown to be involved in the assembly of protein complexes that promote caspase processing and activation in the context of apoptosis. However, as the family of CARD-containing proteins has grown, it has become apparent that the majority of these proteins do not recruit caspases or promote caspase activation. Instead, many participate in NF-kappaB signalling pathways associated with innate or adaptive immune responses. Here, we suggest a simplified classification of the CARD proteins based upon their domain structures and discuss the divergent roles of these proteins in the context of host defence.     

Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.     

Engagement of Nucleotide-binding Oligomerization Domain-containing Protein 1 (NOD1) by Receptor-interacting Protein 2 (RIP2) Is Insufficient for Signal Transduction

Following activation, the cytoplasmic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1) interacts with its adaptor protein receptor-interacting protein 2 (RIP2) to propagate immune signaling and initiate a proinflammatory immune response. This interaction is mediated by the caspase recruitment domain (CARD) of both proteins. Polymorphisms in immune proteins can affect receptor function and predispose individuals to specific autoinflammatory disorders. In this report, we show that mutations in helix 2 of the CARD of NOD1 disrupted receptor function but did not interfere with RIP2 interaction. In particular, N43S, a rare polymorphism, resulted in receptor dysfunction despite retaining normal cellular localization, protein folding, and an ability to interact with RIP2. Mutation of Asn-43 resulted in an increased tendency to form dimers, which we propose is the source of this dysfunction. We also demonstrate that mutation of Lys-443 and Tyr-474 in RIP2 disrupted the interaction with NOD1. Mapping the key residues involved in the interaction between NOD1 and RIP2 to the known structures of CARD complexes revealed the likely involvement of both type I and type III interfaces in the NOD1·RIP2 complex. Overall we demonstrate that the NOD1-RIP2 signaling axis is more complex than previously assumed, that simple engagement of RIP2 is insufficient to mediate signaling, and that the interaction between NOD1 and RIP2 constitutes multiple CARD-CARD interfaces.     

The tandem CARDs of NOD2: intramolecular interactions and recognition of RIP2

Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-κB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs.     

Structural transformation-mediated dimerization of caspase recruitment domain revealed by the crystal structure of CARD-only protein in frog virus 3

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.     

Crystal Structure of a Complex of NOD1 CARD and Ubiquitin

The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance.     

An induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways

Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich repeats that associates with the CARD-containing kinase RICK and activates nuclear factor kappaB (NF-kappaB). We show that self-association of Nod1 mediates proximity of RICK and the interaction of RICK with the gamma subunit of the IkappaB kinase (IKKgamma). Similarly, the RICK-related kinase RIP associated via its intermediate region with IKKgamma. A mutant form of IKKgamma deficient in binding to IKKalpha and IKKbeta inhibited NF-kappaB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKgamma, IKKalpha, or IKKbeta was sufficient for induction of NF-kappaB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an important role for NF-kappaB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor alpha receptor.     

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.     

CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-kappaB activation and apoptosis.

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.     

Novel Disulfide Bond-Mediated Dimerization of the CARD Domain Was Revealed by the Crystal Structure of CARMA1 CARD

CARMA1, BCL10 and MALT1 form a large molecular complex known as the CARMA1 signalosome during lymphocyte activation. Lymphocyte activation via the CARMA1 signalosome is critical to immune response and linked to many immune diseases. Despite the important role of the CARMA1 signalosome during lymphocyte activation and proliferation, limited structural information is available. Here, we report the dimeric structure of CARMA1 CARD at a resolution of 3.2 Å. Interestingly, although CARMA1 CARD has a canonical six helical-bundles structural fold similar to other CARDs, CARMA1 CARD shows the first homo-dimeric structure of CARD formed by a disulfide bond and reveals a possible biologically important homo-dimerization mechanism.     

L-Ala-γ-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interaction with nucleotide-binding oligomerization domain 1

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 μM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 μM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 μM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.     

Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1

Activation of procaspase-9, a key component of the apoptosis mechanism, requires the interaction of its caspase recruitment domain (CARD) with the CARD in the adaptor protein Apaf-1. Using nuclear magnetic resonance spectroscopy and mutagenesis we have determined the structure of the CARD from Apaf-1 and the residues important for binding the CARD in procaspase-9. Apaf-1's CARD contains seven short alpha-helices with the core six helices arranged in an antiparallel manner. Residues in helix 2 have a central role in mediating interaction with procaspase-9 CARD. This interaction surface is distinct from that proposed based on the structure of the CARD from RAIDD, but is coincident with that of the structurally similar FADD death effector domain and the Apaf-1 CARD interface identified by crystallographic studies.