Joseph P. Boyle

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Background: Nucleotide binding and oligomerization domain-containing protein 2 (NOD2) is a protein involved in the recognition of bacterial pathogens through detection of muramyl dipeptide. Results: Purified recombinant NOD2 was found to bind ATP and muramyl dipeptide. Conclusion: NOD2 is an intracellular signaling receptor for muramyl dipeptide. Significance: These results help to define the molecular events involved in NOD2 signaling. Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.

Insights into the molecular basis of the NOD2 signalling pathway

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1β, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohns disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more ‘basic’ elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.

Comparative Genomic and Sequence Analysis Provides Insight into the Molecular Functionality of NOD1 and NOD2.

Amino acids with functional or key structural roles display higher degrees of conservation through evolution. The comparative analysis of protein sequences from multiple species and/or between homologous proteins can be highly informative in the identification of key structural and functional residues. Residues which in turn provide insight into the molecular mechanisms of protein function. We have explored the genomic and amino acid conservation of the prototypic innate immune genes NOD1 and NOD2. NOD1 orthologs were found in all vertebrate species analyzed, whilst NOD2 was absent from the genomes of avian, reptilian and amphibian species. Evolutionary trace analysis was used to identify highly conserved regions of NOD1 and NOD2 across multiple species. Consistent with the known functions of NOD1 and NOD2 highly conserved patches were identified that matched the Walker A and B motifs and provided interaction surfaces for the adaptor protein RIP2. Other patches of high conservation reflect key structural functions as predicted by homology models. In addition, the pattern of residue conservation within the leucine-rich repeat (LRR) region of NOD1 and NOD2 is indicative of a conserved mechanism of ligand recognition involving the concave surface of the LRRs.

International Union of Basic and Clinical Pharmacology. XCVI. Pattern Recognition Receptors in Health and Disease

Since the discovery of Toll, in the fruit fly Drosophila melanogaster, as the first described pattern recognition receptor (PRR) in 1996, many families of these receptors have been discovered and characterized. PRRs play critically important roles in pathogen recognition to initiate innate immune responses that ultimately link to the generation of adaptive immunity. Activation of PRRs leads to the induction of immune and inflammatory genes, including proinflammatory cytokines and chemokines. It is increasingly clear that many PRRs are linked to a range of inflammatory, infectious, immune, and chronic degenerative diseases. Several drugs to modulate PRR activity are already in clinical trials and many more are likely to appear in the near future. Here, we review the different families of mammalian PRRs, the ligands they recognize, the mechanisms of activation, their role in disease, and the potential of targeting these proteins to develop the anti-inflammatory therapeutics of the future.

Blau syndrome polymorphisms in NOD2 identify nucleotide hydrolysis and helical domain 1 as signalling regulators

Understanding how single nucleotide polymorphisms (SNPs) lead to disease at a molecular level provides a starting point for improved therapeutic intervention. SNPs in the innate immune receptor nucleotide oligomerisation domain 2 (NOD2) can cause the inflammatory disorders Blau Syndrome (BS) and early onset sarcoidosis (EOS) through receptor hyperactivation. Here, we show that these polymorphisms cluster into two primary locations: the ATP/Mg2+‐binding site and helical domain 1. Polymorphisms in these two locations may consequently dysregulate ATP hydrolysis and NOD2 autoinhibition, respectively. Complementary mutations in NOD1 did not mirror the NOD2 phenotype, which indicates that NOD1 and NOD2 are activated and regulated by distinct methods.

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

Background: Protein/protein interactions are critical for signal transduction. Results: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) helix 2 mutants impair signaling but not interaction with receptor-interacting protein 2 (RIP2). Conclusion: NOD1 and RIP2 interaction is necessary, but not sufficient, for NOD1 signaling. Significance: NOD1 signaling is more complex than previously assumed and is likely to involve multiple CARD interfaces. 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.

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.

Computational analysis predicts the Kaposi's sarcoma‐associated herpesvirus tegument protein ORF63 to be alpha helical

The innate immune response provides our first line of defence against infection. Over the course of evolution, pathogens have evolved numerous strategies to either avoid activating or to limit the effectiveness of the innate immune system. The Kaposis sarcoma‐associated herpesvirus (KSHV) contains tegument proteins in the virion that contribute to immune evasion and aid the establishment of viral infection. For example, the KSHV tegument protein ORF63 modulates inflammasome activation to inhibit the innate immune response against the virus. Understanding the likely structure of proteins involved in immune evasion enables potential mechanisms of action to be proposed. To understand more fully how ORF63 modulates the innate immune system we have utilized widely available bioinformatics tools to analyze the primary protein sequence of ORF63 and to predict its secondary and tertiary structure. We found that ORF63 is predicted to be almost entirely alpha‐helical and may possess similarity to HEAT repeat containing proteins. Consequently, ORF63 is unlikely to be a viral homolog of the NLR protein family. ORF63 may inhibit the innate immune response by flexibly interacting with its target protein and inhibiting the recruitment of protein co‐factors and/or conformational changes required for immune signaling. Proteins 2012;

Cell Swelling and the NLRP3 Inflammasome

Multiple chemically and biologically diverse stimuli activate the NLRP3 inflammasome, but the precise mechanism for activation remains to be determined (Davis et al., 2011xDavis, B.K., Wen, H., and Ting, J.P.-Y. Annu. Rev. Immunol. 2011; 29: 707–735Crossref | PubMed | Scopus (433)See all ReferencesDavis et al., 2011). Cellular perturbation and homeostatic destabilization also activate NLRP3. Consistent with this, in the September 2012 issue of Immunity, Compan et al. (2012)xCompan, V., Baroja-Mazo, A., Lopez-Castejon, G., Gomez, A.I., Martinez, C.M., Angosto, D., Montero, M.T., Herranz, A.S., Bazan, E., Reimers, D. et al. Immunity. 2012; 37: 487–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (67)See all ReferencesCompan et al. (2012) presented data suggesting that the NLRP3 inflammasome is activated by cell swelling. Cell swelling followed by a restorative reduction in volume resulted in caspase-1 activation in murine bone-marrow-derived macrophages and macrophages from the teleost fish Sparus aurata (gilthead seabream) (Figure 2 in Compan et al., 2012xCompan, V., Baroja-Mazo, A., Lopez-Castejon, G., Gomez, A.I., Martinez, C.M., Angosto, D., Montero, M.T., Herranz, A.S., Bazan, E., Reimers, D. et al. Immunity. 2012; 37: 487–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (67)See all ReferencesCompan et al., 2012). Murine cells lacking NLRP3 (or ASC or caspase-1) did not process interleukin-1β in response to cell swelling, leading to the conclusion that the NLRP3 inflammasome mediates the cellular response to cell swelling and that this is evolutionarily conserved from fish to mammals.Various NLR-specific expansions have been identified in fish. Generally they cannot be identified as full orthologs of specific mammalian NLRs. However, a number show greatest similarity to the NACHT domain of NLRC3 (Huang et al., 2008xHuang, S., Yuan, S., Guo, L., Yu, Y., Li, J., Wu, T., Liu, T., Yang, M., Wu, K., Liu, H. et al. Genome Res. 2008; 18: 1112–1126Crossref | PubMed | Scopus (166)See all ReferencesHuang et al., 2008; Laing et al., 2008xLaing, K.J., Purcell, M.K., Winton, J.R., and Hansen, J.D. BMC Evol. Biol. 2008; 8: 42Crossref | PubMed | Scopus (90)See all ReferencesLaing et al., 2008). To date there are, to our knowledge, no published reports of direct NLRP3 orthologs in fish.We have consequently performed a detailed search of the currently available fish genome assemblies in ENSEMBL for evidence of NLRP3 orthologs. These encompassed Takifugu rubripes (fugu), Xiphophorus maculatus (platyfish), Gasterosteus aculeatus (stickleback), Gadus morhua (cod), Latimeria chalumnae (coelacanth), Tetraodon nigroviridis (tetraodon), Oreochromis niloticus (tilapia), and Danio rerio (zebrafish).BLASTP searches with the amino acid sequence of human NLRP3 (NCBI accession number NP_004886.3) identified a gene on zebrafish chromosome 17 (ENSEMBL gene ID ENSDARG00000090946) that, upon reciprocal BLASTP analysis against the nonredundant human protein database, returned NLRP3 as the most significant hit. However, the domain organization of the zebrafish protein differs from that of mammalian NLRP3, consisting of an NTPase domain, leucine-rich repeats, and a C-terminal PRY-SPRY domain. Homology searches failed to identify a specific N-terminal effector domain. Hence it is questionable whether this protein can be described as a true NLRP3 ortholog. Importantly, we were unable to identify any other putative NLRP3 orthologs in the other fish genomes with human NLRP3, or indeed the supposed zebrafish NLRP3, as search terms. Instead, NLR expansions with greatest similarity to the NLRC3 NTPase were returned as hits. All the fish, except the tetraodon, possessed clear orthologs of ASC.It is plausible that NLRP3-like functionality is provided by a fish-specific NLR expansion, such as NLR-B, the NACHT of which phylogentically clusters with the mammalian NLRPs (Laing et al., 2008xLaing, K.J., Purcell, M.K., Winton, J.R., and Hansen, J.D. BMC Evol. Biol. 2008; 8: 42Crossref | PubMed | Scopus (90)See all ReferencesLaing et al., 2008). However, Sparus aurata macrophages do not respond to classical NLRP3 activators including ATP, nigericin, alum crystals, and monosodium urate crystals (Figure 2E in Compan et al., 2012xCompan, V., Baroja-Mazo, A., Lopez-Castejon, G., Gomez, A.I., Martinez, C.M., Angosto, D., Montero, M.T., Herranz, A.S., Bazan, E., Reimers, D. et al. Immunity. 2012; 37: 487–500Abstract | Full Text | Full Text PDF | PubMed | Scopus (67)See all ReferencesCompan et al., 2012), suggesting that this may not be the case. The absence of direct NLRP3 orthologs and the lack of classical NLRP3 activity in the teleost indicate that reconsideration of the evolutionary and mechanistic basis for the observed inflammatory response to cell swelling is needed.

Structure-based alignment of human caspase recruitment domains provides a framework for understanding their function

Intracellular signalling is driven by protein-protein interactions. Members of the Death Domain superfamily mediate protein-protein interactions in both cell death and innate immune signalling pathways. They drive the formation of macromolecular complexes that act as a scaffold for protein recruitment and downstream signal transduction. Death Domain family members have low sequence identity, complicating their identification and predictions of their structure and function. We have taken all known human caspase recruitment domains (CARDs), a subfamily of the Death Domain superfamily, and generated a structure-guided sequence alignment. This alignment has enabled the identification of 14 positions that define the hydrophobic core and present a template for the identification of novel CARD sequences. We identify a conserved salt bridge in over half of all human CARDs and find a subset of CARDs likely to be regulated by tyrosine phosphorylation in their type I interface. Our alignment highlights that the CARDs of NLRC3 and NLRC5 are likely to be pseudodomains that have lost some of their original functionality. Together these studies demonstrate the benefits of structure-guided sequence alignments in understanding protein functionality.