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Evaluation of Nod-like receptor (NLR) effector domain interactions.

ABSTRACT: Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-kappaB activation.In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well as kinase RIPK2 and inflammatory caspases (C1, C2, C4, C5) under identical conditions. Our results confirmed the interaction of NOD1 and NOD2 with RIPK2, and between NLRP3 and ASC, but most importantly, our studies revealed hitherto unrecognized interactions of NOD2 with members of the NLRP subfamily. We found that NOD2 specifically and directly interacts with NLRP1, NLRP3 and NLRP12. Furthermore, we observed homodimerization of the RIPK2 CARD domains and identified residues in NOD2 critical for interaction with RIPK2.In conclusion, our work provides further evidence for the complex network of protein-protein interactions underlying NLR function.

SUBMITTER: Wagner RN

PROVIDER: S-EPMC2660581 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Evaluation of Nod-like receptor (NLR) effector domain interactions.

Wagner Roland N RN Proell Martina M Kufer Thomas A TA Schwarzenbacher Robert R

PloS one 20090401 4

Members of the Nod-like receptor (NLR) family recognize intracellular pathogens and recruit a variety of effector molecules, including pro-caspases and kinases, which in turn are implicated in cytokine processing and NF-kappaB activation.In order to elucidate the intricate network of NLR signaling, which is still fragmentary in molecular terms, we applied comprehensive yeast two-hybrid analysis for unbiased evaluation of physical interactions between NLRs and their adaptors (ASC, CARD8) as well ...[more]

PMID: 19337385

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Project description:Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.

Project description:Plant nucleotide-binding leucine-rich repeat (NLR) receptors mediate immune responses by directly or indirectly sensing pathogen-derived effectors. Despite significant advances in the understanding of NLR-mediated immunity, the mechanisms by which pathogens evolve to suppress NLR activation triggered by cognate effectors and gain virulence remain largely unknown. The agronomically important immune receptor RB recognizes the ubiquitous and highly conserved IPI-O RXLR family members (e.g., IPI-O1) from Phytophthora infestans, and this process is suppressed by the rarely present and homologous effector IPI-O4. Here, we report that self-association of RB via the coiled-coil (CC) domain is required for RB activation and is differentially affected by avirulence and virulence effectors. IPI-O1 moderately reduces the self-association of RB CC, potentially leading to changes in the conformation and equilibrium of RB, whereas IPI-O4 dramatically impairs CC self-association to prevent RB activation. We also found that IPI-O1 associates with itself, whereas IPI-O4 does not. Notably, IPI-O4 interacts with IPI-O1 and disrupts its self-association, therefore probably blocking its avirulence function. Furthermore, IPI-O4 enhances the interaction between RB CC and IPI-O1, possibly sequestering RB and IPI-O1 and subsequently blocking their interactions with signaling components. Taken together, these findings considerably extend our understanding of the underlying mechanisms by which emerging virulent pathogens suppress the NLR-mediated recognition of cognate effectors. The virulence effector IPI-O4 blocks NLR RB-mediated recognition of its cognate effector IPI-O1 by suppressing the self-association of both the RB coiled-coil domain and IPI-O1. IPI-O1 and IPI-O4 exhibit striking differences in subcellular localization, self-association status, and effects on RB self-association. IPI-O4 probably sequesters the RB-IPI-O1 complex and subsequently blocks the interactions of these proteins with signaling components to prevent RB activation.

Dataset Information

Evaluation of Nod-like receptor (NLR) effector domain interactions.

Publications

Evaluation of Nod-like receptor (NLR) effector domain interactions.

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