Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.
Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. V. cholerae bacteria belonging to the O1 and O139 serogroups cause cholera epidemics in many developing countries (10, 23), whereas strains belonging to non-O1 non-O139 V. cholerae (NOVC) serogroups have been associated with endemic gastroenteritis and extraintestinal infections in humans (10, 23). Unlike the case for the O1 and O139 strains of V. cholerae, little is known about the virulence and pathogenicity of NOVC strains. Identification and characterization of the NOVC strains carrying virulence genes is important, as these serogroups may emerge as potential epidemic strains in the future. Recently, we found one of these strains, V:5/04, a clinical isolate that caused a severe sporadic outbreak in Sweden in 2004, to express virulence factors such as the type VI secretion system component Hcp (20). Here we further characterized this strain and found that it produced outer membrane vesicles with intrinsic inflammatory potential. Outer membrane vesicles (OMVs) are spherical fragments of bacterial membrane that are produced by a wide variety of Gram-negative bacteria during normal growth (5). These vesicles are formed by protrusions of bacterial outer membrane that are released into the environment. As these vesicles are released from the surface, they can also entrap parts of the underlying bacterial periplasm. OMVs have important functions in host-pathogen interactions, such as the delivery to host cells of active bacterial toxins, such as ClyA cytotoxin, ␣-hemolysin, and CNF1 of Escherichia coli (3,25,44) and hemolysin (Hly) of enterohemorrhagic E. coli (EHEC) (2). Moreover, biologically active H. pylori vacuolating cytotoxin A is associated with OMVs that bind to and are internalized by epithelial cells, as shown by the detection of OMVs in human gastric mucosa from Helicobacter pylori-infected individuals (11,24). Furthermore, different studies provided evidence that OMVs influence inflammation and disease in vivo. In one example, it was shown that epithelial cells produce interleukin-8 (IL-8), a cytokine that is pivotal for neutrophil and monocyte recruitment, in response to H. pyloriand Pseudomonas aeruginosa-derived OMVs (4, 21). It was recently revealed that this effect is, at least partly, dependent on the delivery of nucleotide-binding domain-containing protein 1 (NOD1) active peptidoglycan (PGN) (22). Moreover, during meningococcal septicemia, meningococci are known to release OMVs in the circulation, which contributes to the high endotoxin levels characteristic of these infections (34). OMVs are thus expected to contain several physiologically relevant pathogen-associated molecular patterns (PAMPs) that can be recog-
NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.
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.
NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.
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