NOD2 Protein Research Articles

Down-regulation of NOD1 in neutrophils of periparturient dairy cows

Increased susceptibility of periparturient dairy cows to infectious diseases has been linked to neutrophil dysfunction. Cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) mediate numerous aspects of innate immunity in humans and experimental animals. The authors hypothesized that neutrophils in periparturient cows have reduced NLR expression, resulting in declined immune responses to NLR activation. In this study, blood neutrophils from periparturient and mid-lactating cows were isolated and assessed for mRNA and protein expression of NOD1 and NOD2. Neutrophil phagocytosis and oxidative burst activity following NLR agonist-induced activation were also investigated. Periparturient cows demonstrated weak-to-absent NOD1 mRNA expression along with a significant reduction in NOD1 protein in their neutrophils. However, no difference in NOD2 protein expression was observed between groups although NOD2 mRNA abundance in periparturient cows was significantly reduced. Phagocytosis and oxidative burst activity measured by production of reactive oxygen species (ROS) were diminished in periparturient cows. NOD1 activation enhanced phagocytosis and ROS production in both isolated cell populations, regardless of the lactation stage. Nevertheless, NOD1-activated neutrophils showed reduced phagocytosis and ROS generation in periparturient cows with respect to mid-lactating subjects. Collectively, our findings suggest that NOD1 down-regulation in neutrophils may be a mechanism involved in periparturient neutrophil dysregulation.

Peptidoglycan and muramyl dipeptide from Staphylococcus aureus induce the expression of VEGF-A in human limbal fibroblasts with the participation of TLR2-NFκB and NOD2-EGFR

Keratitis caused by Staphylococcus aureus often leads to Vascular Endothelial Growth Factor (VEGF)-dependent neovascularization, but contribution of peptidoglycan (PGN), muramyl dipeptide (MDP) and lipoteichoic acid (LTA) from S. aureus to VEGF-dependent neovascularization has not been well-studied. This work was focused on the analysis of S. aureus cell wall components in the production of VEGF family members (VEGF-A, VEGF-B, VEGF-C and VEGF-D) in ocular limbal fibroblasts. Primary culture of human limbal fibroblasts (PCHLFs) were stimulated with PGN, MDP, and LTA, and VEGF family; toll-like receptor 2 (TLR2), nucleotide-binding oligomerization domain 1 (NOD1), and NOD2 expression were determined by RT-PCR. Anti-TLR2 antibody, epidermal growth factor receptor (EGFR) signaling inhibitors (AG1478 and PD98059), and NFκB activation were used to analyze VEGF-A by ELISA. TLR2 and NOD1 expression were analyzed by flow cytometry. The stimulation of PCHLFs with PGN and MDP increased the levels of VEGF-A expression (mRNA and protein) in a time-dependent and dose-dependent manner. VEGF-B, VEGF-C and VEGF-D were expressed constitutively, and no further induction was observed in stimulated PCHLFs. LTA did not increase the expression levels of the VEGF family. TLR2 mRNA and protein were increased only when PCHLFs were stimulated with PGN. Treatment with an anti-TLR2 antibody blocked the interaction of PGN with the receptor, inhibiting VEGF-A over-expression; the presence of anti-TLR2 antibodies did not affect the over-production of VEGF-A after MDP treatment. PCHLFs stimulated with PGN and MDP, but not with LTA, activated NFκB. MDP stimulated the production of NOD1 and NOD2 mRNAs in a time-dependent and dose-dependent manner, and NOD2 protein was only increased by MDP. Treatment of PCHLFs with AG1478 and PD98059 inhibitors prior to stimulation with MDP resulted in the inhibition of VEGF-A over-production, compared with PCHLFs stimulated with MDP alone. Taken together, these results suggest that limbal fibroblasts produce VEGF-A through PGN-TLR2-NFκB and MDP-NOD2-EGFR.

The Innate Immune Protein Nod2 Binds Directly to MDP, a Bacterial Cell Wall Fragment

Mammalian Nod2 is an intracellular protein that is implicated in the innate immune response to the bacterial cell wall and is associated with the development of Crohn’s disease, Blau syndrome, and gastrointestinal cancers. Nod2 is required for an immune response to muramyl dipeptide (MDP), an immunostimulatory fragment of bacterial cell wall, but it is not known whether MDP binds directly to Nod2. We report the expression and purification of human Nod2 from insect cells. Using novel MDP self-assembled monolayers (SAMs), we provide the first biochemical evidence for a direct, high-affinity interaction between Nod2 and MDP.

The c-Jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process

NOD2 is one of the best characterized members of the cytosolic NOD-like receptor family. NOD2 is able to sense muramyl dipeptide, a specific bacterial cell wall component, and to subsequently induce various signaling pathways leading to NF-κB activation and autophagy, both events contributing to an efficient innate and adaptive immune response. Interestingly, loss-of-function NOD2 variants were associated with a higher susceptibility for Crohn disease, which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-Jun N-terminal kinase-binding protein 1 (JNKBP1), a scaffold protein characterized by an N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following muramyl dipeptide activation. This interaction attenuates NOD2-mediated NF-κB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signaling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and in immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis.

Blau Arteritis Resembling Takayasu Disease with a Novel NOD2 Mutation

To put forward a new concept--Blau arteritis, a form of large-vessel vasculitis phenotypically related to Takayasu disease but genetically and clinically part of an expanded phenotype of Blau syndrome. We provide a clinical description of a new case and summarize previously published cases of arteritis associated with Blau syndrome. Genetic testing was performed by direct sequencing of exon 4 of the NOD2 gene. The case described and those reviewed from the literature demonstrate the emerging phenotype of Takayasu-like arteritis in patients with Blau syndrome. Although most patients described to date depict an otherwise classic Blau syndrome phenotype, the current case was atypical in that the predominant features were arteritic. A novel substitution, G464W, in a highly conserved position near the nucleotide oligomerization domain of the NOD2 protein is also described. Blau arteritis can be observed in the context of both typical and atypical (incomplete) Blau syndrome. The associated mutation in the NOD2 gene raises the question of the potential importance of this gene among patients with "primary" forms of Takayasu arteritis.

Study on protective mechanism of kushenin injection on colonic mucosa of experimental colitis rats

To study the effect of NOD2 on colitis pathogenesis in experimental rats, and discuss therapeutical effect and mechanism of kushenin injection (OMT) on colitis in experimental rats. Fourty Sprague-Dawley (SD) rats were randomly divided into four groups: the normal control group, the model group, the SASP group, and the OMT group, with 10 rats in each group. Except the normal control group, models were established in the remaining three groups with TNBS. The OMT group was injected with kushenin injection, the SASP group was orally administered with mesalazine suspension, the model group and the normal group were orally administered with distilled water for 15 days. Colon lesion score and histological score of experimental rats were observed. Expression of NOD2, NF-kappaB p65 protein in rats colonic mucous was detected by immunohistochemistry. Expression of IL-6 in rat colon mucous was detected by ELISA. Compared with normal control group, the expression of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa of the model group were significantly increased (P < 0.01). The SASP group and the OMT group showed lower expressions of NOD2, NF-kappaB p65 and IL-6 in colonic mucosa than the model group (P < 0.01, P < 0.05). The over expression of colonic mucosa proteins NOD2 and NF-kappaB p65 and increasing secretion of IL-6 take part in the appearance and development of ulcerative colitis. OMT can attenuate ulcerative colitis and protect colonic mucosa by inhibiting expression of NOD2, NF-kappaB p65 and decreasing IL-6.

Silencing TLR4 Down-regulates NOD1-mediated Inflammatory Responses by &amp;lt;I&amp;gt;Aspergillus fumigatus&amp;lt;/I&amp;gt; Conidia Stimulation in RAW264.7 Macrophages

This paper aims to study the role of NOD1 and TLR4 signal transduction in RAW264.7 monocyte-macrophages in the state of normal or immunosuppression when stimulated by <i>Aspergillus fumigatus</i> conidia and to investigate the expression of NOD1 signal transduction after silencing the gene of TLR4. First, we established an immune suppression model, and then assayed expression levels of NOD1, TLR4, RIP2, MyD88 mRNA and TNF-α protein using methods of RT-PCR and Western blot, when macrophages were stimulated for 12 h by <i>Aspergillus fumigatus</i> conidia after tranfected for 24 h with TLR4-siRNA by using technology of RNAi. RAW264.7 cells were cultured for 12 h with RPMI1640 culture medium containing immunosuppressive agents (methylprednisolone sodium succinate) at a concentration of 0.04mg/mL. The immune suppression model of macrophages was established with 90% survival rate. The signaling pathway of NOD1 and TLR4 in macrophages was activated by given stimulus of <i>Aspergillus fumigatus</i> conidia and exerted the effect of anti-<i>Aspergillus fumigatus</i> spores stimulation through releasing pro-inflammatory cytokines TNF-α. The macrophages cannot start their effective immune response when they were in the state of immunosuppression. Silencing efficiency of macrophates was up to 83% after transfection with TLR4-siRNA. Silencing the gene of TLR4 down-regulates the effect of NOD1 signal transduction in RAW264.7 cells on anti-<i>Aspergillus fumigatus</i> conidia stimulation.

PTU-092 Crohn's disease associated NOD2 variants show differential activation of NF-κb in response to auto-signalling and muramyl dipeptide

IntroductionSingle nucleotide polymorphisms (SNPs) located of the NOD2/CARD15 gene (nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family, member 15) are associated with increased susceptibility to Crohn's disease (CD). These SNPs...

The pattern‐recognition receptor nucleotide‐binding oligomerization domain–containing protein 1 promotes production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts

Pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-containing protein 2 (NOD-2), have been shown to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to analyze the expression, regulation, and function of the PRR NOD-1 in RA synovial fibroblasts (RASFs), and to examine its interaction with other PRRs. Expression of NOD-1 was analyzed by immunohistochemistry in synovial tissue from RA patients, psoriatic arthritis patients, gout patients, and osteoarthritis (OA) patients. RASFs and human monocyte-derived macrophages (HMDMs) were stimulated with L-alanyl-γ-D-glutamyl-meso-diaminopimelic acid, palmitoyl-3-cysteine-serine-lysine-4, poly(I-C), lipopolysaccharide, heat-inactivated bacteria, tumor necrosis factor α (TNFα), or interleukin-1β (IL-1β). Expression levels of IL-6, CCL5, matrix metalloproteinases (MMPs), NODs, and TLRs were measured by real-time reverse transcription-polymerase chain reaction and/or enzyme-linked immunosorbent assay. NOD-1 and NOD-2 were silenced with target-specific small interfering RNA. Phosphorylation of IL-1 receptor-associated kinase 1 (IRAK-1) was measured by Western blotting. Expression of NOD-1 protein was significantly increased in RA synovium compared to OA synovium. The basal expression of NOD-1 was similar in RASFs, OASFs, healthy control peripheral blood mononuclear cells, and healthy control HMDMs. Stimulation of RASFs with TLR-3 up-regulated the expression of NOD-1. Expression of IL-6, CCL5, MMPs, TLR-2, and NOD-2 was significantly up-regulated in RASFs by stimulation with the NOD-1 ligand. A synergistic effect on IL-6 production was observed in cells stimulated with NOD-1 and TLR-2 ligands or NOD-1 and TLR-4 ligands. Silencing of NOD-1, but not NOD-2, decreased the levels of IL-6 in RASFs after stimulation with TLR-2 and IL-1β, and blocked the phosphorylation of IRAK-1. NOD-1 is strongly expressed in different cell types in the synovial tissue of patients with RA. These results indicate that NOD-1, either alone or interacting with other inflammatory mediators, can play an important role in the chronic and destructive inflammation of the joints in RA.

Sa1996 Crohn's Disease Associated NOD2 Variants Show Differential Activation of NFkB in Response to Autosignaling and Muramyl Dipeptide

Introduction Single nucleotide polymorphisms (SNPs) located of the NOD2/CARD15 gene (nucleotide-binding oligomerization domain containing 2/caspase recruitment domain family, member 15) are associated with increased susceptibility to Crohn9s disease (CD). These SNPs are thought to disrupt the sensing of bacterial muramyl dipeptide (MDP) at the C-terminus of the NOD2 protein. The precise contribution of each of these SNPs (SNP5, 8, 12 and 13) to NF-κB activation by means of NOD2-auto-signalling and stimulation with MDP has not been investigated at low levels of NOD2 expression. Data regarding the linkage disequilibrium (LD) between these CD-associated SNPs are scarce. Methods NOD2 variant constructs (rs2066842 (SNP5), rs2066844 (SNP8), rs2066845 (SNP12) and rs2066847 (SNP13), SNP5+8, SNP5+12 and SNP5+13) were created by site-directed mutagenesis of a pCMV plasmid containing wild-type N-terminal FLAG-NOD2. NF-κB luciferase assays were performed on HEK293 cells following transient transfection (20 h) with wildtype (WT) and NOD2 variant constructs, titrating NOD2 from 1 to 100 ng/well. The NF-κB luciferase response of NOD2 (1 ng)-transfected HEK293 cells to MDP (10 μg/well) was measured. Two-way ANOVA and unpaired t-tests were used. By means of Haploview-analysis of sequencing data of the exons and exon-intron boundaries in 24 paediatric Caucasian Crohn9s disease patients, we assessed the LD between SNP5 and SNP8, 12 and 13. Results Two-way ANOVA demonstrated an effect of NOD2 genotype and concentration on auto-signalling at low levels of expression (p 2 Conclusion Our combined genetic and functional analyses demonstrate that the association of SNP5 with Crohn9s disease is unlikely due to LD with other SNPs. At low levels of NOD2 expression, NOD2 variant constructs differ from WT in their auto-signalling and MDP-stimulated activation of NF-κB. Competing interests None declared.

A Novel Single Nucleotide Polymorphism within the NOD2 Gene is Associated with Pulmonary Tuberculosis in Chinese

The immune system relies on pattern recognition receptors for the identification of invasion by exogenous pathogenic microorganisms. Nucleotide‐binding oligomerization domain2 is a member of the NLR family and is considered as non‐redundant recognition mechanism of Mycobacterium tuberculosis. The present study was conducted on the fourth exon of the NOD2 gene in 179 cases and 177 healthy controls using PCR sequencing, and found a synonymous mutation Arg587Arg which was located in the ultra‐coil encoding region (Winged Helix and Superhelical Domain) of the NOD2 protein. The synonymous mutation was CGT to CGG encoding the Arginine (OR: 2.06; CI: 1.18–3.60). There was a significant difference between tuberculosis patients and the healthy control group (P&lt;0.05), indicating the Arg587Arg site susceptibility to pulmonary tuberculosis. By focusing on the Pro268Ser, Arg708His and Aal725Val locus in the NOD2 gene, we found that the probability of this series was very low in a Chinese Han population and there was no significant difference (P=0.267). We concluded that the single nucleotide polymorphism Arg587Arg in the NOD2 gene was significantly associated with tuberculosis in the Chinese population that provided new data for the world tuberculosis susceptibility research.

Mutated Major Histocompatibility Complex Class II Transactivator Up-regulates Interleukin-33-dependent Differentiation of Th2 Subset through Nod2 Binding for NLR (NOD-like Receptor) Signaling Initiation

Dominant-negative mutants of class II transactivator (mCIITAs) with N-terminal depletion have been used to repress the transcription of class II genes in xenotransplantation. Here, we report that mCIITA overexpressing myeloid cell line Ana-1 (Ana-1-mCIITA) derived from a C57BL/6 mouse was able to down-regulate the MHC class II expression and reverse immune responses from Th1 (IL-2(+)IFN-γ(+)STAT4(+)) to Th2 (IL-4(+)IL-5(+)IL-10(+)IL-13(+)STAT6(+)) when cocultured with T cells. Mechanism analysis indicated that the mCIITA protein is able to initiate a NOD-like receptor-related signaling pathway via binding of the cytoplasmic Nod2 protein, which was followed by activating RIP2, caspase 1, and IKK-α/β. This ensures the expression of the genes encoding the cytokines IL-33, IL-1β, and TNF-α; however, only the highly expressed IL-33 is responsible for inducing the type 2 response, with a skewed Th2 cytokine secretion (IL-4(+)IL-5(+)IL-10(+)IL-13(+)IL-2(-)IFN-γ(-)), which was completely prevented by the deactivation of the Nod2 gene with siRNA or by the blockage of the IL-33-related signaling using the mAb ST2L against the IL-33 receptor. mCIITA-mediated Th2 conversion was also successfully induced in vivo in a mCIITA-transgenic C57BL/6 mouse model. These results indicate that the Th1/Th2 balance could be regulated by an N terminus-depleted CIITA molecule via NOD-like receptor-related signaling, a property valuable for disease control, especially for inducing transplantation tolerance via the repression of class II expression and the attenuation of a Th1-dominant response.

Crohn's Disease Risk Alleles on the NOD2 Locus Have Been Maintained by Natural Selection on Standing Variation

Risk alleles for complex diseases are widely spread throughout human populations. However, little is known about the geographic distribution and frequencies of risk alleles, which may contribute to differences in disease susceptibility and prevalence among populations. Here, we focus on Crohn's disease (CD) as a model for the evolutionary study of complex disease alleles. Recent genome-wide association studies and classical linkage analyses have identified more than 70 susceptible genomic regions for CD in Europeans, but only a few have been confirmed in non-European populations. Our analysis of eight European-specific susceptibility genes using HapMap data shows that at the NOD2 locus the CD-risk alleles are linked with a haplotype specific to CEU at a frequency that is significantly higher compared with the entire genome. We subsequently examined nine global populations and found that the CD-risk alleles spread through hitchhiking with a high-frequency haplotype (H1) exclusive to Europeans. To examine the neutrality of NOD2, we performed phylogenetic network analyses, coalescent simulation, protein structural prediction, characterization of mutation patterns, and estimations of population growth and time to most recent common ancestor (TMRCA). We found that while H1 was significantly prevalent in European populations, the H1 TMRCA predated human migration out of Africa. H1 is likely to have undergone negative selection because 1) the root of H1 genealogy is defined by a preexisting amino acid substitution that causes serious conformational changes to the NOD2 protein, 2) the haplotype has almost become extinct in Africa, and 3) the haplotype has not been affected by the recent European expansion reflected in the other haplotypes. Nevertheless, H1 has survived in European populations, suggesting that the haplotype is advantageous to this group. We propose that several CD-risk alleles, which destabilize and disrupt the NOD2 protein, have been maintained by natural selection on standing variation because the deleterious haplotype of NOD2 is advantageous in diploid individuals due to heterozygote advantage and/or intergenic interactions.

Activation of Nod1 and Nod2 induces innate immune responses of prostate epithelial cells

Nod1 and Nod2 are cytosolic receptors which are responsible for sensing bacterial peptidoglycan derivatives. In this study, we determined whether Nod1 and Nod2 are involved in the innate immune responses of prostate epithelial cells. The expression of Nod1 and Nod2 was examined by RT-PCR and immunohistochemistry. ELISA was performed to determine the production of cytokines/chemokines. Activation of NF-κB and MAPK was examined using western blot analysis. The Nod1 gene was distinctly expressed in all tested cells including DU145, PC3, and TRAMP-C2 cells, whereas Nod2 expression was weak. Both Nod1 and Nod2 proteins were expressed in normal mouse prostate epithelia with difference of expression levels. Tri-DAP (Nod1 agonist), but not MDP (Nod2), increased the production of IL-8 (or KC) and IL-6 in prostate epithelial cells. Tri-DAP and MDP could upregulate the gene expression of COX-2 and activate NF-κB and MAPK. In addition, Tri-DAP and MDP synergized with TLR agonists to induce the production of IL-8/KC or IL-6 in PC3 and TRAMP-C2 cells. We finally showed that Nod1 and Nod2 were also expressed in a wide range of prostate lesions including prostate intraepithelial neoplasm (PIN), phyllodes-like tumor, and adenocarcinoma in TRAMP (transgenic adenocarcinoma of the mouse prostate) mice, even though the expression level of Nod1 and Nod2 was different. These results indicate that Nod1 and Nod2 may play important roles in the innate immune response of prostate epithelial cells and the development and progression of prostate cancer.

TLR2 and RIP2 Pathways Mediate Autophagy of Listeria monocytogenes via Extracellular Signal-regulated Kinase (ERK) Activation

Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2(-/-) and Nod2(-/-) macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2(-/-) mice. Tlr2(-/-) and Nod2(-/-) cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes.

Intrinsic expression of Nod2 in CD4+ T lymphocytes is not necessary for the development of cell‐mediated immunity and host resistance to Toxoplasma gondii

Nod2 belongs to the nucleotide-binding domain leucine-rich repeat family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently, it has been reported that T-cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here, we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon TCR stimulation in vitro, Nod2-deficient CD4(+) T cells showed normal activation, IL-2 production, proliferation, and Th1/2 differentiation. Nod2 mRNA and protein were expressed in CD4(+) T and CD8(+) T cells at substantial levels. Therefore, Nod2, although expressed in CD4(+) T cells, does not have an intrinsic function in T-cell activation and differentiation.

Synthesis and Biological Evaluation of Biotinyl Hydrazone Derivatives of Muramyl Peptides

Muramyl peptides derived from bacterial peptidoglycan have long been known for their ability to trigger host innate immune responses, including inflammation and antimicrobial defense. Muramyl peptides have also been widely studied for their role as immune adjuvants. In mammals, the nucleotide-binding oligomerization domain (Nod) proteins Nod1 and Nod2 detect distinct muramyl peptide structures and mediate their biological activity. Because of the poor immunogenicity of these small peptidoglycan derivatives, research in this field is currently limited by the lack of reagents to track or immobilize specific muramyl peptides. We present here the generation and initial biological characterization of synthetic muramyl peptides covalently coupled to dansyl or biotinyl derivatives and demonstrate that biotinyl coupling on the muramyl moiety results in derivatives that can be tracked by immunofluorescence and maintain full biological activity, as observed by their capacity to trigger Nod signaling. Moreover, using digitonin-mediated permeabilization techniques on live cells, we also demonstrate that biotinylated muramyl peptides efficiently reach the host cytosol, where they activate Nod signaling. Therefore, these derivatives represent useful probes to study the cell biology and the biochemistry of host responses to muramyl peptides.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Expression, purification, and characterization of recombinant NOD1 (NLRC1): A NLR family member

NOD1 (NLRC1) is a member of the NLR family of innate immunity proteins, which are important cellular sensors of various pathogens. Deregulated NOD1 signaling is involved in various autoimmune, inflammatory, and allergic diseases, making it a potential target for drug discovery. However, to date, the successful high-yield purification NOD1 protein has not been reported. Here we describe the large-scale expression of recombinant NOD1 protein in non-adherent mammalian cells. One-step immunoaffinity purification was carried out, yielding highly pure protein with excellent yields. Gel-sieve chromatography studies showed that the purified NOD1 protein eluted almost exclusively as a monomer. Addition of the NOD1 ligand (γ-Tri-DAP) stimulated NOD1 protein oligomerization. Using purified NOD1 protein for nucleotide binding studies by the Fluorescence Polarization Assay (FPA) method, we determined that NOD1 binds preferentially to ATP over ADP and AMP or dATP. We also documented that purified NOD1 protein binds directly to purified pro-apoptotic protein Bid, thus extending recent data that have identified Bid as an enhancer of NOD1 signaling. This expression and purification strategy will enable a wide variety of biochemical studies of mechanisms of NOD1 regulation, as well as laying a foundation for future attempts at drug discovery.