WT that encompasses a regulation of key genes involved in protein folding, DNA repair, cellular redox homeostasis, and metabolism was observed both under normal growth conditions and after stimulation with MDP. By using the comparison of NOD2WT and disease-associated NOD2 SNP13 variant, we have identified a proteomic signature pattern that may further our understanding of the influence of genetic variations in the NOD2 gene in the pathophysiology of chronic inflammatory bowel disease.NOD2 belongs to a growing family of regulatory nucleotide-binding oligomerization domain proteins with a central nucleotide-binding oligomerization domain and N-terminal caspase recruitment domains that are involved in programmed cell death and immune responses. The domain structure consists of two adjacent N-terminal caspase recruitment domains, a central nucleotide binding domain, and 10 C-terminal leucine-rich repeats (1). The leucine-rich repeats of NOD2 are homologous to those seen in R proteins and Toll-like receptors, which recognize pathogen-associated molecular patterns, thus enabling an innate cellular response to pathogens. Muramyl dipeptide (MurNAc-L-Ala-Diso-Gln, MDP-LD) 2 derived from peptidoglycan was recently identified as the ligand of NOD2 (2, 3). Upon MDP stimulation, NOD2 has been shown to activate the transcription factor nuclear factor B (NF-B) by interacting with the RIP-like interacting CLARP kinase (RICK/RIP2) (4).A frameshift mutation in the leucine-rich repeats of NOD2 (L1007fsinsC, SNP13), which leads to a partial truncation of the leucinerich repeats in the protein, has been associated with the development of Crohn disease, a human chronic relapsing-remitting inflammatory bowel disease (5-7). The truncation leads to a decreased MDP responsiveness and subsequent NF-B activation. Expression of NOD2 sensitizes intestinal epithelial cells to release the chemotactic cytokine IL-8 upon stimulation with bacterial cell wall components (8, 9). It has been shown recently that tumor necrosis factor-␣ up-regulates NOD2 in intestinal epithelial cells via an NF-B-dependent mechanism (8 -10). Nod2Ϫ/Ϫ mice are deficient in epithelial cryptdin expression and exhibit a higher susceptibility to oral Listeria infection (11). Thus, NOD2 has been implicated as a sentinel in maintaining the integrity of the intestinal barrier against luminal pathogens (12). However, the complex changes in protein expression subsequent to an activation of NF-B induced by NOD2 remain to be elucidated. We have addressed this task using a global proteomic approach. HEK293 cells were stably transfected with expression constructs encoding for the wild type form of NOD2 (NOD2 WT ) or the disease-associated NOD2 L1007fsinsC variant (NOD2 SNP13 ) stimulated with MDP-LD for 4 and 24 h, respectively, and protein extracts were analyzed by two-dimensional GE. Differentially regulated protein spots were detected by a semi-automated digital image analysis system, sampled from the gel, and the contained proteins subsequently were identified by MALDI-TOF pepti...

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