Analysis of NOD2-mediated Proteome Response to Muramyl Dipeptide in HEK293 Cells

Sina Klopfleisch,Hans Lehrach,Philip Rosenstiel,Dieter Weichart,Dirk Seegert,Susanne Billmann,Johan Gobom,Stefan Schreiber,Robert Häsler,Niklas Gustavsson

doi:10.1074/jbc.m505986200

Abstract

NOD2, a cytosolic receptor for the bacterial proteoglycan fragment muramyl dipeptide (MDP), plays an important role in the recognition of intracellular pathogens. Variants in the bacterial sensor domain of NOD2 are genetically associated with an increased risk for the development of Crohn disease, a human chronic inflammatory bowel disease. In the present study, global protein expression changes after MDP stimulation were analyzed by two-dimensional PAGE of total protein extracts of human cultured cells stably transfected with expression constructs encoding for wild type NOD2 (NOD2(WT)) or the disease-associated NOD2 L1007fsinsC (NOD2(SNP13)) variant. Differentially regulated proteins were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) peptide mass fingerprinting and MALDI MS/MS. The limited overlap in the responses of the NOD2-overexpressing cell lines to MDP included a down-regulation of heat shock 70-kDa protein 4. A complex pro-inflammatory program regulated by NOD2(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 NOD2(WT) 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.

Highlights

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
HEK293 cells were stably transfected with expression constructs encoding for the wild type form of NOD2 (NOD2WT) or the disease-associated NOD2 L1007fsinsC variant (NOD2SNP13) stimulated with muramyl dipeptide (MDP)-LD for 4 and 24 h, respectively, and protein extracts were analyzed by two-dimensional GE
This study presents for the first time an analysis of the changes in the cellular proteome upon activation of NOD2 by using its ligand, the bacterial cell wall component MDP-LD, as a stimulus

Summary

MATERIALS AND METHODS

Cell Culture and Generation of Stable Transfectants—Human HEK293 and myelomonocytic THP-1 cells were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). After selection and verification of protein expression, six independent stable clones were pooled per plasmid to generate polyclonal cell lines in order to avoid experimental bias from stable plasmid integration. For detection of protein spots with significant up- or down-regulation in any of the comparisons between cell lines or treatments, the minimal regulation factors were set to 1.5 and 0.667, respectively. This requirement was combined with the following settings. The gel samples containing the protein spots were delivered into the wells of the modified MTPs. In situ tryptic digestion was performed according to Shevchenko et al [16] with some modifications. All amplified DNA fragments were analyzed on 1% agarose gels and subsequently documented by a BioDoc Analyzer (Biometra, Gottingen, Germany)

RESULTS AND DISCUSSION

SERC PPCM

After MDP