Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells.
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.
Weichart
Dieter
D
Max Planck Institute of Molecular Genetics, D-14195 Berlin-Dahlem, Germany.
Gobom
Johan
J
Klopfleisch
Sina
S
Häsler
Robert
R
Gustavsson
Niklas
N
Billmann
Susanne
S
Lehrach
Hans
H
Seegert
Dirk
D
Schreiber
Stefan
S
Rosenstiel
Philip
P
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000737:chemistry*; D002460:Cell Line; D002478:Cells, Cultured; D016000:Cluster Analysis; D004260:DNA Repair; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D020868:Gene Silencing; D014644:Genetic Variation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation; D016209:Interleukin-8 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D053473:Nod2 Signaling Adaptor Protein; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D015854:Up-Regulation
D018840:HSP70 Heat-Shock Proteins; D016209:Interleukin-8; D047908:Intracellular Signaling Peptides and Proteins; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D010455:Peptides; D034741:RNA, Small Interfering; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D012313:RNA; D008156:Luciferases; D014357:Trypsin