Related Experiment Video
Updated: Aug 15, 2026
Comparative Strategies for Ubiquitination Detection in Mammalian Cell Lysates Using SMAD2/SMURF2 as a Model
Published on: April 17, 2026
Analysis of NOD2-mediated proteome response to muramyl dipeptide in HEK293 cells
Dieter Weichart1, Johan Gobom, Sina Klopfleisch
1Max Planck Institute of Molecular Genetics, D-14195 Berlin-Dahlem, Germany.
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.
Insights
NOD2 receptor variants are linked to Crohn's disease. This study analyzed protein changes in cells with normal or variant NOD2, revealing a proteomic signature that may explain disease development.
Area of Science:
- Immunology
- Genetics
- Proteomics
Background:
- NOD2 recognizes bacterial muramyl dipeptide (MDP), crucial for detecting intracellular pathogens.
- Genetic variants in NOD2 are associated with increased Crohn's disease risk.
Purpose of the Study:
- To analyze global protein expression changes in response to MDP stimulation in cells with wild-type (WT) NOD2 versus a disease-associated NOD2 variant (SNP13).
- To identify a proteomic signature linked to NOD2 genetic variations and their role in Crohn's disease pathophysiology.
Main Methods:
- Two-dimensional PAGE of total protein extracts from human cultured cells.
- Stable transfection with NOD2(WT) and NOD2(SNP13) constructs.
- Protein identification using MALDI-TOF mass spectrometry (MS) and MALDI MS/MS.
Main Results:
- Limited overlap in MDP-induced responses between NOD2(WT) and NOD2(SNP13) cells, including downregulation of heat shock 70-kDa protein 4.
- NOD2(WT) regulates a pro-inflammatory program involving protein folding, DNA repair, redox homeostasis, and metabolism.
- A distinct proteomic signature was identified when comparing NOD2(WT) and NOD2(SNP13) variants.
Conclusions:
- NOD2 variants influence cellular responses to bacterial stimuli.
- The identified proteomic signature provides insights into the genetic basis of Crohn's disease.
- Understanding NOD2's role in immune response and metabolism is key to Crohn's disease research.
