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THE NOD2 PROTEIN IS REGULATED VIA THE UBIQUITIN-PROTEASOME PATHWAY

I Schoultz¹, M Lerm², P Söderkvist³, JD Söderholm^1
1Department of Biomedicine and Surgery, Division of Surgery and ³Division of Cell Biology, ²Department of Molecular and Clinical Medicine, Division of Medical Microbiology, Faculty of Health Sciences, Linköping University, Linköping, Sweden

BACKGROUND: Crohn's disease (CD) has long been an enigma. Predisposing mutations have been mapped to the CARD15/Nod2 locus, which encodes a cytoplasmic receptor recognising bacterial muramyl dipeptide (MDP). This recognition event triggers an activation of nuclear factor-kB (NFkB). Despite active research, little is still known about how Nod2 is regulated.
METHODS: Immunoprecipitation experiments were performed in the colorectal cancer cell line SW480 with a polyclonal Nod2 antibody. IkBa, known to be degraded following uibquitination, was used as a positive control. Coimmunoprecipitation with ubiquitin was confirmed by immunoblotting with a monoclonal ubiquitin antibody. The turn over of Nod2 was measured through immunoprecipitation following inhibition of the protein synthesis with cyclohexamide. To further study the involvement of the proteasome in the degradational process, the level of Nod2 was measured through immunoprecipitation after inhibition of the proteasome with MG-132.
RESULTS: Experiments showed that Nod2 has a rapid turn over, with 50% of the protein being degraded after 30 minutes, and co-immunoprecipitates with ubiquitin. Immunoprecipitation assays also reviled accumulation of Nod2 in cells treated with MG-132.
CONCLUSIONS: Our results show that Nod2 is regulated via the ubiquitin-proteasome pathway. The present findings may impact on our current understanding of the aetiology of Crohn's disease and give new clues about how the disease might emerge.

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