P063. Investigation of the function of NOD2 in Crohn's disease using phosphoproteomics
P. Allan1, O. Brain2, J. Baker3, B. Owens1, T. Steevels1, P. Simpson4, T. Pichulik3, E. Khatamzas3, A. Leslie3, B. Kessler5, A. Simmons3
1IMM, Translational Gastroenterology Unit, Oxford, United Kingdom; 2John Radcliffe Hospital, Oxford, United Kingdom; 3Weatherall Institute Of Molecular Medicine, MRC Human Immunology Unit, Oxford, United Kingdom; 4John Radcliffe Hospital, Translational Gastroenterology Unit, Oxford, United Kingdom; 5Henry Wellcome Building for Molecular Physiology, Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom
Background: NOD2 is an intracellular pattern recognition receptor that induces autophagy, important for bacterial handling and antigen presentation, in human monocyte-derived dendritic cells (hMDDCs). Variants of NOD2 associated with terminal ileal Crohn's disease fail to induce autophagy normally on NOD2 stimulation and display delayed bacterial handling and defective antigen presentation. We have mapped the NOD2 signalling cascade in hMDDCs by phosphoproteomics to identify proteins involved in NOD2-mediated autophagy and inflammatory pathway induction in these cells.
Aim: To determine the molecular basis of NOD2 signalling and its effect on autophagy induction and hMDDC maturation.
Methods: Primary human monocytes expressing wild type NOD2 were used to generate hMDDCs. The effect of stimulation of NOD2 with its ligand muramyl dipeptide (MDP) and of cross-talk with TLR2 by stimulation with MDP and synthetic triacylated lipoprotein (Pam3CSK4) was assessed using phosphoproteomics. Phosphorylated proteins were extracted from cell lysates in cells unstimulated or post MDP or MDP+Pam3CSK4 triggering, differentially weighted labelled (for MDP+Pam3CSK4 stimulated), off-gel fractionated and subject to mass spectrometry analysis by LC-MS/MS and Q‑TOF. The function of differentially phosphorylated proteins on hMDDC maturation, autophagy induction and bacterial handling was then investigated.
Results: The NOD2 signalling cascade is diverse and a number of proteins have been identified; functional experiments to ascertain their role within the signalling pathway have been carried out.
Conclusions: Phosphoproteomics casts light on how NOD2 signals, how it might control hMDDC maturation, interact with the autophagy pathway, handle bacteria and present antigen in hMDDCs.