Characterization of IKK-phosphorylated ITCH in TNF signaling

Case Western Reserve University, Cleveland OH

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Abstract

?DESCRIPTION (provided by applicant): Sarcoidosis is a genetic inflammatory disorder that can manifest in tissues of the lymph node, eye and skin, but most commonly manifests in lung. This chronic granulomatous disorder is still a disease of unknown etiology, but exhibits characteristics of overactive T cells, increased IL-17 production, and increased lung neutrophil to macrophage ratios. Two reoccurring genetic components in sarcoidosis are NOD2, an intracellular pattern recognition receptor, and ITCH, a HECT E3 ubiquitin ligase. NOD2 detects muramyl dipeptide (MDP), a breakdown product from peptidoglycan in gram-positive bacterial cell walls. Upon MDP sensing and activation, NOD2 forms a complex with RIP2 to signal into the IKK complex, and ultimately lead to NF-?B signaling. GWAS studies have shown gain of function mutations in NOD2 contribute to overactivation of the pathway. While this pathway is most suspect in predisposition to sarcoidosis, human loss of ITCH has been linked to early onset sarcoidosis. ITCH has a role in down regulation of NOD2-mediated NF-?B signaling through K63 ubiquitination of Rip2. Overactivation of IKKs has furthermore been suspect in contributing to other disease states. We have discovered a novel phosphorylation site on ITCH, and on a similar HECT E3 ubiquitin ligase, Nedd4. Phosphorylation at these sites impairs ubiquitin ligase activity. Taken together, these findings suggest that gain of function NOD2 polymorphisms may result in ITCH impairment through posttranslational modification by IKKs. We hypothesize that IKK-mediated disruption of ITCH leads to hyper-activation of inflammatory status. We are proposing biochemical evaluation of phosphorylation at this site. We also propose a genetic experiment to determine the role of aberrant TNF signaling in the pathogenesis of ITCH-driven sarcoidosis in a murine model. These studies will provide mechanistic insights in ITCH and NEDD4 that may facilitate a better understanding of sarcoidosis and treatment efficacies.