Rescuing Nod2, an innate immune receptor of bacterial cell wall fragments, in Crohn Disease

Abstract

Nucleotide binding oligomerization domain‐containing protein 2 (NOD2, CARD15) is an intracellular host pattern recognition receptor that recognizes fragments of bacterial cell wall. Nod2 is important to human biology, as when it is mutated, it loses the ability to respond properly to bacterial cell wall fragments. In order to determine the mechanisms of misactivation in the Nod2 Crohn's mutants, we developed a cell based system to screen for protein‐protein interactors of Nod2. We identified Hsp70 (heat shock protein 70) as a protein interactor of both wild‐type and mutant Nod2. The objective of this study is to determine the mechanism by which Hsp70 stabilizes Nod2. Induced Hsp70 expression in cells increased Nod2 mediated NF‐kB activation in response to bacterial cell wall stimulation. We found Hsp70 to regulate Nod2′s half‐life, as increasing the Hsp70 level in cells increased Nod2′s half‐life, and down‐regulating Hsp70 decreased Nod2′s half‐life. The expression level of the Crohn's associated Nod2 variants were less compared to wild‐type. The over‐expression of Hsp70 significantly increased Nod2 levels as well as the signaling capacity of the mutants. Thus our study shows that restoring the stability of the Nod2 Crohn's mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. Further, we have identified the minimal region of Hsp70 that facilitates the interaction with Nod2. Studies are currently under way to identify pharmacoperones that can potentially stabilize the Crohn's associated Nod2 variants.Funding: This project was supported by the Delaware COBRE program, with a grant from the NIH: NIGMS (1 P30 GM110758‐01) from the National Institutes of Health.