Biochemical Characterization of the Interaction between Innate Immune Receptor Nod2 and its Chaperones

Abstract

The human body houses over 10 trillion bacterial cells, of which our innate immune system must delineate between pathogenic and commensal. Disease can arise during misregulation of this bacteria, and one example is the debilitating inflammatory bowel disorder, Crohn's disease (CD). The molecular pathology of CD is known to be a mutation in Nod2, an innate immune receptor responsible for binding directly to bacterial cell wall fragment, muramyl dipeptide (MDP). MDP has recently been shown to stabilize Nod2 in a cell based assay, and after binding to MDP, Nod2 signaling proteins activate the NF‐κB pathway, a protective inflammatory response. In Crohn's variants, however, Nod2 is rapidly degraded, thereby the downstream signaling is disrupted. Our initial work identified Hsp70 as a chaperone molecule that binds to and stabilizes Nod2. The purpose of this study is to characterize this interaction and stabilization effect, such that the natural stabilization of Nod2 within the cell can be extrapolated to a possible treatment option that mimics Hsp70's activity on unstable Nod2 Crohn's variants. Hsp70 is a highly ubiquitous protein that is able to confer selective stability by tuning its specific activity via a number of different cochaperones. Our data shows that Hsp40 contributes to the chaperone complex, and through limited proteolysis, we show that both Hsp70 and Hsp40 mediate Nod2 stability in vitro, and that this stability is further enhanced with the addition of Nod2's ligand, MDP. Furthermore, we have biochemically characterized the importance of Hsp70's ATPase activity for Nod2's stability. Significance of this study extends beyond CD, as understanding Hsp70's action and determining a method of control for Nod2 could be extrapolated to stabilize a large number of disease‐relevant mutated proteins.Support or Funding InformationThe authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.