Elucidation of Molecular Mechanism of NOD2 Innate Immune Receptor Stabilization by Chaperone HSP70

Abstract

The innate immune system is the body's first line of defense against invading pathogens such as bacteria. Innate immune receptors such as nucleotide-binding oligomerization domain-containing protein 2 (NOD2) are crucial in recognizing conserved patterns derived from bacterial cell walls as bacteria break down, which induces immune responses via signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Variations in NOD2 have been linked to Crohn's disease (CD), and our recent biophysical and biochemical studies have revealed that these variants are inherently unstable, and hence, they might be subject to unwanted degradation in cells. This leads to insufficient “sensors” available for recognizing bacterial invasion followed by uncontrollable inflammation in CD. Interestingly, these negative outcomes could be lessened or even reversed by overexpression of chaperone heat shock protein 70 (HSP70), a novel regulator of NOD2, to enhance the half-lives of NOD2 variants and enable their activation of immune signaling pathways. The ability of HSP70 to “correct” NOD2 variants’ function indicates this chaperone has the potential to become a therapeutic drug against CD. Therefore, we hypothesize that elucidating the mechanism of HSP70-NOD2 interaction will provide useful information for developing a peptide-based drug that can stabilize NOD2 variants in CD patients and rescue their function. We have designed several constructs serving as small peptide analogs of HSP70 for characterizing the HSP70-NOD2 interactions. From our trypsin-mediated degradation assay, we show that these analogs retain HSP70's ability to stabilize and protect NOD2 against trypsin digestion. We then measure the binding affinities of NOD2-HSP70 constructs interactions in the absence or in the presence of different cofactors (ATP, ADP, NOD2 ligand MDP) using surface plasmon resonance (SPR). In the future, we plan to determine the mechanistic details of NOD2-HSP70 interaction utilizing cross-linking/mass spectroscopy experiment. The findings from this study will enhance our understanding of NOD2 regulation and open up opportunities for expanding the development of effective therapeutic drugs against CD.