Chaperoning mechanism of innate immune receptor NOD2 by HSP70

Abstract

Host pattern‐recognition receptors (PRRs) are the frontline members of the innate immune system. They are responsible for recognizing molecular structures derived from bacteria or damaged cells and producing appropriate innate immune responses against potential threats from pathogenic agents. However, their defective forms can lead to the development of inflammatory diseases. Crohn’s disease (CD), a prevalent and incurable inflammatory bowel disease, has been shown to closely link to three polymorphisms of the intracellular innate receptor nucleotide‐binding oligomerization domain containing 2 (NOD2). Biophysical and biochemical data show that CD‐associated NOD2 mutants have a much shorter half‐life and reduced signaling function compared to the wild‐type, contributing to the downstream chronic inflammation condition found in CD patients. Given NOD2’s involvement in disease development, it has become an attractive target for drug development against CD. Besides the existing effort in developing anti‐inflammatory drugs and immune suppressors, a novel approach focuses on designing peptidomimetics targeting NOD2 based on its positive modulator protein ‐ stress‐induced heat shock protein 70 kDa (HSP70). Overexpression of both full‐length and truncated HSP70 in cells stabilizes NOD2 mutants and rescues their function, indicating that HSP70 can be used as a platform for developing NOD2 peptide agonists. We first determined the direct interactions at a low micromolar affinity between HSP70 and NOD2 by surface plasmon resonance (SPR) and biolayer interferometry (BLI) technologies. The optimized binding assays will further allow screening of potential HSP70‐derived peptides that retain the same affinity for NOD2. To identify the interacting regions on the amino acid level, we will utilize cross‐linking mass spectrometry (XLMS) together with different docking algorithms. The results will give us a glance into the regulatory mechanisms of HSP70 on NOD2 and provide beneficial structural information for structure‐based peptide drug design. In the future, we plan to study the effects of HSP70‐NOD2 interactions on NOD2‐specific immune responses via RNA sequencing and RT‐PCR.