Biochemical and structural characterization of NOD-like receptors and insights into their function in innate immune signaling

Abstract

The human immune system is composed of two main parts, the innate and the adaptive immune system. Pattern recognition receptors (PRRs) present in the innate immune system are capable of recognizing pathogens via pathogen-associated molecular patterns (PAMPs). Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) belong to the cytosolic PRRs and can be divided into two major subfamilies, pyrin domain-containing receptors (NLRPs) or caspase recruitment domain-containing receptors (NLRCs). This thesis focusses on the NOD-like receptors NLRP10, NLRP12 and NOD2. It covers different biochemical approaches and techniques regarding protein design, purification, interaction and activation. <br/> Nucleotide-binding domain, leucine-rich repeat and pyrin domain containing receptor 10 (NLRP10) is a PRR that is activated by mitochondrial damage, which leads to NLRP10 inflammasome activation, ASC speck formation, and caspase-1-dependent cytokine release (Próchnicki et al., 2023; D. Zheng et al., 2023). Also, poly (I:C) has been hypothesized to be an activator of the NLRP10 inflammasome (Masters, 2023). In this thesis was shown that truncated constructs of NLRP10 offer a different behavior during purification from recombinant expression by forming a monomer-dimer equilibrium in solution. Through mutation of several lysine and arginine residues to glutamines in the C-terminal region, the overall positive charge was removed, decreasing the interaction with cellular membranes and resulting in a highly reduced activity of NLRP10. This was shown by decreased ASC speck formation leading to the hypothesis that either nucleotide or membrane binding is involved in the NLRP10 inflammasome activation. These results are consistent with the observation that mitochondrial damage and release of distinct molecular entities leads to NLRP10 activation. <br/> The chapter about NLRP12 deals with the off-target effect of small molecule inhibitors that interact with NLRP3, the closest neighbor in the NLRP family (Hochheiser, Pilsl, et al., 2022; Keuler et al., 2022). Therefore, various interaction studies using surface plasmon resonance (SPR) spectroscopy, thermal shift assays, and crystallization experiments were performed. In addition, an effort was made to obtain novel NLRP12-specific binders in collaboration with the Core Facility Nanobodies at the University Clinics Bonn. <br/> The receptor NOD2 belongs to the CARD containing subfamily and was shown to be activated by sensing muramyl dipeptide (MDP) to induce subsequent signaling (Girardin et al., 2003; Inohara et al., 2003; Stafford et al., 2022). During the expression and purification of NOD2, the binding partner valosin-containing protein (VCP) was identified and characterized. It was endogenously co-purified from Sf-9 insect cells and formed stable complexes with NOD2. The regulatory effect of VCP on the inflammatory response in NOD2 activation has been described previously (Ghalandary et al., 2022). <br/> Since the function and structure of these NOD-like receptors are poorly described, they are valuable targets for future research to gain more insights and develop possible approaches for pharmaceutical applications.