Multifaceted roles and regulation of nucleotide-binding oligomerization domain containing proteins

Abstract

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.

Keywords: Crohn’s; NF-κB; NOD1; NOD2; inflammation; peptidoglycan.

Figure 1

Domain architecture and structure of NOD1 and NOD2. (A) Schematic representation of NOD1 and NOD2 highlighting the domain boundaries and amino acid stretches of the CARD, NBD, and LRRs. AlphaFold structures of (B) NOD1 and (C) NOD2.

Figure 2

Putative assembly for the NOD2 containing NODosome. Interactions with heat shock proteins, HSP70 and HSP90, stabilize monomeric NOD2. Binding to its ligands ATP via the NBD domain and phosphorylated MDP to the LRR results in conformation changes transitioning from a closed to an open state. Ligand-bound NOD2 is then predicted to assemble into a 7-mer or 8-mer base structure that then serves as a platform for RIPK2 recruitment and poly-ubiquitination, resulting in the functional NODosome. NOD2 is used in this illustration, but it is presumed that a similar activation pathway occurs with NOD1.

Figure 3

RIPK2 dependent and independent signal transduction in response to pathogen-associated molecular patterns. RIPK2-dependent – activation of NOD1 and NOD2 by peptidoglycan (PGN) components typically leads to recruitment of RIPK2. PGN can enter the cytosol via several mechanisms. This includes co-delivery with injected bacterial effector proteins or the fusion of bacterial outer membrane vesicles with host cells. PGN shed by bacteria can also be delivered to the cytosol by a variety of solute carrier channels (SLC15A family members) residing in the plasma membrane, endosomes, and phagosomes. Binding of RIPK2 to NOD1 and NOD2 results in its phosphorylation and polyubiquitination by TRAF2 and other E3 ligases. This, in turn, can result in the polyubiquitination of NEMO, leading to NF-κB phosphorylation and transit to the nucleus. Tab2/3 and Tak1 recruitment and activation transduces a signal to map kinase family members, further potentiating pro-inflammatory gene induction through c-Jun and AP-1. Invasive bacterial species can induce damage to the bacteria containing vacuole and release PGN. This event has been described as recruiting NOD proteins and the autophagy scaffold ATG16L1. RIPK2-independent – this response is described as occurring in the absence of RIPK2. In addition to PGN, NOD1 and NOD2 bind to ssRNA from RNA viruses and induce upregulation of interferon through MAVS and the transcription factors IRF3 and IRF7.

Figure 4

ER stress pathways and NOD activation. Several small molecules and bacterial proteins are known to induce ER stress and activate NOD proteins. Depletion of the ER calcium stores using thapsiagargin, fatty acids, and dithiothreitol is known to activate NOD1/2. However, this may be explained, at least in part, by the sustained increase in cytosolic calcium resulting in enhanced uptake of PGN from the extracellular fluid. Sphingosine 1-phosphate a molecule produced in response to numerous cell stresses, binds to NOD1 and NOD2 leading to upregulation of IL-6 and IL-8.

Figure 5

Aberrant S-palmitoylation of NOD2 in Crohn’s disease and Blau Syndrome. Loss-of-function mutations in NOD2 predispose individuals to developing Crohn’s disease, whereas gain-of-function mutations result in Blau syndrome and early-onset sarcoidosis. NOD2 loss-of-function mutations manifests in a variety of ways, including an inability to bind RIPK2 or PGN. NOD2 also contains two essential S-palmitoylation sites for its functionality, and several of the Crohn’s associated NOD2 mutant proteins are hypo-palmitoylated. Conversely, one extensively studied Blau syndrome mutation, NOD2^C495Y displays enhanced levels of S-palmitoylation. S-palmitoylation of NOD2, and NOD1, is catalyzed by the protein palmitoyltransferase zDHHC5. S-palmitoylation is a reversible post-translational modification, however the acyl thioesterase(s) that mediate fatty acid removal from NODs have not been identified. The current evidence suggests that both hypo- and hyper-palmitoylation of NOD2 results in aberrant signal transduction.