Molecular basis of NOD1 and NOD2 signaling.

摘要

NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2) are related innate immune receptors responsible for initiating a response to bacterial infection. They belong to a class of receptors known as Pattern Recognition Receptors (PRRs), which are germline encoded immune receptors that mediate various innate immune responses. These receptors recognize conserved microbial motifs known as Pathogen-Associated Molecular Patterns (PAMPs). The PRR-PAMP paradigm forms the bedrock of how innate immunity is understood today. As two of the first intracellular PRRs discovered, NOD1 and NOD2 came to define an entire subclass of PRRs, the NOD-like receptors (NLRs). PRRs relay their signals through protein:protein interaction motifs that typically adopt a characteristic Death Domain (DD) fold. NOD1 and NOD2 signal through their respective CAspase Recruitment Domains (CARDs), which are part of a DD subfamily. The CARDs of NOD1 and NOD2 interact with multiple downstream effectors and are thus situated at a key point for regulation and coordination of NOD1 and NOD2 signaling.;To better understand this regulation, I structurally and functionally characterized interactions made by the CARDs of NOD1 and NOD2. Receptor Interacting Protein kinase 2 (RIP2) is an effector of both NOD1 and NOD2 that activates the NF-kappaB pathway to elicit an inflammatory response. I discovered a new binding interaction between the CARDs of NOD1 and NOD2 and ubiquitin. Furthermore, I elucidated a role for this interaction by showing that ubiquitin binds NOD1 and NOD2 CARDs competitively with the CARD of RIP2. Through biophysical and biochemical investigation, I identified mutants of NOD1 CARD that did not bind ubiquitin and were thus insensitive to its competitive effect on RIP2 binding. Utilizing this mutant in functional studies defined ubiquitin as a negative regulator of NOD1 signaling. Characterizing NOD1 allowed rational design of mutations that uncovered a similar role for ubiquitin in the NOD2 pathway. This introduces the potential for broader application of these findings in other DD-mediated pathways.;NOD1 and NOD2 also bind the autophagy protein ATG16L. I investigated the molecular mechanisms of this interaction and found that NOD1 and NOD2 bind ATG16L through their CARDs. I also found that the domain on ATG16L responsible for binding NOD1 and NOD2 is the C-terminal WD40 beta-propeller. Furthermore, the CARD:beta-propeller interaction is sufficient to mediate interaction between NOD1 or NOD2 and ATG16L. The finding that the ATG16L beta-propeller also binds ubiquitin leaves open the possibility that ubiquitin regulates pathway selection by NOD1 and NOD2. Together, these studies advance our understanding of NOD1 and NOD2 signaling and lay the groundwork for further mechanistic investigations into coordination of inflammatory and autophagic signaling pathways by the immune system in general.