Probing the peptidoglycan molecular recognition mechanisms of innate immune receptors NOD2 and NLRP1 leucine rich repeat domains
Date
2021
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Publisher
University of Delaware
Abstract
The innate immune system is the body’s first line of defense against invading microbial pathogens. A critical component of this immune defense are the cytosolic NOD-like receptors (NLRs), which sense bacterial derived peptidoglycan (PG) fragments to elicit a downstream immune response. NLRs of particular interest to human health are NLRP1 and NOD2, as mutations in both are associated with increased susceptibility to a host of autoimmune and inflammatory diseases, most notably Vitiligo and Crohn’s disease. Though the leucine rich repeat (LRR) domain is commonly proposed as a site of molecular recognition for these receptors, the mechanisms by which these proteins sense and respond to bacterial signals remains poorly understood. Limited knowledge of this interaction makes the development of targeted therapeutics of inflammation very challenging. To gain insight into the mechanisms of NLR activation, this thesis details the development of biophysical tools to better understand how these receptors respond to signals that generate an immune response. Using surface plasmon resonance (SPR) to probe the promiscuity of these proteins for peptidoglycan fragments has given us insight for the first time into the orientation requirements of these ligands within NLR binding pockets, as well as revealed their unique low nanomolar carbohydrate binding affinity. The mechanisms of recognition of these proteins are also extensively studied using a library of synthetic photoactivatable peptidoglycan derivatives. Incorporating photoactivatable groups at different positions around the carbohydrate allows for covalent trapping of these PG-LRR interactions. These techniques have allowed for strategic magnification of NLR molecular recognition, from whole receptor, to LRR domain, to amino acid level details of this first critical recognition step of innate immune activation. The tools developed and used in this thesis reveal new details about what types of ligands can be sensed by NOD2 and NLRP1 and have opened the door for potential development of probes to directly target uncontrolled inflammation.
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Keywords
Peptidoglycan, Nanomolar carbohydrate, Leucine rich repeat, Microbial pathogens, Inflammation