Regulating the stabilities of NOD1 and NOD2, two innate immune receptors, with sugars and chaperones
Date
2019
Authors
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Publisher
University of Delaware
Abstract
A healthy human body contains as many bacterial cells as it does human cells, and while some of these bacteria are known to cause diseases, many are beneficial and vital for our well-being, comprising the microbiome. Therefore, it is extremely important for us to distinguish the harmful, pathogenic bacteria from the helpful, commensal ones. Our body’s first line of defense against invading pathogens is the innate immune system, which is composed of a number of pattern recognition receptors (PRRs) that respond to microbe associated molecular patterns (MAMPs) unique to specific microorganisms. Two well characterizes PRRs are nucleotide- binding oligomerization domain containing proteins 1 and 2 (NOD1 and NOD2), members of the NOD-like receptor family (NLRs). These NLRs recognize different fragments from bacterial cell wall peptidoglycan (PG) using their C-terminal leucine rich repeat (LRR) domains and then signal the body to deploy an immune response. These proteins are naturally very unstable and mutations that further destabilize them often impair their functions, leading to a variety of diseases. NOD1 mutations have been correlated with gastric adenocarcinoma and lung cancer, while three single- nucleotide polymorphisms of NOD2 have a strong correlation with Crohn’s disease. ☐ Presented in this dissertation are approaches developed to express and purify NOD1-LRR recombinantly from E. coli. The protein was purified from the soluble fraction of the lysate with the use of detergents, or from the insoluble fraction when denatured with urea. After denaturation, the protein refolded properly according to circular dichroism (CD) experiments. Additionally, NOD1, NOD2, and NOD2 Crohn’s associated variants are post-translationally O-GlcNAcylated by the enzyme O-GlcNAc transferase (OGT). A cellular thermal shift assay (CETSA) typically used to detect protein-ligand binding was adapted in order to monitor the stability changes caused by O-GlcNAcylation, and it was found that wild type NOD1 and NOD2 were stabilized when modified by OGT. Lastly, NOD1 and NOD2 each form a complex with OGT and the 70 kDa heat shock protein (HSP70), which both function to stabilize these NLRs. Immunoprecipitations revealed that the three proteins were bound simultaneously, and protein docking simulations predicted a potential complex between NOD2, OGT, and HSP70. The fundamental findings in this dissertation are that the innate immune system utilizes multiple molecular mechanisms, particularly post-translational O-GlcNAc modification and HSP70 binding, to protect NOD1 and NOD2, enabling them to properly recognize PG fragments and initiate the appropriate immunity pathways. This research gives insights into potential therapeutic developments that can combat the variety of diseases that result from the instability of these NLRs.
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Keywords
GlcNAc, HSP70, Immunity, NOD-like receptors, O-GlcNAc transferase, Peptidoglycan