Studying The Effect of O‐GlcNAc Modification and Other Stabilizers on Nod1 and Nod2

Abstract

In order to distinguish infectious pathogens from the trillions of commensal bacteria on the body, humans utilize their innate immune systems, which deploy pattern recognition receptors (PRRs) to detect microbe associated molecular patterns (MAMPs) specific to certain microorganisms. The PRRs Nod1 and Nod2 detect MAMPs found in the peptidoglycan (PG) of bacterial cell walls, and upon binding initiate an immune response via the NF‐kB and other pathways, leading to the expression of pro‐inflammatory cytokines and chemokines. Misregulation of Nod1 signaling is associated with various diseases, including gastric ulcers, stomach cancer, and lung cancer. Improper Nod2 signaling has been linked to Crohn's disease, and three different Crohn's associated variants of Nod2 lead to increased susceptibility to the disease and are unstable relative to the wild type. The enzyme O‐GlcNAc Transferase (OGT), which adds a GlcNAc monosaccharide to certain serines/threonines, post‐translationally modifies both Nod1 and Nod2. This post‐translational modification (PTM) is common among thousands of proteins in the nucleus, membrane, and cytosol, and is mediated by only two enzymes: OGT, which adds GlcNAc, and O‐GlcNAcase (OGA), which removes it. The effect of this PTM is diverse among the numerous client proteins. Targets include transcription factors, including oncogenes and tumor suppressors, as well as insulin receptors, kinases, chaperones, and others. We investigate the role of O‐GlcNAcylation on Nod1 and Nod2. Modification increases the stability of both PRRs according to multiple assays, including a cellular thermal shift assay (CETSA) that measures thermal stability and a cycloheximide‐chase assay that measures cellular proteosomal degradation rates. O‐GlcNAc modification also leads to increased PG‐induced NF‐kB activity. The Crohn's associated variants of Nod2 are modified by OGT as well, and the modification rescues the particularly unstable 1007fs variant to near‐wild type. Additionally, we measure the stability effects gained from PG fragment binding and chaperone proteins.Support or Funding InformationMITZUTANI GLYCOSCIENCE FOUNDATIONNATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967)NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153 to NEZ)NIH NIGMS COBRE P20GM104316‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.