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J Bioenerg Biomembr. 2018 Jun;50(3):231-240. doi: 10.1007/s10863-018-9754-z. Epub 2018 Apr 18.

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

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Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.
Department of Biological Sciences, University of Delaware, Newark, DE, 19716, USA.


O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.


CETSA; Crohn’s disease; NLRs; O-GlcNAcylation; OGT; Peptidoglycan

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