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

• Published in: Journal of bioenergetics and biomembranes 2018-06, Vol.50 (3), p.231-240
• Main Authors: Drake, Walter R., Hou, Ching-Wen, Zachara, Natasha E., Grimes, Catherine Leimkuhler
• Format: Article
• Language: English
• Subjects: Animal Anatomy; Animal Biochemistry; Assaying; Binding; Biochemistry; Bioorganic Chemistry; Chemistry; Chemistry and Materials Science; Crohn's disease; Cycloheximide; Dynamic stability; Histology; Morphology; Mutation; NF-κB protein; Nod1 protein; NOD2 protein; O-GlcNAcylation; Oligomerization; Organic Chemistry; Post-translation; Proteins; Stability analysis; Transcription; Translation
• ISSN: 0145-479X; 1573-6881
• DOI: 10.1007/s10863-018-9754-z

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.