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

Walter R. Drake, Ching Wen Hou, Natasha E. Zachara, Catherine Leimkuhler Grimes

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)231-240
Number of pages10
JournalJournal of Bioenergetics and Biomembranes
Volume50
Issue number3
DOIs
StatePublished - Jun 1 2018

Keywords

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

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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