Format

Send to

Choose Destination
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.

Author information

1
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA.
2
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
3
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA. cgrimes@udel.edu.
4
Department of Biological Sciences, University of Delaware, Newark, DE, 19716, USA. cgrimes@udel.edu.

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.

KEYWORDS:

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

PMID:
29671171
PMCID:
PMC6126917
DOI:
10.1007/s10863-018-9754-z
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Springer Icon for PubMed Central
Loading ...
Support Center