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

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.

Fig. 1

NLR Activators (A) MDP consists of N-acetylmuramic acid with an L-Ala D-isoGln dipeptide chain. It is a ligand derived from peptidoglycan detected by Nod2 (B) iE-DAP is a dipeptide derived from peptidoglycan detected by Nod1 (C) GlcNAc, a carbohydrate, leads to inflammasome formation for NLRP3 and is also O-linked to protein serines and threonines by OGT, with UDP-GlcNAc serving as the donor

Fig. 2

CETSA of wild type Nod2. HEK293T-Nod2-Myc/Tet-op cells were incubated with 1 µM Thiamet-G or DMSO for 8 h, and CETSA and immunoblots were performed (see materials and methods). Relative amounts of Nod2-Myc to SOD1 were plotted from three independent experiments as the means ± S.D. using GraphPad Prism (Boltzmann Sigmoidal model)

Fig. 3

CETSA of Nod2 Crohn’s associated variants and Nod1 (A) HEK293T-Nod2–1007fs-Myc/Tet-op and (B) HEK293T-Nod2-G908R-Myc/Tet-op cells were incubated with 1 µM Thiamet-G or DMSO for 4 h, and CETSA and immunoblots were performed (see materials and methods). (C) HEK293T-Nod1-Flag/Tet-op cells were incubated with 1 µM Thiamet-G or DMSO for 8 h, and CETSA and immunoblots were performed. Relative amounts of Nod2 Myc/Nod1 Flag to SOD1 were plotted from three independent experiments as the means ± S.D. using GraphPad Prism (Boltzmann Sigmoidal model (D) Melting temperature comparison between DMSO and Thiamet-G in Nod1, Nod2 and Nod2 variant cells obtained by CETSA. * indicates significance according to F-test

Fig. 4

O-GlcNAcylation regulates the half-life of Nod1. (A) HEK293T-Nod1-expressing cells were incubated with 1 μM Thiamet-G or DMSO for 4 h, prior to cycloheximide treatment (50 μg/mL) and cells were collected at the indicated time intervals. Nod1 was detected by anti-Flag western blotting. (B) Half-lives were determined by plotting relative Flag intensities vs. time assuming first order decay, and calculating T_1/2 = ln(2)/k

Fig. 5

OGT modifies Nod2 mutants and Nod1. HEK293T-Nod2-Myc, HEK293T-Nod2-G908R-Myc, HEK293T-Nod2-S933A-Myc, and HEK293T-Nod2–1007fs-Myc cell lines were immunopurified with Myc antibody and blotted with CTD110.6 and Myc antibodies; control cells do not express Myc-Nod2. HEK293T-Nod1-Flag cell line was immunopurified with Flag antibody and blotted with CTD110.6 and Flag antibodies. Control cells do not express Flag-Nod1. Thiamet-G inhibits OGA, elevating O-GlcNAcylation

Fig. 6

NF-κB assay of Nod2 variants and Nod1. (A) Dual-luciferase assay performed on HEK293T cells transfected with 0.1 ng Nod2, 702, 908 or 1007fs plasmid in the presence of 100 nM Thiamet-G or DMSO for 4 h. After 4 h, cells were incubated with 20 µM MDP for 5h, harvested, and tested for luciferase activity (B) Dual-luciferase assay performed on HEK293T transfected with 0.1 ng Nod1 in the presence of 100 nM Thiamet-G or DMSO for 4 h. After 4 h, cells were incubated with 20 µM iE-DAP for 8h, harvested, and tested for luciferase activity

Fig. 7

Residue 908 is in close proximity to S933. In the wild type G908 Nod2, S933 is free and solvent exposed. In the Crohn’s associated variant, Nod2-G908R, the bulky arginine side chain can be situated directly nearby S933. Images were created using MacPyMol and PDB 5IRN of crystallized rabbit Nod2