Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response
Kishu Ranjan, … , Xuchen Zhang, Clara Abraham
Kishu Ranjan, … , Xuchen Zhang, Clara Abraham
Published September 1, 2021
Citation Information: J Clin Invest. 2021;131(17):e145472. https://doi.org/10.1172/JCI145472.
View: Text | PDF
Research Article Immunology

Ubiquitination of ATF6 by disease-associated RNF186 promotes the innate receptor-induced unfolded protein response

Abstract

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate–induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.

Authors

Kishu Ranjan, Matija Hedl, Saloni Sinha, Xuchen Zhang, Clara Abraham

×

Figure 6

The rare RNF186 A64T IBD risk variant demonstrates lower levels of NOD2-induced, UPR-dependent outcomes.

Options: View larger image (or click on image) Download as PowerPoint
The rare RNF186 A64T IBD risk variant demonstrates lower levels of NOD2-...
(A) In vitro ubiquitination with purified HA-ubiquitin with/without purified FLAG-ATF6 with/without purified myc-RNF186-A64 or myc-RNF186-T64. Ubiquitin protein (α-HA) was detected by Western blot. Representative of 2 independent experiments. Marker positions are shown (kDa). (B–H) MDMs (rs6426833 AA low RNF186-expressing carriers) were transfected with empty vector (EV) or myc-tagged RNF186-A64 (WT) or RNF186-T64 (risk variant). RNF186 protein expression as detected by (B) flow cytometry (n = 6) or (C) Western blot. (D–H) Transfected MDMs were treated with 100 μg/mL MDP (n = 6) ± 10 μM CPA. (D) Fold phospho-PERK and phospho-IRE1α induction at 30 minutes. (E) Fold change mRNA expression at 6 hours. (F and G) Fold phospho-protein induction at 15 minutes. (H) Cytokines at 12 hours. Mean + SEM. *P < 0.05; **P < 0.01; ***P < 0.001; †P < 1 × 10−4 determined by 2-tailed Student’s t test with a Bonferroni-Holm correction for multiple comparisons. Vec, vector.