Fig. 1: G6374 induces efficient ubiquitination and degradation of IRE1. | Nature Communications

Fig. 1: G6374 induces efficient ubiquitination and degradation of IRE1.

From: Chemically-induced degradation of the endoplasmic-reticulum stress sensor IRE1 by a VHL-recruiting chimera

Fig. 1: G6374 induces efficient ubiquitination and degradation of IRE1.The alternative text for this image may have been generated using AI.

a Chemical structure of the heterobifunctional IRE1 degrader G6374. b Immunoblot (IB) analysis of endogenous IRE1 depletion in AMO1 cells (17 h) with a titration of G6374 compared to CPD-2828 (left) and IB quantification (right; n = 4 biological replicates, mean ± SEM). c Selectivity of G6374 (1 μM, 1 h) determined by SelectScreen Profiling against 220 kinases. d IB assessment of kinases inhibited by 90% or more for depletion by G6374 (4 h) in AMO1 and KMS27 cells. e Global proteomics analysis of G6374- versus G2642-induced protein depletion (1 µM, 4 h) in AMO1 cells. f IRE1 depletion in AMO1 cells treated with G6374 (3 µM, 2 h), with or without E1 inhibitor TAK-243 (E1i; 2 µM) or Neddylation inhibitor TAS4464 (NAEi; 0.2 µM). E1i and NAEi were added 4 h prior to G6374. Samples were analyzed by IB for IRE1 or K48-linked ubiquitin (Ub). g IRE1 depletion by G6374 (3 µM, 2 h) in AMO1 cells treated with bafilomycin A1 (BafA1; 30 nM), chloroquine (Chl; 100 μM), or bortezomib (Btz; 100 nM), added 2 h prior to G6374. Samples were analyzed by IB for IRE1, and respectively for LC3 or K48-Ub to confirm lysosome or proteasome inhibition. h Immunoprecipitation (IP) of endogenous IRE1 from AMO1 cells expressing doxycycline (Dox)-inducible IRE1 shRNA, treated with G6374 (3 µM, 2 h) or DMSO. Dox-induced IRE1 depletion controls for IP-IB specificity. i In vitro ubiquitination of recombinant IRE1.KR incubated with G6374 plus recombinant components of the CRL2VHL complex and native ubiquitin. Samples were analyzed by IRE1 IB. j In vitro ubiquitination of recombinant IRE1.KR as in (i) but using Me-Ub instead of Ub. Samples were analyzed by IRE1 IB. The number of Ub sites is based on relative molecular mass. k Distribution of IRE1 protein with 1, 2, 3, or 4 attached Me-Ub, quantified from (j). “% Ub-IRE1 species” was calculated from the band intensity of each species divided by the sum of IRE1 Ub1, Ub2, Ub3, and Ub4 intensities. l Reaction processivity calculated from (j) as the probability-weighted average Ub number in each respective species.

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