Journal: iScience

Article Title: Small molecule SJ572946 activates BAK to initiate apoptosis

doi: 10.1016/j.isci.2022.105064

Figure Lengend Snippet: Auto-activation-impaired R127A BAK is extensively unfolded and degraded at the mitochondria (A) Titration of 15 N-labeled R127A(s) BAK with SJ572946, monitored by 15 N- 1 H HSQC 2D-NMR. (B) Per-residue CSPs induced by SJ572946 in the amide backbone of R127A(s) BAK, calculated from the spectra shown in panel (A). Dotted red line represents CSPs average +SD. (C) Mapping of the CSPs in panel (B) onto the model of R127A(s) built using the crystal structure of apo BAK identifies SJ572946 binding to the activation groove. (D) Results of liposome permeabilization assays, quantified as the AUCs of the kinetic traces from Figure S6 C, revealing the cooperation of BID BH3 peptides with SJ572946 in direct activation of R127A(s) BAK. Data are presented as the mean +SE of n = 2 experiments, each comprising n = 3 technical replicates. ∗∗∗∗p <0.0001, ∗∗∗p <0.0002, ∗∗p <0.0021, ∗p <0.0332; Tukey–Kramer one-way ANOVA. (E) Results of mitochondrial poration assays measuring cyt c release after incubation with WT BID BH3 ± SJ572946 (bottom), followed by limited proteolysis with calpain (middle) and BMH crosslinking (top) of mitochondria purified from BCL2allKO HCT116 cells constitutively expressing R127A(s) BAK. The aberrant patterns of proteolysis and crosslinking indicate that R127A(s) BAK is inactivated by adopting a conformation different from that of active BAK. Remarkably, R127A(s) BAK was susceptible to calpain proteolysis even in the absence of BH3 peptide activators. R127A(s) BAK was extensively degraded in these cells, as shown in the BMH crosslinking blots. The BMH crosslinking pattern did not change in the presence of BH3 peptide activators, consistent with BAK being in an altered unfolded conformation at the mitochondria.

Article Snippet: Briefly, cells were suspended in mannitol experimental buffer (MEB; 10 mM HEPES (pH 7.5), 150 mM mannitol, 50 mM KCl, 0.02 mM EGTA, 0.02 mM EDTA, 0.1% BSA, and 5 mM succinate) with 0.001% digitonin and treated with compound H6 alone or in combination with BIM or BID peptides (sequences Ac-MRPEIWIAQELRRIGDEFNA-NH2 and Ac-EDIIRNIARHLAQVGDSMDRY-NH2 respectively; New England Peptide).

Techniques: Activation Assay, Titration, Labeling, Residue, Binding Assay, Incubation, Purification, Expressing