Fig. 7: ERX-41 binds LAL and is independent of lipase activity.
a–c, Cellular thermal shift assays show heat stability of LAL or vinculin protein in SUM-159 cells (a); relative levels of LAL (b) and vinculin (c) were quantitated and graphed. d, Predicted binding of ERX-41 (colored in green) to LAL (PDB code: 6V7N, LXXLL domain colored in orange). e–f, Representation of ERX-41 as a stick model (e) or space-filled model (f) bound to LAL in ribbon diagram. g–h, Representation of ERX-41 as a stick model (g) or space-filled model (h) bound to the modeled surface of LAL. i, PDB structure of LAL protein showing relative positions of catalytic (with H274Y MT) and LXXLL domains (L242P MT). j, Lysates from described SUM-159 cells were incubated with biotinylated ERX-11/ERX-41, subjected to streptavidin binding and eluates evaluated for LAL pulldown by immunoblotting. k, Dose–response curve of ERX-41 in described SUM-159 cells was measured by CellTiter-Glo. Data presented as mean ± s.e.m.; n = 4 for all groups except KO + L242P, with n = 3 biologically independent samples. l, Top, evaluation of basal lipase activity in parental SUM-159 (WT), clones with LIPA KO (KO) or with reconstitution of WT-LIPA (KO + WT), H274Y MT-LIPA (KO + H274Y), ΔLXXLL MT-LIPA (KO + ΔLXXLL) or L242P MT-LIPA (KO + L242P). Data presented as mean ± s.e.m.; n = 3 biologically independent samples. Bottom, protein expression of LAL and vinculin control. m–o, Effect of vehicle or 1 μM ERX-41 on induction of UPR genes at protein (m) or RNA level (n,o) in recombinant SUM-159 cells (n, sXBP1; o, DDIT3). Data presented as mean ± s.e.m.; n = 3 independent biological samples. Significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Adjusted P values are shown. p–s, Recombinant cDNAs with WT-LIPA, lacking signal peptide (WT-∆SP) or with ER-retention peptide (WT-KDEL) were synthesized (p) and stably transduced in SUM-159 cells with LIPA KO. q, Sensitivity of expressed recombinant LAL proteins to Endo H or PNGase F cleavage. r, Effect of vehicle or 1 μM ERX-41 on induction of UPR genes at protein level in recombinant SUM-159 cells. s, Dose–response curves to ERX-41 in these SUM-159 cells were performed by CellTiter-Glo and graphed. Data presented as mean ± s.e.m.; n = 3 biologically independent samples. Immunoblot studies in a–c,j,m,q,r were performed twice independently, with similar results. Numerical source data for d,g–i,k and uncropped blots for c are provided.
