Fig 1: Structure-based screen yields an hRpn13-binding compound.a Emission at 350 nm for 1 µM hRpn13 Pru with addition of XL5 (black) or RA190 (gray). The plots depict mean ± SD from three parallel recordings against compound concentration and were fit by using the equation [inhibitor] vs. response—Variable slope (four parameters) in GraphPad Prism8, above which chemical structures are included. b 1H, 15N HSQC spectra of 20 µM 15N-hRpn13 Pru (black) or 250 µM 15N-hRpn13 Pru with twofold molar excess XL5 (orange) in NMR buffer at 10 °C, with an expansion for clarity. Arrows highlight the shifting of hRpn13 signals from their free state to their XL5-bound state. Residue signals that disappear (italicized gray) or V38 (red), which appears, following XL5 addition are labeled. c hRpn13 amino acids significantly affected by XL5 addition in b are highlighted (light blue) on a secondary structure diagram of the hRpn13 Pru (purple):hRpn2 (940–953) (light orange) complex (PDB 6CO4). hRpn13 residues shifted by greater than one standard deviation above average or that appear (V38) or disappear following XL5 addition are highlighted. Prolines or residues not observed for free and XL5-bound hRpn13 are colored gray; hRpn2 side-chain heavy atoms are displayed with nitrogen and oxygen colored blue and red respectively. d ITC analysis of hRpn13 binding to XL5. Raw ITC data (top) from titration of 200 µM hRpn13 Pru into 20 µM XL5 with the binding isotherm and fitted thermodynamic values (bottom). e Illustration of hRpn13-encoding ADRM1 gene displaying exons, the hRpn13 Pru and DEUBAD domains colored in purple, the hRpn13 binding sites for ubiquitin (Ub), hRpn2, and UCHL5, the binding epitopes of the two anti-hRpn13 antibodies, and the trRpn13 protein expressed in HCT116 trRpn13. f HCT116 WT (black), HCT116 trRpn13 (blue), or RPMI 8226 WT (orange) cells were treated with the indicated concentration of XL5 for 48 h and cell metabolism measured by an MTT assay; data represent mean ± SD of n = 6 biological replicates. Viability is calculated as (?570)sample/(?570)control*100 (%).
Fig 2: Engineered cell lines establish hRpn13 requirement for XL5-PROTAC-induced apoptosis.a Chemical structures of XL5 (orange)-PROTACs (VHL, blue; CRBN, green, IAP, burgundy). b RPMI 8226 cells were treated with the indicated concentration of XL5 (orange), XL5-VHL (navy), XL5-VHL-2 (blue), XL5-CRBN (green), XL5-IAP (burgundy), VHL-ligand (light blue), thalidomide (light green), or IAP-ligand (pink) for 48 h and cell metabolism measured by an MTT assay; data represent mean ± SD of n = 6 biological replicates. Viability is plotted as (?570)sample/(?570)control*100 (%). IC50 values are listed for XL5 and XL5-PROTAC. c Immunoblot of whole-cell extract from RPMI 8226 WT, trRpn13-MM1, or trRpn13-MM2 cells probing hRpn13 (1 s and 20 min exposure) or ß-actin. d Sanger sequencing analyses of hRpn13 cDNA from RPMI 8226 WT, trRpn13-MM1, or trRpn13-MM2 cells denoting the location of the two sgRNAs (red arrow) on hRpn13-encoding gene ADRM1 Exon 2 with cDNA sequence (CDS) labeled. Allele is abbreviated as “A”. e RPMI 8226 WT (blue), trRpn13-MM1 (black) or trRpn13-MM2 (gray) cells were treated with the indicated concentrations of XL5-VHL-2 for 48 h and cell metabolism measured by an MTT assay; data represent mean ± SD of n = 6 biological replicates. Viability is calculated as (?570)sample/(?570)control*100 (%). f Immunoblots of whole cell lysate from RPMI 8226 WT, trRpn13-MM1, or trRpn13-MM2 cells treated for 24 h with 40 µM XL5-VHL-2 with comparison to DMSO (vehicle control) immunoprobing for cleaved caspase-9 (top panel), hRpn13 (two middle panels with 1 min or 10 min exposure), or ß-actin (as a loading control, bottom panel). A black asterisk indicates cleaved caspase-9 in the 1-min immunoblot for hRpn13, as hRpn13 was probed following cleaved caspase-9 and without stripping the membrane.
Supplier Page from Abcam for Anti-ADRM1/ARM-1 antibody [EPR11449(B)]