Fig. 7: XL5-VHL-2 triggers hRpn13Pru ubiquitination.
a Lysates from RPMI 8226 WT cells treated for 24 h with 100 nM carfilzomib or DMSO were immunoprobed for hRpn13 with 1 or 30 min exposure times and β-actin, as indicated. b Lysates from RPMI 8226 WT cells treated with 50 μg/mL cycloheximide (CHX) for the indicated time were immunoprobed for hRpn13 and β-actin. The immunoblots are representative of three independent experiments. Quantitation of hRpn13 (from the 15-second immunoblot, blue) and hRpn13Pru (from the 30-minute immunoblot, brown) level plotted as mean ± SE in the bottom panel. c Lysates from RPMI 8226 WT cells treated with 100 nM carfilzomib, 40 μM XL5-VHL-2, or DMSO (control) for 24 h were immunoprobed for hRpn13 with anti-hRpn13 antibodies recognizing amino acids 100–200 (left panel) or 350–407 (right panel) and for β-actin. A faint band above hRpn13 is marked by a green dashed arrow. d Immunoblots with antibodies against ubiquitin, hRpn13, GST, or β-actin as indicated of GST pulldowns (left panel) and whole-cell lysates (right panel) from RPMI 8226 WT cells treated for 24 h with 40 μM XL5-VHL-2 or DMSO. Pulldowns were done with GST-hRpn2 (940–953) or GST (control). Ubiquitinated hRpn13 species are marked by green, orange, or purple dashed arrows for expected molecular weight of modified hRpn13Pru, hRpn13, or either, respectively. e Molecular weight of hRpn13 or hRpn13Pru with conjugation of 0–4 ubiquitin molecules. A band indicated in d with an orange arrow matches monoubiquitinated hRpn13 (indicated in orange) whereas other hRpn13 species (d green arrow) migrate at molecular weights consistent with ubiquitination of hRpn13Pru (green). Two bands (d purple arrow) could be derived by ubiquitination of either hRpn13 or hRpn13Pru (purple). f Schematic illustration of our structure-based pipeline whereby XL5 was identified by in silico and biophysical screening using the hRpn13 Pru:hRpn2 structure. The experimentally determined structure of XL5-bound hRpn13 Pru was then used to guide placement of a VHL-targeting module to generate XL5-VHL-2 for greater potency in cellular assays. Treatment of cells with XL5-VHL-2 led to the discovery of hRpn13Pru, the targeting of which leads to apoptosis.
