Fig. 5: HURP binds the vinca domain of β-tubulin and competes for vinorelbine.
From: HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance
a GFP-HURP (200-620) wild type and the Patch 1&2 mutant were analyzed by SDS-PAGE and stained with Coomassie blue. b–e Quantification of microtubule dynamicity, including (b) polyermization rate, (c) depolymerization rate, (d) catastrophe frequency, and (e) microtubule length, in the presence of GFP-HURP (200-620) wild type or the Patch 1&2 mutant. Data are mean ± SD from three independent experiments (n = 25 (b–d) and n = 19 (e) microtubules). Two-tailed Student’s t test; Statistical differences: *, P < 0.05; n.s., not significant. f–j Quantification of microtubule dynamicity, including (f) polyermization rate, (g) depolymerization rate, (h) catastrophe frequency, (i) rescue frequency and (j) microtubule length, in the presence of GFP-HURP FL wild type, and the GFP-HURP FL Patch 1 or GFP-HURP FL Patch 1&2 mutants. Data are mean ± SD from three independent experiments (n = 20 microtubules for each condition). Two-tailed Student’s t test; Statistical differences: **, P < 0.01; ***, P < 0.001; n.s., not significant. k Superimposed structures of HURP (423-534) (green), DARPin (pink), and PN2-3 (purple) with β-tubulin (gray). l Overlapping regions in β-tubulin that contact HURP (428-534), PN2-3 and vinorelbine. Protein secondary structures of the contact regions in β-tubulin are shown. Interfacing residues in β-tubulin that facilitate HURP (428-534), PN2-3 and vinorelbine interaction are highlighted in green, purple and brown, respectively. m Superimposition of the HURP (428-534) (green), vinblastine (orange) and vinorelbine (brown) structures. n SDS-PAGE analysis of vinblastine-mediated tubulin association with or without HURP-TBD. Supernatant and pellet fractions are indicated as S and P, respectively. o The bar graph of tubulin in supernatant. Data are mean ± SD from four independent experiments. Two-tailed Student’s t test; Statistical differences: **, P < 0.01; ****, P < 0.0001. p GMPCPP-stabilized microtubules were incubated with GFP-HURP FL with or without vinorelbine. Scale bars, 10 μm. q GFP signal intensity for each condition (arb. units). Data are mean ± SD from three independent experiments (n = 50 microtubules). r Schematic representation of how HURP prevents vinorelbine-induced tubulin aggregation. HURP contains a microtubule-binding domain (MBD; black) and a tubulin-binding domain (TBD; green).
