Figure 1
From: The chaperone HSPB1 prepares protein aggregates for resolubilization by HSP70
HSPB1-3D co-aggregates with substrate forming smaller species. (a) Domain organization of HSPB1 constructs. Full-length HSPB1 is composed of the α-crystallin domain (ACD) flanked by the N- and C-terminal regions (NTR and CTR). Amino acid numbers, and the IXI/V motif are labeled. Three phosphorylatable serine residues found in the NTR (Ser15, Ser78 and Ser82) are highlighted. The phosphomimetic mutant, HSPB1-3D, was obtained by the substitution of those serines to aspartates. The IXI/V motif (181IPV183) of the HSPB1-3D mutant was further mutated into 181GxG183, generating the dimeric HSPB1-3D-GxG mutant. (b) Oligomerization state of 20 µM HSPB1 and its mutants was assessed by SEC analysis. Aggregation of 2 µM (c) luciferase or (d) LDH under heat shock (45 °C or 55 °C, respectively) in the absence or presence of 20 µM HSPB1 or HSPB1-3D. The substrate aggregation was assessed by measurement of optical density at 320 nm. Optical densities of HSPB1 and HSPB1-3D alone under heat shock were measured as controls. Error bars show standard deviations, n ≥ 3. (e, f) The size distributions of HSPB1 and HSPB1-3D, as well as native pre-heat shock (preHS) and aggregated (agg) (e) luciferase and (f) LDH were estimated by dynamic light scattering (DLS).
