Fig. 7: HSPA8 restrains initiator RHIM-fibril formation.
a The RHIM-based fibril growth was monitored by the ThT staining. RIP1(497–582), ZBP1(150–293) and TRIF(677–698) amyloid assembled in the presence or absence of HSPA8. The newly formed fibrils were stained with ThT (50 μM). The fluorescence intensity was measured every 2 min during the fibril growth process. Excitation wavelength: 430 nm; emission wavelength: 485 nm. b Representative negative stain EM showing that HSPA8 disassembled the pre-formed RHIM-fibrils. The pre-formed RHIM-based fibrils were incubated with or without HSPA8 (5 μM) and ATP (4 mM) for 2 h and analyzed by negative stain EM. Scale bar, 100 nm. c Immunoblotting analysis of the RHIM-containing proteins disassembled by HSPA8. After the disassembly reaction, the disassembled and the insoluble fibrils were separated by centrifugation and analyzed by immunoblotting with the indicated antibodies. d The RHIM-fibril disassembly process was monitored by ThT staining assay. The disassembled fibrils were stained with ThT (50 μM). The fluorescence intensity was measured every 2 min during the disassembly process. ThT signals of sonicated fibrils were subtracted from ThT time course data as baseline signals. Excitation wavelength: 430 nm; emission wavelength: 485 nm. e The hybrid working model for HSPA8 reversing functional RHIM-based amyloids. HSPA8 directly targets the RHIM-containing proteins and prevents the RHIM-amyloid formation; HSPA8 disassembles the pre-formed RHIM-amyloids coupled with ATP hydrolysis. All results are reported from one representative experiment from at least three independent repeats.
