HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation

Abstract

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8 (HSPA8) as a new type of enzyme - which we name as 'amyloidase' - that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X₁)φ(X₃). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

Fig. 1. HSPA8 suppresses necroptosis.

a Hspa8 was identified as a necroptosis-inhibitory gene by a genome-wide siRNA screening. WT and MLKL-KO L929 cells were seeded in 384-well plates and transfected with siRNA pools in parallel. After 72 h, cell viability was determined by measuring intracellular ATP levels as described in Materials and methods. The hit siRNA oligos (targeting 458 genes) were ranked, and the top 23 genes were further confirmed individually with two or more lentivirus-shRNAs (listed in Supplementary information, Fig. S1a) as shown in scatter plot (ii). siCaspase-8 was the positive control that induced spontaneous necroptosis in the WT L929 but not in the MLKL-KO L929 cells, as shown in (i). b Knocking down HSPA8 promoted RIP3-dependent necroptosis in mouse L929 cells. HSPA8 was knocked down by siRNA transfection for 36 h, and necroptosis was induced by treating cells with T/Z for 3 h. The siHSPA8-enhanced necroptosis was blocked by treating cells with RIP3 kinase inhibitor GSK872 (2 mM) or knocking out RIP3. Two oligos, siHSPA8-1 and siHSPA8-2, were used to confirm the necroptosis-inhibitory role of HSPA8 by knocking down the expression of HSPA8. Cell viability was determined by measuring intracellular ATP levels. Necroptosis inducer T/Z: T, TNFα (20 ng/mL); Z, z-VAD (20 μM). The data are represented as mean ± SD of duplicate wells. The knockdown efficiency of HSPA8 was tested by immunoblotting analysis (right panel). c Inhibition of HSPA8 promoted necroptosis signaling. The WT L929 cells and RIP3-KO L929 cells were pretreated with HSPA8 inhibitor AZ for 2 h. Then necroptosis was induced by treating cells with T/Z for 3 h. Cell viability was determined by measuring intracellular ATP levels. The data are represented as mean ± SD of duplicate wells. The necroptosis molecular markers (p-RIP1, p-RIP3, and p-MLKL) were analyzed with the indicated antibodies by immunoblotting (right panel). d HSPA8 inhibitor injection induced hypothermia. The WT and Rip3^−/−mice were injected with HSPA8 inhibitor PES (90 mg/kg). Body temperature was measured at the indicated intervals. n = 6 mice for each group. e Representative intestinal histological images of WT and Rip3^−/− mice after HSPA8 inhibitor injection. WT and Rip3^−/− mice were treated with the HSPA8 inhibitor PES (90 mg/kg) once daily for two consecutive days. Afterward, the intestines were collected and fixed in 4% paraformaldehyde at 4 °C for 48 h. Paraffin sections were prepared following standard protocols, and hematoxylin was used for counterstaining. Scale bar, 50 μm. f Representative immunohistochemistry images of intestine tissue showing the p-MLKL signal in WT mice after HSPA8 inhibitor injection. The WT and Rip3^−/−mice were injected with HSPA8 inhibitor PES (90 mg/kg) as detailed in e. Scale bar, 50 μm. g The Kaplan–Meyer survival curve showed that HSPA8 protected TNF-induced SIRS. Before TNF (7 μg) injection, the WT, Rip3^−/−, and Mlkl^−/− mice were pre-injected with vehicle control or HSPA8 inhibitor PES (45 mg/kg) for 30 min. Mouse survival was monitored at 120-min intervals. n ≥ 5 mice for each group. P values were determined by unpaired two-tailed Student’s t-test with Welch’s correction. ns, no significance. *P < 0.05; **P < 0.01; ***P < 0.005. All results are reported from one representative experiment from at least three independent repeats.

Fig. 2. HSPA8 targets RIP3 to prevent the execution of necroptosis.

a HSPA8 blocked RIP3 oligomerization-induced necroptosis. After 36 h of transfection of siHSPA8 oligos into the mRIP3-2×FKBP-3T3 cells, RIP3 oligomerization was induced by adding 20 nM FKBP dimerizer (AP20187, C₈₂H₁₀₇N₅O₂₀). The dimerizer competitor Tac (10 μM) was used as a negative control to block RIP3 oligomerization-induced necroptosis. The HSPA8 knockdown efficiency was tested by immunoblotting (right panel). b Schematic representation of the full-length and truncation variants of RIP3. FL, full-length; NT, N-terminal; CT, C-terminal; KD, kinase domain; ID, intermediate domain; RHIM, RIP homotypic interaction motif. c, d Mapping the HSPA8 binding region of RIP3 by co-immunoprecipitation. The Flag-tagged truncated RIP3 (as shown in b) and Myc-tagged HSPA8 cDNAs were co-transfected into 293FT cells. After 24 h of transfection, the whole-cell lysates were immunoprecipitated with anti-Myc beads, and immunoblotted with the indicated antibodies. Results are reported from one representative experiment from at least three independent repeats. e Mutations of the RHIM-core of RIP3 did not disrupt the interaction with HSPA8. Flag-tagged WT or the RHIM-core mutant, RIP3-4A mutant (VQVG into AAAA), was co-transfected with Myc-tagged HSPA8 into 293FT cells. Immunoprecipitation with anti-Myc beads was carried out as described in c and d. f In vitro His pull-down assay verified the interaction between HSPA8 and RIP3-RHIM. GST-tagged HSPA8-SBD domain (GST-HSPA8-SBD_385–647) and His-tagged RIP3-RHIM region with the mutation of the RHIM-core (His-Sumo-RIP3_388–518-4A) were purified from E. coli separately and mixed at the ratio of 1:1 (50 μM each). The His-tagged RIP3-RHIM was used as bait; the GST-tagged HSAP8 was used as prey and was pulled down by His-tagged RIP3-RHIM. g Co-immunoprecipitation of endogenous RIP3 and HSPA8. The L929 cells were transfected with the indicated siRNAs. After 48 h, the whole-cell lysate was incubated with anti-RIP3 antibody and protein A/G beads. The immunoprecipitated complexes were probed with the indicated antibodies. P values were determined by unpaired two-tailed Student’s t-test with Welch’s correction. **P < 0.01; ***P < 0.005. All results are reported from one representative experiment from at least three independent repeats.

Fig. 3. HSPA8 inhibits RIP3-amyloid fibrillation in vitro and in cells.

a HSPA8 inhibited RIP3 spontaneous polymerization in vivo. Flag-RIP3-expressing HeLa cells were transfected with HSPA8 siRNA. Necroptosis was induced for 10 h. Whole-cell lysates were subjected to either non-reducing gel electrophoresis (to detect RIP3 polymerization as pointed by arrows) or SDS-PAGE (as loading control) as detailed in Materials and methods. RIP3 and HSPA8 levels were analyzed by immunoblotting with the indicated antibodies. GAPDH served as the loading control. b HSPA8 blocked RIP3 self-interaction in vivo. HeLa cells were co-transfected with Myc-tagged RIP3 and Flag-tagged RIP3, followed by knocking down the expression of HSPA8. Thirty-six hours later, the whole-cell lysates were subjected to immunoprecipitation with anti-Myc beads. The co-immunoprecipitated RIP3 was verified by immunoblotting. c Representative negative stain EM of newly formed RIP3 fibrils. HSPA8 and RIP3_(418–518) were purified from E. coli system. RIP3 fibril growth assay was carried out by incubating the fibrillation ‘seed’ RIP3 with freshly purified RIP3 monomers in the presence or absence of HSPA8 as described in Materials and methods. In short, the fibrillation ‘seeds’ (0.5 μM) were prepared by sonication of the pre-formed RIP3_(418–518) fibrils, and then incubated with freshly purified RIP3_(418–518) monomers (5 μM) in the presence or absence of HSPA8 (5 μM) at 37 °C for 2 h. The newly formed fibrils were precipitated for EM observation. Scale bars, 200 nm. d The growth process of RIP3 fibrils was monitored by ThT staining assay. The newly assembled RIP3 fibrils (as described in c) were stained with ThT (50 μM). The fluorescence intensity was measured every 2 min during the fibril growth process. Emission wavelength: 485 nm; excitation wavelength: 430 nm. e Schematic representation of the truncation strategy of HSPA8. NBD, nucleotide-binding domain; SBD, substrate-binding domain. FL, full-length; NT, N-terminal; CT, C-terminal. f Mapping of HSPA8 domain that mediated the interaction with RIP3. Myc-tagged truncated HSPA8 (as shown in e) was co-transfected with Flag-tagged RIP3 into 293FT cells. Twenty-four hours after transfection, the whole-cell lysates were subjected to immunoprecipitation with anti-Myc beads. The co-precipitated RIP3 and HSPA8 were quantified by immunoblotting with the indicated antibodies. Results are reported from one representative experiment from at least three independent repeats. g The SBD domain of HSPA8 is sufficient for blocking RIP3 fibril growth. RIP3 fibril growth was carried out in the presence of HSPA8-FL or HSPA8-CT as described in c. The newly formed RIP3 fibrils were pelleted and quantified by immunoblotting. Results are reported from one representative experiment from at least three independent repeats.

Fig. 4. HSPA8 disassembles the pre-formed RIP3 amyloidal fibrils coupled with ATP hydrolysis.

a Representative EM analysis of HSPA8-mediated disassembly of RIP3 fibrils. The pre-formed α-syn (upper panels) or RIP3 (bottom panels) fibrils were incubated with HSPA8 (5 μM), along with the ATP regeneration system, and ATP (4 mM) for 2 h, and analyzed by negative stain EM as detailed in Materials and methods. Scale bar, 100 nm. b Immunoblotting analysis of the disassembled RIP3 fibrils. After the disassembly reaction as described in a, the supernatant fraction (the disassembled soluble proteins) and pellet fraction (the insoluble fibrils) were separated by centrifugation and analyzed by immunoblotting with the indicated antibodies. c The disassembly process of RIP3 fibrils was monitored by ThT staining assay. The disassembled RIP3 fibrils (described in a) were stained with ThT (50 μM). The fluorescence intensity was measured every 2 min during the disassembly process; the ThT signals of sonicated fibrils were subtracted as baseline signals from the ThT time course data. The collected data were normalized with the ThT fluorescence intensity of the initial RIP3 fibrils before the disassembly reaction. Excitation wavelength: 430 nm; emission wavelength: 485 nm. d The stoichiometric ratio of the HSPA8 to RIP3 in the disassembly reaction system. The curves showed the real-time kinetics of the disassembled RIP3 fibrils in the presence of different ratios of HSPA8. e Partial trypsin digestion analysis of the aggregated RIP3 fibrils. Left: assay design of partial trypsin digestion. After the disassembly reaction, the RIP3 fibrils were incubated with trypsin (50 μg/mL) at 37 °C for the indicated times. The undigested RIP3 was detected by immunoblotting with anti-RIP3 antibody (upper panel). Lower chart: quantification of the RIP3 signals in the upper panel by image J. f ATP is required for the amyloidase activity of HSPA8. Left: Assay design of analyzing the RIP3 fibrils by centrifugation. RIP3 fibrils were disassembled by incubating with HSPA8 (as described in a) in the presence or absence of ATP. After the disassembly reaction, the disassembled RIP3 and the insoluble fibrils were separated by centrifugation and analyzed by immunoblotting with the indicated antibodies. g The ATP hydrolysis activity of HSPA8 is required for its amyloidase activity. The WT full-length, C-terminal fragment (385–647aa), and the full-length ATPase-deficient mutant (K71A) of HSPA8 were purified from E. coli and incubated with the pre-formed RIP3 fibrils, respectively. After the disassembly reaction, the soluble RIP3 and the insoluble fibrils were separated by centrifugation and analyzed by immunoblotting with the indicated antibodies. h Negative staining immunogold EM of HSPA8 binding to RIP3 fibrils. The microscope images shown here are representative examples of HSPA8-SBD bound to RIP3 fibrils. Anti-HSPA8 antibody was used at a dilution of 1:100. The binding of the antibody was visualized by adding an anti-rabbit gold-conjugated antibody. All results are reported from one representative experiment from at least three independent repeats.

Fig. 5. A consensus hexapeptide motif of the RHIM-containing proteins is required for HSPA8 recognition.

a Comparison of the peptide substrate recognition modes in SBD of HSPA8 (orange) and DnaK (purple). HSPA8 β-subdomain bound mouse RIP3 peptide NSLVAP (red); DnaK bound its substrate peptide NRLILT (pink). Structure of the DnaK/NRLILT complex referenced in PDB (ID: 4EZY). Model of the HSPA8/NSLVAP complex derived from AlphaFold prediction. b Surface view of the mRIP3 peptide bound to the HSPA8-SBD hydrophobic pocket. Gray, β-subdomain; orange, α-subdomain. c The hydrophobic pockets of DnaK (left) and HSPA8 (right), and the bound peptides. d The hydrophobic residues of mouse RIP3 hexapeptide are required for the interaction with HSPA8. The hydrophobic residues of the mRIP3 hexapeptide were individually mutated to aspartic acid (D). Flag-tagged mutant RIP3 was co-transfected with Myc-tagged mHSPA8 into 293 T cells. The whole-cell lysates were subjected to immunoprecipitation with anti-Myc beads. e Sequence alignment of hexapeptides of the RHIM-containing proteins. mRIP3: mouse RIP3 (448–459aa); hRIP1: human RIP1 (539–550aa); hRIP3: human RIP3 (458–469aa); hZBP1-1: the first RHIM motif of human ZBP1 (206–217aa); hZBP1-2: the second RHIM motif of human ZBP1 (264–275aa); hTRIF: human TRIF (687–698aa). The RHIM-core tetrapeptide was highlighted in green; the hydrophobic residues within the hexapeptides were colored in cyan. f Interactions between HSPA8 and the initiator RHIM-containing proteins. The Myc-tagged HSPA8 and the individual Flag-tagged RHIM cDNAs were co-transfected into 293 T cells. Twenty-four hours after transfection, the whole-cell lysates were subjected to immunoprecipitation using anti-Flag beads and immunoblotted with the indicated antibodies. Results are reported from one representative experiment from at least three independent repeats. g–i The hydrophobic residues in hexapeptides of the RHIM-containing proteins were required for the interaction with HSPA8. The hydrophobic residues in hexapeptides of the RHIM-containing proteins were individually mutated (g for RIP1, h for ZBP1, and i for TRIF) and co-transfected with Myc-tagged HSPA8 into 293 T cells. The whole-cell lysates were subjected to immunoprecipitation with anti-Myc beads.

Fig. 6. HSPA8 inhibits necroptosis initiation by blocking the initiator RHIM-containing protein activation.

a, d, g Knocking down HSPA8 promoted RIP1-, ZBP1-, or TRIF-initiated necroptosis. Necroptosis was initiated by Dox-induced RIP1-∆DD, ZBP1, or TRIF overexpression in the RIP1-KO, WT, or TRIF-KO HT-29 cells, respectively (a for RIP1-∆DD; d for ZBP1; g for TRIF). The cells were transfected with HSPA8 siRNA oligos for 36 h, and then necroptosis was induced by Dox (0.4 μg/mL) plus z-VAD (20 μM) for the indicated time. Cell viability was determined by measuring intracellular ATP levels. The data are represented as mean ± SD of duplicate wells. b, e, h HSPA8 could not block the RHIM-hydrophobic residue mutant RIP1/ZBP1/TRIF-initiated necroptosis. Necroptosis was induced as indicated above (b for RIP1-∆DD; e for ZBP1; h for TRIF). The cells were transfected with HSPA8 siRNA oligos for 36 h, and then necroptosis was induced by Dox (0.4 μg/mL) plus z-VAD (20 μM) for the indicated time. Cell viability was determined by measuring intracellular ATP levels as described in Materials and methods. The data are represented as mean ± SD of duplicate wells. c, f, i HSPA8 could not inhibit the RHIM-hydrophobic residue mutant RIP1/ZBP1/TRIF-directed necroptosis signaling to MLKL. The cells were transfected with HSPA8 siRNA oligos for 36 h, and then necroptosis was induced by adding Dox (0.4 μg/mL) plus z-VAD (20 μM). The whole-cell lysates were subjected to non-reducing gel electrophoresis for analyzing MLKL oligomerization and immunoblotting for detecting phosphorylated MLKL. c for RIP1-∆DD; f for ZBP1; i for TRIF. P values were determined by unpaired two-tailed Student’s t-test with Welch’s correction. ns, no significance. *P < 0.05; **P < 0.01; ***P < 0.005. All results are reported from one representative experiment from at least three independent repeats.

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.