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

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

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

Fig. 5

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.

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