Molecular chaperone ability to inhibit amyloid-derived neurotoxicity, but not amorphous protein aggregation, depends on a conserved pH-sensitive Asp residue

Abstract

Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but the respective mechanisms are not fully understood. The BRICHOS domain constitutes a disease-associated small heat shock protein-like chaperone family, with activities against both amyloid toxicity and amorphous protein aggregation. Here, we show that the activity of two BRICHOS domain families against Alzheimer’s disease associated amyloid-β neurotoxicity to mouse hippocampi in vitro depends on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The conserved Asp in its ionized state promotes structural flexibility of the BRICHOS domain and has a pKa value between pH 6.0–7.0, suggesting that chaperone effects against amyloid toxicity can be affected by physiological pH variations. Finally, the Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families and animal species, indicating independent evolution of molecular chaperone activities against amyloid fibril formation and non-fibrillar amorphous protein aggregation.

ATP‐independent molecular chaperone activity generated under reducing conditions

“…The previously described vector pT7HisNT*‐Bri2 BRICHOS 8 containing the Bri2 BRICHOS sequence (corresponding to residues 113–231 of full‐length human Bri2) was subjected to Cys to Ser mutations (C164S and C223S) using the QuickChange XL site‐directed mutagenesis kit (Agilent Technologies, CA, USA). Expression and purification of the different constructs, including the Bri2 BRICHOS Thr to Trp (T206W) mutant, and their assembly states have been carried out as previously described 8,14 …”

“…SDS-PAGE and native PAGE analyses show that these dimers and tetramers are largely noncovalently linked. Using characteristic tryptophan fluorescence emission changes upon dimer formation of a Thr to Trp Bri2 BRICHOS mutant, 14 an apparent K D of 1.0 ± 0.2 μM was calculated for the assembly formation (Figure S1). However, adding a 200-fold molar excess of the reductant TCEP converts monomers entirely into a polydisperse mixture of HMW assemblies, with a similar size compared to oligomers isolated from E. coli S1) are indicated with # (intracellular) or * (extracellular).…”