ATP‐independent molecular chaperone activity generated under reducing conditions

Abstract

Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra‐ to intermolecular disulfide bond relay mechanism. Chaperone‐active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol‐containing compounds and proteins, and appear in parallel with reduction‐induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space. Significance Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction‐induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.

Amyloid inhibition by molecular chaperones in vitro can be translated to Alzheimer's pathology in vivo

“…14 The Bri2 BRICHOS oligomers, on the contrary, act more as classical chaperones, preventing most effectively amorphous aggregation of client proteins. 14,52 The mechanism of action to retard Aβ42 fibril formation was assigned to inhibition of secondary nucleation in addition to Please do not adjust margins Please do not adjust margins fibril-end elongation. 14 A single-point mutant (R221E) that stabilizes the monomeric state of Bri2 BRICHOS, has shown similar effects as the Bri2 BRICHOS monomer, inhibiting the same microscopic nucleation events.…”

OAF: a new member of the BRICHOS family

“…2 and Supplementary Fig. S3 ); (iv) a predicted disulphide bridge between α-helix 2 and β-strand 4, whose conserved cysteines have been involved in a homopolymerization mechanism in reducing conditions, key for the ATP-independent chaperone function of these domains ( Leppert et al , 2022 ); and (v) a highly conserved aspartic acid (Asp74) located at the end of β-strand 2 ( Supplementary Figs S2 and S3 ). This residue has been recently implicated in a pH-dependent regulatory mechanism of the BRICHOS domain's chaperone activity ( Chen et al.…”

Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation

“…As shown here, BRICHOS needs to form larger oligomers that bring together and expose hydrophobic motifs to gain efficient canonical chaperone activity, while crystallins generally get more active upon dissociation into smaller subunits 27 . The apparently simple strategy to enable BRICHOS-substrate interactions by bringing together short loop motifs can be a manner to regulate chaperone activity under physiological conditions 46 and mutagenesis of the BRICHOS domain might be a way to generate novel chaperone functions.…”