An ankyrin repeat chaperone targets toxic oligomers during amyloidogenesis

Abstract

Numerous age‐linked diseases are rooted in protein misfolding; this has motivated the development of small molecules and therapeutic antibodies that target the aggregation of disease‐linked proteins. Here we explore another approach: molecular chaperones with engineerable protein scaffolds such as the ankyrin repeat domain (ARD). We tested the ability of http://firstglance.jmol.org/fg.htm?mol=3DEO, a small, robust, ATP‐ and cofactor‐independent plant chaperone built from an ARD, to antagonize disease‐linked protein aggregation. http://firstglance.jmol.org/fg.htm?mol=3DEO delays the aggregation of multiple proteins including the amyloid beta peptide (Aβ) associated with Alzheimer's disease and α‐synuclein associated with Parkinson's disease. Kinetic modeling and biochemical analyses show that http://firstglance.jmol.org/fg.htm?mol=3DEO targets early oligomers during Aβ aggregation, preventing their transition to a self‐propagating nucleus on the fibril surface. Accordingly, http://firstglance.jmol.org/fg.htm?mol=3DEO rescued neuronal cells from the toxicity of extracellular Aβ42 aggregates. The substrate‐binding domain of http://firstglance.jmol.org/fg.htm?mol=3DEO, composed primarily of the ARD, is necessary and sufficient to prevent Aβ42 aggregation and protect cells against Aβ42 toxicity. This work provides an example in which an ARD chaperone non‐native to mammalian cells harbors anti‐amyloidal activity, which may be exploited for bioengineering.

Updates on Aβ Processing by Hsp90, BRICHOS, and Newly Reported Distinctive Chaperones

“…Fibrillation of Aβ42 was significantly slowed by cpSRP43 in a dose-dependent manner [65]. Transmission electron microscopy (TEM) imaging showed that Aβ40 and A syn fibrillation were significantly delayed in the presence of cpSRP43.…”

“…The 43 kD subunit of the chloroplast signal recognition particle (cpSRP43) is an ATP-independent plant chaperone shown to target Aβ oligomers [65]. It contains an engineerable protein scaffold ankyrin repeat domain (ARD).…”

“…Generally, four to six copies of the ankyrin repeat stack on top of each other, forming an elongated structure that has a hydrophobic core throughout it [67]. Each repeat forms two antiparallel a-helices antiparallel to one another to make a helix-turn-helix conformation [65,66]. A loop extends at a 90-degree angle from the structure in order to facilitate the formation of a hairpin-like b-sheet with neighboring motifs [66].…”

“…Consensus sequences both within and in between stacked repeats work to stabilize the ARDs while numerous residues project from the protein surface, providing binding sites for specific protein partners. Using this information, ARDs can be designed to have high specificity and affinity for a number of target proteins [65]. In the case of cpSRP43, biochemical analysis showed that its substrate-binding domain had high anti-amyloid activity against various forms of Aβ such as Aβ42, Aβ40, and ASyn.…”

“…Transmission electron microscopy (TEM) imaging showed that Aβ40 and A syn fibrillation were significantly delayed in the presence of cpSRP43. The mechanism through which cp-SRP43 inhibits Aβ42 and Aβ40 aggregation is the inhibition of secondary nucleation, which was found using the kinetic analysis pipeline [65,68]. To analyze which structural domain of CpSRP43 is involved in anti-fibril activity, Gupta et al generated deletion constructs for testing.…”