Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations

Abstract

. (2016). Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations. Biological Chemistry: official scientific journal of the GBM, 397 (5), 401-415. Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations AbstractLiving systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsCappelli, S., Penco, A., Mannini, B., Cascella, R., Wilson, M. R., Ecroyd, H., Li, X., Buxbaum, J. N., Dobson, C. M., Cecchi, C., Relini, A. & Chiti, F. (2016 measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasises the efficiency and versatility of these protein molecules.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

“…In addition, we cannot discount completely the possibility that oligomer assembly into larger species, with reduced membrane binding and diffusional mobility, could also play a role in the in vivo mechanism of action of trodusquemine. Such a mechanism has been observed previously for a range of oligomers in the presence of a variety of molecular chaperones 30,[51][52][53] and some small molecules 54,55 . Moreover, evidence suggests that more subtle effects than overt physicochemical changes to oligomer structure, such as those observed upon certain chemical changes to fibrils or oligomers, may play a key role in the ability of intrinsically disordered proteins to induce toxicity [56][57][58][59] .…”

“…We used oligomers of Aβ 40 in a recently characterized form stabilized by zinc ions 29 , which are distinct from the Aβ 42 ADDLs described previously 28 . In addition, we also studied oligomeric species produced from the N-terminal domain of the HypF protein from E. coli (HypF-N) 8,30 in order to explore the effects of trodusquemine on another well-characterized model of misfolded protein oligomers. We also explored the effects of trodusquemine on the morphological and structural properties of the oligomers thought to be responsible for mediating their toxicity.…”

Chaperones as Suppressors of Protein Misfolded Oligomer Toxicity

“…Both mechanisms have been indeed observed on a recent report in which the effect of the chaperones αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin known to have a chaperone-like activity, was investigated over a wide range of concentrations, both super- and sub-stoichiometric relative to HypF-N toxic oligomers, ranging from 4:1 to 1:16 (Cappelli et al, 2016). AFM images and light scattering measurements showed that the chaperones increase the size of the aggregates to an extent that correlates with chaperone concentration, ranging from null to remarkable increase.…”

Inhibition of amyloid beta fibril formation by monomeric human transthyretin

“…The potent inhibition of fibril formation by M‐TTR can serve as a model for understanding how amyloid pathways can be reshaped. Indeed, TTR and M‐TTR have been shown to prevent amyloid formation in other amyloid‐prone proteins, including HypF‐N and the curli protein CsgA, a bacterial amyloid forming protein that is responsible for biofilm formation . Human wild‐type tetrameric TTR and the engineered M‐TTR inhibit CsgA amyloid formation in vitro .…”