Living 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, emphasises the efficiency and versatility of these protein molecules.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super- versus sub-stoichiometric chaperone concentrations / Cappelli, S; Penco, A; Mannini, Benedetta; Cascella, Roberta; Wilson, M; Ecroyd, H; Li, X; Buxbaum, J; Dobson, Cm; Cecchi, Cristina; Relini, A; Chiti, Fabrizio. - In: BIOLOGICAL CHEMISTRY. - ISSN 1431-6730. - ELETTRONICO. - (2016), pp. 0-0. [10.1515/hsz-2015-0250]

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

MANNINI, BENEDETTA;CASCELLA, ROBERTA;CECCHI, CRISTINA;CHITI, FABRIZIO
2016

Abstract

Living 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, emphasises the efficiency and versatility of these protein molecules.
2016
0
0
Cappelli, S; Penco, A; Mannini, Benedetta; Cascella, Roberta; Wilson, M; Ecroyd, H; Li, X; Buxbaum, J; Dobson, Cm; Cecchi, Cristina; Relini, A; Chiti, Fabrizio
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1020560
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