RESUMEN

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

Asunto(s)

Cuerpos de Inclusión , Superóxido Dismutasa , Humanos , Cuerpos de Inclusión/metabolismo , Espectroscopía de Resonancia Magnética , Mutación , Agregado de Proteínas , Pliegue de Proteína , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo

RESUMEN

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.

Asunto(s)

Proteínas de Transporte de Catión/deficiencia , Proteínas de la Membrana/deficiencia , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas Transportadoras de Cobre , Transporte de Electrón , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Expresión Génica , Proteína 1 Reguladora de Hierro/genética , Proteína 1 Reguladora de Hierro/metabolismo , Potencial de la Membrana Mitocondrial/genética , Proteínas de la Membrana/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Mitofagia/genética , Chaperonas Moleculares/genética , Fosforilación Oxidativa , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo