Prooxidant properties of p66shc are mediated by mitochondria in human cells.

Evgeny R Galimov,Alena S Sidorenko,Alesya V Tereshkova,Peter M Chumakov,Boris V Chernyak

doi:10.1371/journal.pone.0086521

Abstract

p66shc is a protein product of an mRNA isoform of SHC1 gene that has a pro-oxidant and pro-apoptotic activity and is implicated in the aging process. Mitochondria were suggested as a major source of the p66shc-mediated production of reactive oxygen species (ROS), although the underlying mechanisms are poorly understood. We studied effects of p66shc on oxidative stress induced by hydrogen peroxide or by serum deprivation in human colon carcinoma cell line RKO and in diploid human dermal fibroblasts (HDFs). An shRNA-mediated knockdown of p66shc suppressed and an overexpression of a recombinant p66shc stimulated the production of ROS in the both models. This effect was not detected in the mitochondrial DNA-depleted ρ0-RKO cells that do not have the mitochondrial electron transport chain (ETC). The p66shc-dependent accumulation of mitochondrial ROS was detected with HyPer-mito, a mitochondria-targeted fluorescent protein sensor for hydrogen peroxide. The fragmentation of mitochondria induced by mitochondrial ROS was significantly reduced in the p66shc deficient RKO cells. Mitochondria-targeted antioxidants SkQ1 and SkQR1 also decreased the oxidative stress induced by hydrogen peroxide or by serum deprivation. Together the data indicate that the p66shc-dependant ROS production during oxidative stress has mitochondrial origin in human normal and cancer cells.

Highlights

Mitochondria are multifunctional organelles that play a key role in energy production, apoptosis, thermogenesis, calcium signaling and metabolism of reactive oxygen species (ROS) [1]
Stable knockdown of p66shc expression was achieved by an introduction of a lentiviral construct pLSLP expressing shRNA targeting a unique region in the p66shc mRNA that is absent in the p52shc and p46shc isoforms
A similar level of p66shc mRNA inhibition was observed in the r0 derivative of RKO cells, which does not contain mitochondrial DNA and has no functional electron transport chain (ETC) (Fig. 1B)

Summary

Introduction

Mitochondria are multifunctional organelles that play a key role in energy production, apoptosis, thermogenesis, calcium signaling and metabolism of reactive oxygen species (ROS) [1]. Mitochondrial dysfunction impairs the activity of cells, tissues and organs, and participates in a remarkably wide range of pathologies commonly associated with old age and aging [2,3,4]. Excessive production of ROS in dysfunctional mitochondria is believed to be the major cause of damage that contributes to diseases [5,6,7]. Mitochondrial genome is extremely susceptible to damaging effects of ROS produced in mitochondria as it has a limited DNA repair capacity [8]. Impaired mitochondria with mutated mtDNA tend to accumulate in aging tissues further contributing to the aging process [9,10,11]

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Results

Conclusion