Mitochondrial matrix P53 sensitizes cells to oxidative stress

doi:10.1016/j.mito.2013.03.001

Mitochondrion

Volume 13, Issue 4, July 2013, Pages 277-281

https://doi.org/10.1016/j.mito.2013.03.001 Get rights and content

Highlights

•
Studies utilized a mitochondrial-matrix specific p53 construct.
•
Mitochondrial matrix p53 reduces cell viability following oxidative stress.
•
Mitochondrial p53 reduces mitochondrial DNA (mtDNA) abundance following H₂O₂ exposure.
•
Oxidative stress reduces mitochondrial function in mitochondrial p53 cells.

Abstract

A mitochondrial matrix-specific p53 construct (termed p53-290) in HepG2 cells was utilized to determine the impact of p53 in the mitochondrial matrix following oxidative stress. H₂O₂ exposure reduced cellular proliferation similarly in both p53-290 and vector cells, and p53-290 cells demonstrating decreased cell viability at 1 mM H₂O₂ (~ 85% viable). Mitochondrial DNA (mtDNA) abundance was decreased in a dose-dependent manner in p53-290 cells while no change was observed in vector cells. Oximetric analysis revealed reduced maximal respiration and reserve capacity in p53-290 cells. Our results demonstrate that mitochondrial matrix p53 sensitizes cells to oxidative stress by reducing mtDNA abundance and mitochondrial function.

Introduction

Tumor suppressor protein p53 is a well-characterized modulator of cellular processes (Efeyan and Serrano, 2007, Lanni et al., 2012). From transcriptional control to cell cycle regulation, p53 is an important regulator of cellular homeostasis and damage response in the nucleus (Bieging and Attardi, 2012, Reinhardt and Schumacher, 2012, Tokino and Nakamura, 2000). However, scientific attention has focused a role for p53 outside the nuclear compartment and in the context of the mitochondria.

Compartmentally, protein interactions in the mitochondria can be considered as interactions that occur on the “outside” of the mitochondria and those that occur within the mitochondrial matrix. p53 is an important regulator of the intrinsic apoptotic pathway and thus impacts the outer mitochondrial membrane (Marchenko et al., 2007, Vaseva and Moll, 2009). While the characterization of the outer-mitochondrial membrane p53 interactions has been extensive and ongoing, less attention has been focused on the function of p53 within the mitochondrial matrix. p53 is capable of crossing the mitochondrial membranes and entering the matrix (Achanta et al., 2005, Mahyar-Roemer et al., 2004). There, p53 can interact with mitochondrial DNA (mtDNA) directly at the site of DNA damage (Achanta et al., 2005, Bakhanashvili et al., 2008). Studies have suggested that p53 can augment mtDNA repair but they did not characterize the impact of this intramitochondrial interaction on electron transport. Experimental studies that might elucidate such functions are confounded by p53 effects on the nuclear compartment from the strong nuclear localization signal sequence inherent to p53 (Liang and Clarke, 1999, Liang and Clarke, 2001).

We previously characterized a novel p53 construct that overexpresses p53 exclusively within the mitochondrial matrix (Koczor et al., 2012). This construct contains: 1) the mitochondrial targeting sequence from ornithine transcarbamylase attached to the N-terminus of WT-p53, and 2) truncation of the nuclear localization sequence (amino acids 291–393) in the C-terminus of WT-p53. This construct, termed p53-290, demonstrated that mitochondrial matrix p53 sensitizes cells to antiretroviral compounds ddC (2′,3′-dideoxycytidine) and ddI (2′,3′-dideoxyinosine) (Koczor et al., 2012). Due to the increased risk of oxidative stress-induced mtDNA damage as a result of leakage from the electron transport chain, we next explored the impact of mitochondrial p53 in presence of chemically-induced oxidative stress with H₂O₂ exposure (McKenzie et al., 2004). Results demonstrate that mitochondrial p53 decreased mtDNA abundance and mitochondrial function following H₂O₂. We conclude that translocation of p53 to the mitochondria sensitizes cells to oxidative stress, decreases mitochondrial electron transport reserve capacity, and facilitates cell death.

Section snippets

Mitochondrial p53 construct design

Mitochondrial matrix-targeted p53 was designed as previously described (Koczor et al., 2012). Briefly, site-directed mutagenesis (Stratagene/Agilent, Santa Clara, CA) was utilized to introduce BsiWI restriction sites around the nuclear localization sequence contained in the C-terminal portion of WT p53 (amino acids 291–393) of a commercially available WT p53 plasmid (Clontech, Mountain View, CA). The C-terminal region was restriction digested, ligated, and sequenced to ensure fidelity. The

Results

This study exploited a novel p53 construct that overexpresses p53 that is targeted within the mitochondrial matrix (Koczor et al., 2012). This was accomplished because the construct capitalized on the addition of a mitochondrial targeting sequence on the N-terminal side of human p53 while simultaneously removing the region encoding amino acids 291–393 to eliminate intrinsic nuclear localization of native p53 polypeptide (Fig. 1). This novel construct, termed p53-290, was stably transfected into

Discussion

This work expands on our previous report of the function of p53 within the mitochondria and focuses on the effects of p53 within the matrix (Koczor et al., 2012). The novel p53-290 construct enables elucidation of p53's role on mitochondrial function following oxidative stress. This study documents that increased mitochondrial concentrations of p53, which has been shown to occur following oxidative stress (Vaseva et al., 2012), can enhance the sensitivity of cells to the oxidative stress. Our

References (20)

K.T. Bieging et al.
Deconstructing p53 transcriptional networks in tumor suppression
Trends Cell Biol.
(2012)
C.A. Koczor et al.
p53 and mitochondrial DNA: their role in mitochondrial homeostasis and toxicity of antiretrovirals
Am. J. Pathol.
(2012)
C. Lanni et al.
p53 at the crossroads between cancer and neurodegeneration
Free Radic. Biol. Med.
(2012)
S.P. LeDoux et al.
Mitochondrial DNA repair: a critical player in the response of cells of the CNS to genotoxic insults
Neuroscience
(2007)
H.C. Reinhardt et al.
The p53 network: cellular and systemic DNA damage responses in aging and cancer
Trends Genet.
(2012)
T. Tokino et al.
The role of p53-target genes in human cancer
Crit. Rev. Oncol. Hematol.
(2000)
A.V. Vaseva et al.
The mitochondrial p53 pathway
Biochim. Biophys. Acta
(2009)
A.V. Vaseva et al.
p53 opens the mitochondrial permeability transition pore to trigger necrosis
Cell
(2012)
G. Achanta et al.
Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma
EMBO J.
(2005)
S. Altilia et al.
TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells
Aging
(2012)

There are more references available in the full text version of this article.

Cited by (12)

Mitochondrial matrix-localized p53 participates in degradation of mitochondrial RNAs
2021, Mitochondrion
Citation Excerpt :
Upon multitude pro-apoptotic stimuli, p53, located at the outer mitochondrial membrane facing the cytosolic compartment, may directly regulate apoptosis (Marchenko et al., 2000; Marchenko and Moll, 2014). Apart from its role in cell death, the intra-mitochondrial p53 displays other critical functions: DNA repair, respiration (Achanta et al., 2005; Kulawiec et al., 2009; Bakthavatchalu et al., 2012; Koczor et al., 2013; Vasileiou et al., 2017). The p53, located within the mitochondrial matrix, exhibits physical and functional interaction with mtDNA and DNA polymerase γ (pol γ) in response to mtDNA damage (Achanta et al., 2005; Koczor et al., 2013; Safdar et al., 2016).
Mitochondrial RNA degradation plays an important role in maintenance of the mitochondria genetic integrity. Mitochondrial localization of p53 was observed in non-stressed and stressed cells. p53, as an RNA-binding protein, exerts 3′→5′ exoribonuclease activity. The data suggest that in non-stressed cells, mitochondrial matrix-localized p53, with exoribonuclease activity, may play a housekeeping positive role. p53, through restriction the formation of new RNA/DNA hybrid and processing R-loop, might serve as mitochondrial R-loop suppressor. Conversely, stress-induced matrix-p53 decreases the amount of mitochondrial single-stranded RNA transcripts (including polyA- and non-polyA RNAs), thereby leading to the decline in the amount of mitochondria-encoded oxidative phosphorylation components.
The mitochondrial genome in aging and senescence
2014, Ageing Research Reviews
Citation Excerpt :
These experiments showed a possible detrimental role of p53 on mtDNA. In fact, whereas mild oxidative stress had no effect on the cell, high levels of H2O2 caused a 50% mtDNA loss and 10% viability reduction but only in presence of mitochondrial p53 (Fig. 2) (Koczor et al., 2013). The role of p53 in cell homeostasis is complex as it exerts its “genome-protective” function in multiple ways and in subcellular compartments, including the nucleus and the mitochondria.
Aging is characterized by a progressive decline in organism functions due to the impairment of all organs. The deterioration of both proliferative tissues in liver, skin and the vascular system, as well as of largely post-mitotic organs, such as the heart and brain could be attributed at least in part to cell senescence.
In this review we examine the role of mitochondrial dysfunction and mtDNA mutations in cell aging and senescence. Specifically, we address how p53 and telomerase reverse transcriptase (TERT) activity switch their roles from cytoprotective to detrimental and also examine the role of microRNAs in cell aging. The proposed role of Reactive Oxygen Species (ROS), both as mutating agents and as signalling molecules, underlying these processes is also described.
Mitochondria-targeted Ogg1 and aconitase-2 prevent oxidant-induced mitochondrial DNA damage in alveolar epithelial cells
2014, Journal of Biological Chemistry
Citation Excerpt :
Our results herein showing p53 mitochondrial expression in the setting of mtDNA damage concur with our earlier study showing that p53 mediates asbestos-induced AEC p53 mitochondrial translocation and intrinsic apoptosis (19). In accord with our findings, p53 activation is required for H2O2-induced apoptosis in hOgg1-deficient human fibroblast cells (28), and mitochondrial matrix p53 sensitizes HepG2 cells to oxidative stress by reducing mtDNA (43). Several lines of evidence demonstrate a potentially key association between p53, Ogg1, and Aco-2 including; 1) p53 regulates the transcription of the OGG1 gene in colon and renal epithelial cells (28), 2) p53-deficient cells have reduced Ogg1 protein expression and activity (28), and 3) p53 can reduce Aco-2 gene expression as well as Aco-2 activity (44, 45).
Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.
Impact of senescence on bone quality: Lessons from animal models of aging
2014, Drug Discovery Today: Disease Models
Citation Excerpt :
Therefore, although the ability of p53 to induce senescence in response to stress may be considered a pro-survival event, it would seem that under certain conditions, it could also have a detrimental effect on longevity. Indeed, more recent studies have demonstrated that p53 has the potential to increase mitochondrial sensitivity to high oxidative stress and may therefore represent one potential means through which p53 mediates its pro-aging effects [31]. Increases in oxidative stress are considered to be linked to aging and associated age-related bone loss, being mediated through elevations in the levels of intracellular reactive oxygen species (ROS) [32,33].
The association of osteoporotic bone loss and fracture risk with age is becoming increasingly more apparent as the population pyramid continues to shift toward old age. Elucidation of the underlying mechanisms controlling skeletal aging is therefore considered to be of paramount importance in the development of effective therapeutic strategies with which to treat age-related osteoporosis. In this regard, mouse models of premature aging represent an ideal means by which to study the development and pathophysiology of bone loss in the elderly, as well as to test new and novel targeted therapies.
Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations
2020, Cellular and Molecular Life Sciences
The guardian of the genome p53 regulates exercise-induced mitochondrial plasticity beyond organelle biogenesis
2018, Acta Physiologica

View all citing articles on Scopus

^☆: This study was supported by DHHS/NIH/NIDA DA030996 to W.L.

View full text

Mitochondrial matrix P53 sensitizes cells to oxidative stress☆

Highlights

Abstract

Introduction

Section snippets

Mitochondrial p53 construct design

Results

Discussion

Trends Cell Biol.

Am. J. Pathol.

Free Radic. Biol. Med.

Neuroscience

Trends Genet.

Crit. Rev. Oncol. Hematol.

Biochim. Biophys. Acta

Cell

Novel role of p53 in maintaining mitochondrial genetic stability through interaction with DNA Pol gamma

EMBO J.

TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells

Aging