Dynamics of protein nitration in cells and mitochondria

Nitric oxide is a precursor of reactive nitrating species such as peroxynitrite and nitrogen dioxide that modify proteins to generate 3-nitrotyrosine. Many diseases are associated with increased levels of protein-bound nitrotyrosine, and this is used as a marker for oxidative damage. However, the regulation of protein nitration and its role in cell function are unclear. We demonstrate that biological protein nitration can be a specific and dynamic process. Proteins were nitrated in distinct temporal patterns in cells undergoing inflammatory activation, and protein denitration and renitration occurred rapidly in respiring mitochondria. The targets of protein nitration varied over time, which may reflect their sensitivity to nitration, expression pattern, or turnover. The dynamic nature of the nitration process was revealed by denitration and renitration of proteins occurring within minutes in mitochondria that were subject to hypoxiaanoxia and reoxygenation. Our results have implications that are particularly important for ischemia-reperfusion injury.     

Mitochondrial protein tyrosine nitration

Mitochondria are primary loci for the intracellular formation and reactions of reactive oxygen and nitrogen species including superoxide (O???), hydrogen peroxide (H?O?) and peroxynitrite (ONOO?). Depending on formation rates and steady-state levels, the mitochondrial-derived short-lived reactive species contribute to signalling events and/or mitochondrial dysfunction through oxidation reactions. Among relevant oxidative modifications in mitochondria, the nitration of the amino acid tyrosine to 3-nitrotyrosine has been recognized in vitro and in vivo. This post-translational modification in mitochondria is promoted by peroxynitrite and other nitrating species and can disturb organelle homeostasis. This study assesses the biochemical mechanisms of protein tyrosine nitration within mitochondria, the main nitration protein targets and the impact of 3-nitrotyrosine formation in the structure, function and fate of modified mitochondrial proteins. Finally, the inhibition of mitochondrial protein tyrosine nitration by endogenous and mitochondrial-targeted antioxidants and their physiological or pharmacological relevance to preserve mitochondrial functions is analysed.     

Rapid and selective oxygen-regulated protein tyrosine denitration and nitration in mitochondria

Growing evidence connects a cumulative formation of 3-nitrotyrosyl adducts in proteins as a marker for oxidative damage with the pathogenesis of various diseases and pathological conditions associated with oxidative stress. A physiological signaling role for protein nitration has also been suggested. Controlled "denitration" would be essential for such a contribution of protein nitration to cellular regulatory processes. Thus, we further characterized such a potentially controlled, reversible tyrosine nitration that occurs in respiring mitochondria during oxygen deprivation followed by reoxygenation, which we recently discovered. Mitochondria constitute cellular centers of protein nitration and are leading candidates for a "nitrative" regulation. Mitochondria are capable of completely eliminating 3-nitrotyrosyl adducts during 20 min of hypoxia-anoxia and undergoing a selective partial reduction after only 5 min. This denitration is independent of protein degradation but depends on the oxygen tension. Reoxygenation re-establishes protein tyrosine nitration patterns that are almost identical to the pattern that occurs before hypoxia-anoxia, with nitration levels that depend on the duration of hypoxia-anoxia. The identified mitochondrial targets of this process are critical for energy and antioxidant homeostasis and, therefore, cell and tissue viability. This cycle of protein nitration and denitration shows analogies to protein phosphorylation, and we demonstrate that the cycle meets most of the criteria for a cellular signaling mechanism. Taken together, our data reveal that protein tyrosine nitration in mitochondria can be controlled, is target-selective and rapid, and is dynamic enough to serve as a nitrative regulatory signaling process that likely affects cellular energy, redox homeostasis, and pathological conditions when these features become disturbed.     

Aspects, mechanism, and biological relevance of mitochondrial protein nitration sustained by mitochondrial nitric oxide synthase

The goal of this study was to explore the occurrence of nitrated proteins in mitochondria given that these organelles are endowed with a mitochondrial nitric oxide (NO.-) synthase and considering the important role that mitochondria have in energy metabolism. Our hypothesis is that nitration of proteins constitutes a posttranslational modification by which NO.- exhibits long-term effects above and beyond those bioregulatory ones mediated through the interaction with cytochrome c oxidase. Our studies are aimed at understanding the mechanisms underlying the nitration of proteins in mitochondria and the biological significance of such a process in the cellular milieu. On promoting a sustained NO.- production by mitochondria, we investigated various aspects of protein nitration. Among them, the localization of nitrated proteins in mitochondrial subfractions, the identification of nitrated proteins through proteomic approaches, the characterization of affected pathways, and depiction of a target sequence. The biological relevance was analyzed by considering the turnover of native and nitrated proteins. In this regard, mitochondrial dysfunction, ensuing nitrative stress, may be envisioned as the result of accumulation of nitrated proteins, resulting from an overproduction of endogenous NO.- (this study), a failure in the proteolytic system to catabolize modified proteins, or a combination of both. Finally, this study allows one to gain understanding on the mechanism and nitrating species underlying mitochondrial protein nitration.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Roles of sirtuins in the regulation of antioxidant defense and bioenergetic function of mitochondria under oxidative stress

Abstract

In addition to serving as the power house of mammalian cells, mitochondria are crucial for the maintenance of cellular homeostasis in response to physiological or environmental changes. Several lines of evidence suggest that posttranslational modification (PTM) of proteins plays a pivotal role in the regulation of the bioenergetic function of mitochondria. Among them, reversible lysine acetylation of mitochondrial proteins has been established as one of the key mechanisms in cellular response to energy demand by modulating the flux of a number of key metabolic pathways. In this article, we focus on the role of Sirt3-mediated deacetylation in: (1) flexibility of energy metabolism, (2) activation of antioxidant defense, and (3) maintenance of cellular redox status in response to dietary challenge and oxidative stress. We suggest that oxidative stress-elicited down-regulation of Sirt3 plays a role in the pathophysiology of diabetes, cardiac hypotrophy, mitochondrial diseases, and age-related diseases. Besides, the physiological role of newly identified lysine acylation mediated by Sirt5 and its biochemical effects on oxidative metabolism are also discussed. Moreover, we have integrated the regulatory function of several protein kinases that are involved in the phosphorylation of mitochondrial enzymes during oxidative stress. Finally, the functional consequence of the synergistic regulation through diverse protein modifications is emphasized on the maintenance of the bioenergetic homeostasis and metabolic adaptation of the animal and human cells. Together, we have provided an updated review of PTM in mitochondrial biology and their implications in aging and human diseases through an intricate regulation of energy metabolism under oxidative stress.     

Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells

Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272–1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.     

Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.     

Altered proteome biology of cardiac mitochondria under stress conditions

Myocardial ischemia-reperfusion induces mitochondrial dysfunction and, depending upon the degree of injury, may lead to cardiac cell death. However, our ability to understand mitochondrial dysfunction has been hindered by an absence of molecular markers defining the various degrees of injury. To address this paucity of knowledge, we sought to characterize the impact of ischemic damage on mitochondrial proteome biology. We hypothesized that ischemic injury induces differential alterations in various mitochondrial subcompartments, that these proteomic changes are specific to the severity of injury, and that they are important to subsequent cellular adaptations to myocardial ischemic injury. Accordingly, an in vitro model of cardiac mitochondria injury in mice was established to examine two stress conditions: reversible injury (induced by mild calcium overload) and irreversible injury (induced by hypotonic stimuli). Both forms of injury had a drastic impact on the proteome biology of cardiac mitochondria. Altered mitochondrial function was concomitant with significant protein loss/shedding from the injured organelles. In the setting of mild calcium overload, mitochondria retained functionality despite the release of numerous proteins, and the majority of mitochondria remained intact. In contrast, hypotonic stimuli caused severe damage to mitochondrial structure and function, induced increased oxidative modification of mitochondrial proteins, and brought about detrimental changes to the subproteomes of the inner mitochondrial membrane and matrix. Using an established in vivo murine model of regional myocardial ischemic injury, we validated key observations made by the in vitro model. This preclinical investigation provides function and suborganelle location information on a repertoire of cardiac mitochondrial proteins sensitive to ischemia reperfusion stress and highlights protein clusters potentially involved in mitochondrial dysfunction in the setting of ischemic injury.     

Role of mitochondria in toxic oxidative stress

Oxidative stress and mitochondrial oxidative damage have been implicated in the etiology of numerous common diseases. The critical mitochondrial events responsible for oxidative stress-mediated cell death (toxic oxidative stress), however, have yet to be defined. Several oxidative events implicated in toxic oxidative stress include alterations in mitochondrial lipids (e.g., cardiolipin), mitochondrial DNA, and mitochondrial proteins (eg. aconitase and uncoupling protein 2). Furthermore, recent findings indicate the enrichment of mitochondrial membranes with vitamin E protects cells against the toxic effects of oxidative stress. This review briefly summarizes the role of these mitochondrial events in toxic oxidative stress, including: 1) the protective role of mitochondrial vitamin E in toxic oxidative stress, 2) the role of mitochondrial DNA in toxic oxidative stress, 3) the interaction between cardiolipin and cytochrome c in mitochondrial regulation of apoptosis, 4) the role of mitochondrial aconitase in oxidative neurodegeneration, and 5) the role of mitochondrial uncoupling protein 2 in the pathogenesis of type 2 diabetes.     

Dual localization of glutathione S-transferase in the cytosol and mitochondria: implications in oxidative stress, toxicity and disease

Glutathione (GSH) conjugating enzymes, glutathione S-transferases (GSTs), are present in different subcellular compartments including cytosol, mitochondria, endoplasmic reticulum, nucleus and plasma membrane. The regulation and function of GSTs have implications in cell growth, oxidative stress as well as disease progression and prevention. Of the several mitochondria localized forms, GSTK (GST kappa) is mitochondria-specific since it contains N-terminal canonical and cleavable mitochondria targeting signals. Other forms like GST alpha, mu and pi purified from mitochondria are similar to the cytosolic molecular forms or 'echoproteins'. Altered GST expression has been implicated in hepatic, cardiac and neurological diseases. Mitochondria-specific GSTK has also been implicated in obesity, diabetes and related metabolic disorders. Studies have shown that silencing the GSTA4 (GST alpha) gene resulted in mitochondrial dysfunction, as was also seen in GSTA4 null mice, which could contribute to insulin resistance in type 2 diabetes. This review highlights the significance of the mitochondrial GST pool, particularly the mechanism and significance of dual targeting of GSTA4-4 under in vitro and in vivo conditions. GSTA4-4 is targeted in the mitochondria by activation of the internal cryptic signal present at the C-terminus of the protein by protein-kinase-dependent phosphorylation and cytosolic heat shock protein (Hsp70) chaperone. Mitochondrial GST pi, on the other hand, has been shown to have two uncleaved cryptic signals rich in positively charged amino acids at the N-terminal region. Both physiological and pathophysiological implications of GST translocation to mitochondria are discussed in the review.     

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.     

Oxidative stress in mitochondria

In mitochondria, oxidative phosphorylation and enzymatic oxidation of biogenic amines by monoamine oxidase produce reactive oxygen and nitrogen species, which are proposed to cause neuronal cell death in neurodegenerative disorders, including Parkinson’s and Alzheimer’s disease. In these disorders, mitochondrial dysfunction, increased oxidative stress, and accumulation of oxidation-modified proteins are involved in cell death in definite neurons. The interactions among these factors were studied by use of a peroxynitrite-generating agent, N-morpholino sydnonimine (SIN-1) and an inhibitor of complex I, rotenone, in human dopaminergic SH-SY5Y cells. In control cells, peroxynitrite nitrated proteins, especially the subunits of mitochondrial complex I, as 3-nitrotyrosine, suggesting that neurons are exposed to constant oxidative stress even under physiological conditions. SIN-1 and an inhibitor of proteasome, carbobenzoxy-l-isoleucyl-γ-t-butyl-l-analyl-l-leucinal (PSI), increased markedly the levels of nitrated proteins with concomitant induction of apoptosis in the cells. Rotenone induced mitochondrial dysfunction and accumulation and aggregation of proteins modified with acrolein, an aldehyde product of lipid peroxidation in the cells. At the same time, the activity of the 20S β-subunit of proteasome was reduced significantly, which degrades oxidative-modified protein. The mechanism was proved to be the result of the modification of the 20S β-subunit with acrolein and to the binding of other acrolein-modified proteins to the 20S β-subunit. Increased oxidative stress caused by SIN-1 treatment induced a decline in the mitochondrial membrane potential, ΔΨm, and activated mitochondrial apoptotic signaling and induced cell death in SH-SY5Y cells. As another pathway, p38 mitogen-activated protein (MAP) kinase and exracellular signal-regulated kinase (ERK) mediated apoptosis induced by SIN-1. On the other hand, a series of neuroprotective propargylamine derivatives, including rasagiline [N-propargyl-1(R)aminoindan]and (−)deprenyl, intervened in the activation of apoptotic cascade by reactive oxygen species-reactive nitrogen species in mitochondria through stabilization of the membrane potential, ΔΨm. In addition, rasagiline induced antiapoptotic Bcl-2 and glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells, which was mediated by the ERK-nuclear factor (NF)-κB pathway. These results are discussed in relation to the interaction of oxidative stress and mitochondria in the regulation of neuronal death and survival in neurodegenerative diseases.     

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress

BackgroundOxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes.MethodsWe constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo.ResultsHepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress.ConclusionsThis study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed.General significanceRoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.     

A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.

The synthesis of 40 polypeptides in mitochondria was found to be stimulated after transient exposure of human endothelial cells to sublethal levels of hydroperoxides, such as H(2)O(2), using comparative two-dimensional polyacrylamide gel electrophoresis. Eleven proteins were identified; these include 60 kDa heat shock protein (HSP60), a mitochondrial type of 70 kDa HSP (mtHSP70), manganese-dependent superoxide dismutase (MnSOD), three metabolic enzymes in citric acid cycle, two components for respiratory chain complexes, a ribosomal protein for translation in mitochondria (RM12), and an unnamed protein. These proteins are involved in reduction-oxidation and protein biogenesis, suggesting that their synthesis, which is triggered under oxidative stress conditions, is aimed at playing a defensive role in mitochondria. Moreover, mtHSP70, HSP60, MnSOD, and RM12 were revealed as their respective precursor proteins with mitochondrial targeting sequences. The preproteins of HSP60 and mtHSP70 were transiently accumulated in mitochondria after the removal of H(2)O(2) in a processing competent state, while the accumulated preprotein of MnSOD localized inside mitochondria and remained unchanged. Membrane potential of mitochondria and cellular ATP levels were unchanged under these conditions. Taken together, these results suggest that hydroperoxide stress leads to preprotein accumulation, possibly due to the impairment of the protein-processing system in mitochondria, independent of membrane potential dissipation and ATP depletion.     

Mitochondrial accumulation under oxidative stress is due to defects in autophagy

Mitochondrial dynamics maintains normal mitochondrial function by degrading damaged mitochondria and generating newborn mitochondria. The accumulation of damaged mitochondria influences the intracellular environment by promoting mitochondrial dysfunction, and thus initiating a vicious cycle. Oxidative stress induces mitochondrial malfunction, which is involved in many cardiovascular diseases. However, the mechanism of mitochondrial accumulation in cardiac myoblasts remains unclear. We observed mitochondrial dysfunction and an increase in mitochondrial mass under the oxidative conditions produced by tert‐butyl hydroperoxide (tBHP) in cardiac myoblast H9c2 cells. However, in contrast to the increase in mitochondrial mass, mitochondrial DNA (mtDNA) decreased, suggesting that enhanced mitochondrial biogenesis may be not the primary cause of the mitochondrial accumulation. Therefore, we investigated changes in a number of proteins involved in autophagy. Beclin1, Atg12–Atg5 conjugate, Atg7 contents decreased but LC3‐II accumulated in tBHP‐treated H9c2 cells. Moreover, the capacity for acid hydrolysis decreased in H9c2 cells. We also demonstrated a decrease in DJ‐1 protein under the oxidative conditions that deregulate mitochondrial dynamics. These results reveal that autophagy became defective under oxidative stress. We therefore suggest that defects in autophagy mediate mitochondrial accumulation under these conditions. J. Cell. Biochem. 114: 212–219, 2012. © 2012 Wiley Periodicals, Inc.     

Interaction between mitochondria and oxidative stress in neuronal injury

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.