Effect of progerin on the accumulation of oxidized proteins in fibroblasts from Hutchinson Gilford progeria patients.

The mutation responsible for Hutchinson Gilford Progeria Syndrome (HGPS) causes abnormal nuclear morphology. Previous studies show that free radicals and reactive oxygen species play major roles in the etiology and/or progression of neurodegenerative diseases and aging. This study compares oxidative stress responses between progeric and normal fibroblasts. Our data revealed higher ROS levels in HGPS cells compared to age-matched controls. In response to oxidative challenge, progeric cells showed increased mRNA levels for mitochondrial superoxide dismutase (SOD) and SOD protein content. However, this did not prevent a drop in the ATP content of progeria fibroblasts. Previous studies have shown that declines in human fibroblast ATP levels interfere with programmed cell death and promote necrotic inflammation. Notably, in our investigations the ATP content of progeria fibroblasts was only approximately 50% of that found in healthy controls. Furthermore, HGPS fibroblast analysis revealed a decrease in total caspase-like proteasome activity and in the levels of two active proteolytic complex subunits (beta(5) and beta(7)). A number of studies indicate that the molecular mechanisms causing accelerated aging in progeric patients also occur in healthy cells of older individuals. Thus, the results of this study may also help explain some of the cellular changes that accompany normal aging.

Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen, carbon, or phosphate starvation.

Using proteomic technologies, we identified 62 proteins that are oxidized to carbonyl derivatives during growth of Escherichia coli under nitrogen starvation (NS), carbon starvation (CS), and phosphate starvation (PS) conditions. The carbonylated proteins were converted to 2,4-dinitrophenylhydrazone derivatives and these were identified using Western blotting and mass spectrometry by searching E. coli proteins in the Swiss-Prot and/or NCBI databases. Fourteen of the oxidized proteins were formed under both NS and CS conditions, and only three proteins were specifically oxidized under PS conditions. Interestingly, the carbonyl content of proteins in crude extracts of cells harvested after 48 h of stationary growth under NS and CS was significantly lower than that observed at mid-log and end-log phases of growth. In contrast, the carbonyl content of proteins in extracts of cells grown under PS conditions was fairly constant during comparable periods of growth.

Synergism between the toxicity of chlorophenols and iron complexes.

Synergistic interactions could prove to be relevant when evaluating the toxicity of environmental pollutants in a complex mixture, especially when organic and inorganic substances co-occur at concentrations currently considered to be low-toxic or sublethal. Escherichia coli cells (SR-9 strain) were used as a model system for studying the cellular toxicity of environmental pollutants. Exposure of bacterial cells to a combination of pentachlorophenol (PCP) and a positively charged complex of iron or copper caused a dramatic inhibition of growth and an increase in cell death. Incubation of bacterial cells with PCP and either ferric-1,10-phenanthroline complex [Fe3+(OP)3]3+ (500 and 5 microM, respectively) or cupric-1,10-phenanthroline complex [Cu2+(OP)2]2+ (400 and 0.05 microM, respectively) showed two and four log units of cell death, respectively, in 30 min. In contrast, only minor amounts of cell death were observed with each component alone. Similar effects have been shown for other positively charged complexes of transition metals and for other biocides. The observed synergism was associated with the formation of novel noncharged and lipophilic ternary complexes, which contain PCP anions (or other polychlorinated anions) and the iron (or copper) complex. The ternary complexes demonstrated effective transport of their components into the cells.

Oxidized messenger RNA induces translation errors.

To investigate the effect of RNA oxidation on normal cellular functions, we studied the translation of nonoxidized and oxidized luciferase mRNA in both rabbit reticulocyte lysate and human HEK293 cells. When HEK293 cells transfected with nonoxidized mRNA encoding the firefly luciferase protein were cultured in the presence of paraquat, there was a paraquat concentration-dependent increase in the formation of luciferase short polypeptides (SPs) concomitant with an increase in 8-oxoguanosine. Short polypeptides were also formed when the mRNA was oxidized in vitro by the Fe-ascorbate-H(2)O(2) metal-catalyzed oxidation system before its transfection into cells. Translation of the in vitro oxidized mRNA in rabbit reticulocyte lysate also led to formation of SPs. The SPs formed by either procedure contained the N-terminal and the C-terminal portions of the tagged luciferase. In addition, the oxidized mRNA was able to associate with ribosomes to form polysomes similar to those formed with nonoxidized mRNA preparations, indicating that the oxidized mRNAs are mostly intact; however, their translation fidelity was significantly reduced. Nevertheless, our results indicate that the SPs were derived from both premature termination of the translation process of the oxidized mRNA and the proteolytic degradation of the modified full-length luciferase resulting from translation errors induced by oxidized mRNA. In light of these findings, the physiological consequences of mRNA oxidation are discussed.

Protein oxidation and aging.

Organisms are constantly exposed to various forms of reactive oxygen species (ROS) that lead to oxidation of proteins, nucleic acids, and lipids. Protein oxidation can involve cleavage of the polypeptide chain, modification of amino acid side chains, and conversion of the protein to derivatives that are highly sensitive to proteolytic degradation. Unlike other types of modification (except cysteine oxidation), oxidation of methionine residues to methionine sulfoxide is reversible; thus, cyclic oxidation and reduction of methionine residues leads to consumption of ROS and thereby increases the resistance of proteins to oxidation. The importance of protein oxidation in aging is supported by the observation that levels of oxidized proteins increase with animal age. The age-related accumulation of oxidized proteins may reflect age-related increases in rates of ROS generation, decreases in antioxidant activities, or losses in the capacity to degrade oxidized proteins.

Age-dependent cell death and the role of ATP in hydrogen peroxide-induced apoptosis and necrosis.

Cell death plays a pivotal role in the body to maintain homeostasis during aging. Studies have shown that damaged cells, which must be removed from the body, accumulate during aging. Decay of the capacity and/or control of cell death during aging is widely considered to be involved in some age-dependent diseases. We investigated the accumulation of protein carbonyls and the role of cell death induced by hydrogen peroxide in human fibroblasts from individuals of various ages (17-80 years). The results showed that levels of oxidatively modified proteins increased with age, not only in whole-cell lysates but also in mitochondrial fractions, and this change correlates with a decline in the intracellular ATP level. Exposure of fibroblasts to hydrogen peroxide led to cell death by apoptosis and necrosis. Younger (<60 years old) cells were more resistant to necrosis induced by hydrogen peroxide than were older cells (>60 years old), which contained lower levels of free ATP than did younger cells. Treatment of cells of all ages with inhibitors of ATP synthesis (oligomycin, 2,4-dinitrophenol, or 2-deoxyglucose) made them more susceptible to cell death but also led to a switch in the death mode from apoptosis to necrosis. Furthermore, hydrogen peroxide treatment led to a greater accumulation of several inflammatory cytokines (IL-6, IL-7, IL-16, and IL-17) and increased necrosis in older cells. These results suggest that age-related decline in the ATP level reduces the capacity to induce apoptosis and promotes necrotic inflammation. This switch may trigger a number of age-dependent disorders.

Knockout of caspase-like gene, YCA1, abrogates apoptosis and elevates oxidized proteins in Saccharomyces cerevisiae.

In our previous study, we established that inhibition of apoptosis by the general caspase inhibitor is associated with an increase in the level of oxidized proteins in a multicellular eukaryotic system. To gain further insight into a potential link between oxidative stress and apoptosis, we carried out studies with Saccharomyces cerevisiae, which contains a gene (YCA1) that encodes synthesis of metacaspase, a homologue of the mammalian caspase, and is known to play a crucial role in the regulation of yeast apoptosis. We show that upon exposure to H(2)O(2), the accumulation of protein carbonyls is much greater in a Delta yca1 strain lacking the YCA1 gene than in the wild type and that apoptosis was abrogated in the Delta yca1 strain, whereas wild type underwent apoptosis as measured by externalization of phosphatidylserine and the display of TUNEL-positive nuclei. We also show that H(2)O(2)-mediated stress leads to up-regulation of the 20S proteasome and suppression of ubiquitinylation activities. These findings suggest that deletion of the apoptotic-related caspase-like gene leads to a large H(2)O(2)-dependent accumulation of oxidized proteins and up-regulation of 20S proteasome activity.

Protein oxidation by the cytochrome P450 mixed-function oxidation system.

This mini-review summarizes results of studies on the oxidation of proteins and low-density lipoprotein (LDL) by various mixed-function oxidation (MFO) systems. Oxidation of LDL by the O2/FeCl3/H2O2/ascorbate MFO system is dependent on all four components and is much greater when reactions are carried out in the presence of a physiological bicarbonate/CO2 buffer system as compared to phosphate buffer. However, FeCl3 in this system could be replaced by hemin or the heme-containing protein, hemoglobin, or cytochrome c. Oxidation of LDL by the O2/cytochrome P450 cytochrome c reductase/NADPH/FeCl3 MFO system is only slightly higher (25%) in the bicarbonate/CO2 buffer as compared to phosphate buffer, but is dependent on all components except FeCl3. Omission of FeCl3 led to a 60% loss of activity. These results suggest that peroxymonobicarbonate and/or free radical derivatives of bicarbonate ion and/or CO2 might contribute to LDL oxidation by these MFO systems.

Effect of bicarbonate on iron-mediated oxidation of low-density lipoprotein.

Oxidation of low-density lipoprotein (LDL) may play an important role in atherosclerosis. We studied the effects of bicarbonate/CO2 and phosphate buffer systems on metal ion-catalyzed oxidation of LDL to malondialdehyde (MDA) and to protein carbonyl and MetO derivatives. Our results revealed that LDL oxidation in mixtures containing free iron or heme derivatives was much greater in bicarbonate/CO2 compared with phosphate buffer. However, when copper was substituted for iron in these mixtures, the rate of LDL oxidation in both buffers was similar. Iron-catalyzed oxidation of LDL was highly sensitive to inhibition by phosphate. Presence of 0.3-0.5 mM phosphate, characteristic of human serum, led to 30-40% inhibition of LDL oxidation in bicarbonate/CO2 buffer. Iron-catalyzed oxidation of LDL to MDA in phosphate buffer was inhibited by increasing concentrations of albumin (10-200 microM), whereas MDA formation in bicarbonate/CO2 buffer was stimulated by 10-50 microM albumin but inhibited by higher concentrations. However, albumin stimulated the oxidation of LDL proteins to carbonyl derivatives at all concentrations examined in both buffers. Conversion of LDL to MDA in bicarbonate/CO2 buffer was greatly stimulated by ADP, ATP, and EDTA but only when EDTA was added at a concentration equal to that of iron. At higher than stoichiometric concentrations, EDTA prevented oxidation of LDL. Results of these studies suggest that interactions between bicarbonate and iron or heme derivatives leads to complexes with redox potentials that favor the generation of reactive oxygen species and/or to the generation of highly reactive CO2 anion or bicarbonate radical that facilitates LDL oxidation.

Methionine oxidation and aging.

It is well established that many amino acid residues of proteins are susceptible to oxidation by various forms of reactive oxygen species (ROS), and that oxidatively modified proteins accumulate during aging, oxidative stress, and in a number of age-related diseases. Methionine residues and cysteine residues of proteins are particularly sensitive to oxidation by ROS. However, unlike oxidation of other amino acid residues, the oxidation of these sulfur amino acids is reversible. Oxidation of methionine residues leads to the formation of both R- and S-stereoisomers of methionine sulfoxide (MetO) and most cells contain stereospecific methionine sulfoxide reductases (Msr's) that catalyze the thioredoxin-dependent reduction of MetO residues back to methionine residues. We summarize here results of studies, by many workers, showing that the MetO content of proteins increases with age in a number of different aging models, including replicative senescence and erythrocyte aging, but not in mouse tissues during aging. The change in levels of MetO may reflect alterations in any one or more of many different mechanisms, including (i) an increase in the rate of ROS generation; (ii) a decrease in the antioxidant capacity; (iii) a decrease in proteolytic activities that preferentially degrade oxidized proteins; or (iv) a decrease in the ability to convert MetO residues back to Met residues, due either to a direct loss of Msr enzyme levels or indirectly to a loss in the availability of the reducing equivalents (thioredoxin, thioredoxin reductase, NADPH generation) involved. The importance of Msr activity is highlighted by the fact that aging is associated with a loss of Msr activities in a number of animal tissues, and mutations in mice leading to a decrease in the Msr levels lead to a decrease in the maximum life span, whereas overexpression of Msr leads to a dramatic increase in the maximum life span.

Molecular cloning and characterization of murine caspase-12 gene promoter.

The activation of caspase-12 is involved in endoplasmic reticulum-mediated apoptosis. To investigate how caspase-12 is transcriptionally and translationally regulated, we isolated and sequenced the 5'-flanking region of mouse caspase-12 gene by a PCR-mediated chromosome-walking technique, using mouse genomic DNA as a template. Two DNA fragments of 3,221 and 800 bp were isolated and cloned into pGL3 promoterless vector upstream of the luciferase gene. The small DNA fragment contains the first intron sequence located downstream of the first exon and 27 bp from the second exon, whereas the large fragment contains the small fragment and the 5'-flanking region. Reporter constructs generated from these DNA fragments showed a substantial promoter activity in mouse NIH 3T3 or human embryonic kidney 293 cells grown in the presence of 10% serum. In the absence of serum, the luciferase activity was drastically reduced. However, the luciferase mRNA was higher in serum-starved cells than in control cells, suggesting that translation of luciferase mRNA was drastically inhibited. However, Western blot analysis revealed that the quantity of procaspase-12 is actually higher in serum-starved cells relative to that cultured in the presence of 10% serum. Progressive deletion analysis of the 3,221-bp sequence revealed that the highest luciferase activity was observed with the construct containing 700 bp upstream of ATG. The transcriptional initiation site was identified by 5' RACE techniques using total RNA from NIH 3T3 cells. Our results should facilitate studies on the mechanism regulating the expression of this important gene.

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans.

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.

Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors.

Stimulation of cells with various peptide growth factors induces the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) through activation of phosphatidylinositol 3-kinase. The action of this enzyme is reversed by that of the tumor suppressor PTEN. With the use of cells overexpressing NADPH oxidase 1 or peroxiredoxin II, we have now shown that H2O2 produced in response to stimulation of cells with epidermal growth factor or platelet-derived growth factor potentiates PIP3 generation and activation of the protein kinase Akt induced by these growth factors. We also show that a small fraction of PTEN molecules is transiently inactivated as a result of oxidation of the essential cysteine residue of this phosphatase in various cell types stimulated with epidermal growth factor, platelet-derived growth factor, or insulin. These results suggest that the activation of phosphatidylinositol 3-kinase by growth factors might not be sufficient to induce the accumulation of PIP3 because of the opposing activity of PTEN and that the concomitant local inactivation of PTEN by H2O2 might be needed to increase the concentration of PIP3 sufficiently to trigger downstream signaling events. Furthermore, together with previous observations, our data indicate that peroxiredoxin likely participates in PIP3 signaling by modulating the local concentration of H2O2.

Inhibition of apoptosis in acute promyelocytic leukemia cells leads to increases in levels of oxidized protein and LMP2 immunoproteasome.

On reaching maturity, animal organs cease to increase in size because of inhibition of cell replication activities. It follows that maintenance of optimal organ function depends on the elimination of oxidatively damaged cells and their replacement with new cells. To examine the effects of oxidative stress and apoptosis on the accumulation of oxidized proteins, we exposed acute promyelocytic leukemia cells to arsenic trioxide (As(2)O(3)) in the presence and absence of a general caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone), which is known to inhibit caspase-induced apoptosis. We confirm that treatment of cells with As(2)O(3) induces apoptosis and leads to the accumulation of oxidized proteins. Furthermore, inhibition of caspase activities prevented As(2)O(3)-induced apoptosis and led to a substantial increase in accumulation of oxidized proteins. Moreover, inhibition of caspase activity in the absence of As(2)O(3) led to elevated levels of the LMP2 immunoproteasome protein. We also show that caspase inhibition leads to increases in the levels of oxidized proteins obtained by treatments with hydrogen peroxide plus ferrous iron. Collectively, these results suggest the possibility that an age-related loss in capacity to carry out apoptosis might contribute to the observed accumulation of oxidized proteins during aging and in age-related diseases.

Role of oxidant species in aging.

Organisms are constantly exposed to many different forms of reactive oxygen species and reactive nitrogen species that damage proteins, nucleic acids, and lipids, leading to loss of biological function. The possibility that reactive oxygen/nitrogen-mediated protein damage contributes to the aging process is supported by results of many studies showing that aging is associated with the accumulation of such protein damage. Summarized here are results of studies, showing that the accumulation of,protein damage is a complex function of a multiplicity of factors that govern the intracellular levels of reactive oxygen/nitrogen species, on the one hand, and a multiplicity of factors that govern the degradation and/or repair of damaged proteins, on the other. Basic mechanisms involved in the modification of proteins by various forms of reactive oxygen/nitrogen species are also discussed.

Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis.

SUMO, a small ubiquitin-related modifier, is known to covalently attach to a number of nuclear regulatory proteins such as p53, IkappaB, promyelocytic leukemia protein and c-Jun. The sumoylation reaction is catalyzed by the SUMO protease, which exposes the C-terminal active glycine residue of the nascent SUMO, the heterodimeric SUMO activating enzyme, the SUMO conjugating enzyme, Ubc9, and SUMO protein ligases, in a manner similar to ubiquitinylation. Identification of SUMO-regulated proteins is hampered by the fact that many sumoylated proteins are present at a level below normal detection limit. This limitation was overcome by either in vivo overexpression of Myc-SUMO or in vitro sumoylation with excess biotin-SUMO and Ubc9. Sumoylated proteins so obtained were affinity purified or isolated by immunoprecipitation. The isolated sumoylated proteins were identified by sequence analysis using mass spectrometric methods. Results reveal that several heterogeneous nuclear ribonucleoproteins (hnRNPs), zinc finger proteins, and nuclear pore complex proteins were sumoylated. The sumoylation of hnRNP A1, hnRNP F, and hnRNP K were confirmed in vivo by coimmunoprecipitation. In view of the facts that hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our findings suggest that sumoylation could play an important role in regulating mRNA metabolism.

Regulation of Glutamine Synthetase Activity.

Detailed studies of the glutamine synthetase (GS) in Escherichia coli and other bacteria have shown that the activity of this enzyme is regulated by at least five different mechanisms: (i) cumulative feedback inhibition by multiple end products of glutamine metabolism, (ii) interconversion between taut and relaxed protein configurations in response to binding and dissociation of divalent cations at one of its two metal binding sites, (iii) dynamic interconversion of the enzyme between covalently modified (adenylylated) and unmodified forms by a novel bicyclic cascade system, (iv) repression and derepression of glutamine synthetase formation by cyclic phosphorylation and dephosphorylation of an RNA factor that governs transcription activities, and (v) regulation of glutamine synthetase turnover by the coupling of site specific metal ion-catalyzed oxidation with proteolytic degradation of the enzyme. Glutamine synthetase activity in E. coli is subject to inhibition by seven different end products of glutamine metabolism, namely, by tryptophan, histidine, carbamyl-phosphate, CTP, AMP, glucose-6-phosphate, and NAD+, and also by serine, alanine, and glycine. The cascade theory predicts that the steady-state level of glutamine synthetase adenylylation and therefore its catalytic activity is determined by the combined effects of all metabolites that affect the kinetic parameters of one or more of the enzymes in the cascade. Furthermore, under conditions where the supplies of ATP and glutamate are not limiting and the production of glutamine exceeds the demand, GS is no longer needed, then it will be converted to the catalytically inactive adenylylated form that is not under protection of ATP and glutamate.