Protein oxidation in aging brain.
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Aging↗
Biomedical subjects
E R Stadtman
Publications and source records attributed to E R Stadtman.
Protein oxidation and aging.
A number of systems that generate oxygen free radicals catalyze the oxidative modification of proteins. Such modifications mark enzymes for degradation by cytosolic neutral alkaline proteases. Protein oxidation contributes to the pool of damaged enzymes, which increases in size during aging and in various pathological states. The age-related increase in amounts of oxidized protein may reflect the age-dependent accumulation of unrepaired DNA damage that, in a random manner, affects the concentrations or activities of numerous factors that govern the rates of protein oxidation and the degradation of oxidized protein.
Aging↗
Selective cleavage of thioether linkage in proteins modified with 4-hydroxynonenal.
The peroxidation of polyunsaturated fatty acids leads to numerous products, including 4-hydroxynonenal (HNE). That 4-hydroxy-2-alkenal compounds react with sulfhydryl groups of proteins to form thioether adducts possessing a carbonyl function has been established [Schauenstein, E. & Esterbauer, H. (1979) Ciba Found. Symp. 67, 225-244]. Taking advantage of the fact that Raney nickel catalyzes cleavage of thioether bonds, we have developed a procedure to quantitate the amount of HNE moiety bound to protein by means of a thioether linkage. Adducts of HNE with N-acetylcysteine and glutathione were prepared, labeled with NaB[3H]H4, and then treated with Raney nickel. The 3H-labeled product was recovered in 85-90% yield from both HNE-N-acetylcysteine and HNE-glutathione adducts in a solvent [10% (vol/vol) methanol/chloroform]-estractable form. Treatment of proteins with HNE led to the disappearance of protein sulfhydryl groups. However, less than 10% of the labeled adducts obtained after subsequent reduction with NaB[3H]H4 could be released in a solvent-extractable form upon treatment with Raney nickel. This and the observation that HNE reacts with proteins lacking a sulfhydryl group attests to the fact that HNE can react with amino acid residues other than cysteinyl residues.
Acetylcysteine↗
Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.
We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined.
Aldehydes↗
Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Structural and functional changes.
As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50%. Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2. Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate. Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes. Our results demonstrate the inherent susceptibility of Glu-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.
Glucosephosphate Dehydrogenase↗
Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.
Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.
Adenosine Triphosphate↗
Protein modification in aging.
During aging a number of enzymes accumulate as catalytically inactive or less active forms. The age-related changes in catalytic activity are due in part to reactions of the protein with "active" oxygen species such as ozone, singlet oxygen, or with oxygen free radicals as are produced during exposure to ionizing radiation or to metal ion catalyzed oxidation (MCO) systems. The levels of oxidized proteins in cultured human fibroblasts from individuals of various ages and in liver and brain extracts of rats of different ages increase progressively with age, and in old rats can represent 30-50% of the total cellular protein. The age-related increase in oxidized protein in rat liver and brain tissue is accompanied by a loss of glutamine synthetase (GS) and glucose-6-P dehydrogenase (G-6-PDH) activities, and to a decrease in the level of cytosolic neutral protease activity which is responsible for the degradation of oxidized (denatured) protein. Of particular significance are the results of experiments showing that similar age-related changes occur in the gerbil brain and that these changes are accompanied by a loss of short-term memory as measured by the radial arm maze technique. Chronic treatment (intraperitoneal injections) of old animals with the free radical spin-trap reagent, N-tert-butyl-alpha-phenylnitrone (PBN) resulted in normalization of the several biochemical parameters to those characteristic of the young animals; coincidentally, the short-term memory index was restored to the young animal values. These results provide the first evidence that there is likely a linkage between the age-dependent accumulation of oxidized enzymes and the loss of physiological function.
Aging↗
Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease.
The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal- and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.
Adult↗
Fenton chemistry. Amino acid oxidation.
The oxidation of amino acids by Fenton reagent (H2O2 + Fe(II] leads mainly to the formation of NH+4, alpha-ketoacids, CO2, oximes, and aldehydes or carboxylic acids containing one less carbon atom. Oxidation is almost completely dependent on the presence of bicarbonate ion and is stimulated by iron chelators at levels which are substoichiometric with respect to the iron concentration but is inhibited at higher concentrations. The stimulatory effect of chelators is not due merely to solubilization of catalytically inactive polymeric forms of Fe(OH)3 nor to the conversion of Fe(II) to complexes incapable of scavenging hydroxyl radicals. The results suggest that an iron chelate and another as yet unidentified form of iron are both required for maximal rates of amino acid oxidation. The metal ion-catalyzed oxidation of amino acids is likely a "caged" process, since the oxidation is not inhibited by hydroxyl radical scavengers, and the relative rates of oxidation of various amino acids by the Fenton system as well as the distribution of products formed (especially products of aromatic amino acids) are significantly different from those reported for amino acid oxidation by ionizing radiation. Several iron-binding proteins, peptides, and hemoglobin degradation products can replace Fe(II) or Fe(III) in the bicarbonate-dependent oxidation of amino acids. In view of their ability to sequester metal ions and their susceptibility to oxidation by H2O2 in the presence of physiological concentrations of bicarbonate, amino acids may serve an important role in antioxidant defense against tissue damage.
Amino Acids↗
Metal-catalyzed oxidation of proteins. Physiological consequences.
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Aging↗
Ascorbic acid and oxidative inactivation of proteins.
A number of active oxygen species are likely implicated in the etiology or manifestation of several pathological conditions, including aging, arthritis, carcinogenesis, atherosclerosis, and muscular dystrophy. Ascorbate plays a key role in protecting cells against oxidative damage. Paradoxically, in the presence of Fe3+ or Cu2+, ascorbate can promote the generation of the same reactive oxygen species (.OH, O2-, H2O2, and ferryl ion) it is known to destroy. This prooxidant activity derives from the ability of ascorbate to reduce Fe3+ or Cu2+ to Fe2+ or Cu+, respectively, and to reduce O2 to O2-. and H2O2. Damage to nucleic acid and proteins results from the binding of either Fe2+ or Cu+ to metal binding sites on these macromolecules followed by reaction of the metal complexes with H2O2; this leads to the production of active oxygen species that attack functional groups at or near the metal binding sites.
Antioxidants↗
Covalent modification reactions are marking steps in protein turnover.
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Acetylation↗
Discovery of glutamine synthetase cascade.
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Escherichia coli↗
Determination of carbonyl content in oxidatively modified proteins.
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Amino Acids↗
Protein that prevents mercaptan-mediated protein oxidation.
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Aerobiosis↗
Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences.
In the presence of O2, Fe(III) or Cu(II), and an appropriate electron donor, a number of enzymic and nonenzymic oxygen free radical-generating systems are able to catalyze the oxidative modification of proteins. Whereas random, global modification of many different amino acid residues and extensive fragmentation occurs when proteins are exposed to oxygen radicals produced by high energy radiation, only one or a few amino acid residues are modified and relatively little peptide bond cleavage occurs when proteins are exposed to metal-catalyzed oxidation (MCO) systems. The available evidence indicates that the MCO systems catalyze the reduction of Fe(III) to Fe(II) and of O2 to H2O2 and that these products react at metal-binding sites on the protein to produce active oxygen (free radical?) species (viz; OH, ferryl ion) which attack the side chains of amino acid residues at the metal-binding site. Among other modifications, carbonyl derivatives of some amino acid residues are formed; prolyl and arginyl residues are converted to glutamylsemialdehyde residues, lysyl residues are likely converted to 2-amino-adipylsemialdehyde residues; histidyl residues are converted to asparagine and/or aspartyl residues; prolyl residues are converted to glutamyl or pyroglutamyl residues; methionyl residues are converted to methionylsulfoxide residues; and cysteinyl residues to mixed-disulfide derivatives. The biological significance of these metal ion-catalyzed reactions is highlighted by the demonstration: (i) that oxidative modification of proteins "marks" them for degradation by most common proteases and especially by the cytosolic multicatalytic proteinase from mammalian cells; (ii) protein oxidation contributes substantially to the intracellular pool of catalytically inactive and less active, thermolabile forms of enzymes which accumulate in cells during aging, oxidative stress, and in various pathological states, including premature aging diseases (progeria, Werner's syndrome), muscular dystrophy, rheumatoid arthritis, cataractogenesis, chronic alcohol toxicity, pulmonary emphysema, and during tissue injury provoked by ischemia-reperfusion. Furthermore, the metal ion-catalyzed protein oxidation is the basis of biological mechanisms for regulating changes in enzyme levels in response to shifts from anaerobic to aerobic metabolism, and probably from one nutritional state to another. It is also involved in the killing of bacteria by neutrophils and in the loss of neutrophil function following repeated cycles of respiratory burst activity.
Aging↗
Manganese-dependent disproportionation of hydrogen peroxide in bicarbonate buffer.
At physiological concentrations of HCO3- and CO2, Mn(II) catalyzes disproportionation of H2O2. This catalase-like activity is directly proportional to the concentrations of Mn(II) and H2O2, and it increases exponentially with increases in pH. The effect of increasing pH is almost completely attributable to the concomitant increase in HCO3- concentration. The rate is proportional to the third power of the HCO3- concentration, suggesting that 3 equivalents of HCO3- combine with 1 equivalent of Mn(II) to form the catalytic complex. It is presumed that the redox potential of the Mn(II) in equilibrium with Mn(III) couple in such a complex permits H2O2 to carry out facile reactions with Mn(II) comparable to those that occur with Fe(III) and Cu(II) chelate complexes, in which OH. and O2-. are established intermediates. The Mn-catalyzed disproportionation of H2O2 does not occur at physiological pH in the absence of HCO3-. Hepes, inorganic phosphate, and inorganic pyrophosphate inhibit the reaction catalyzed by the Mn/HCO3- system. These results are similar to those of Sychev et al. [Sychev, A.Y., Pfannmeller, U. & Isak, V.G. (1983) Russ. J. Phys. Chem. 57, 1690-1693]. The catalase-like activity of Mn(II)-bicarbonate complexes reported here, together with the superoxide dismutase activity of Mn complexes demonstrated by Archibald and Fridovich [Archibald, F.S. & Fridovich, I. (1982) Arch. Biochem. Biophys. 214, 452-463], strengthen the proposition that Mn may play an important role in the protection of cells against oxygen radical-mediated damage.
Bicarbonates↗
Manganese(II) catalyzes the bicarbonate-dependent oxidation of amino acids by hydrogen peroxide and the amino acid-facilitated dismutation of hydrogen peroxide.
In bicarbonate/CO2 buffer, Mn(II) and Fe(II) catalyze the oxidation of amino acids by H2O2 and the dismutation of H2O2. As the Mn(II)/Fe(II) ratio is increased, the yield of carbonyl compounds per mole of leucine oxidized is essentially constant, but the ratio of alpha-ketoisocaproate to isovaleraldehyde formed increases, and the fraction of H2O2 converted to O2 increases. In the absence of Fe(II), the rate of Mn(II)-catalyzed leucine oxidation is directly proportional to the H2O2, Mn(II), and amino acid concentrations and is proportional to the square of the HCO3- concentration. The rate of Mn(II)-catalyzed O2 production in the presence of 50 mM alanine or leucine is about 4-fold the rate observed in the absence of amino acids and accounts for about half of the H2O2 consumed; the other half of the H2O2 is consumed in the oxidation of the amino acids. In contrast, O2 production is increased nearly 18-fold by the presence of alpha-methylalanine and accounts for about 90% of the H2O2 consumed. The data are consistent with the view that H2O2 decomposition is an inner sphere (cage-like) process catalyzed by a Mn coordination complex of the composition Mn(II), amino acid, (HCO3-)2. Oxidation of the amino acid in this complex most likely proceeds by a free radical mechanism involving hydrogen abstraction from the alpha-carbon as a critical step. The results demonstrate that at physiological concentrations of HCO3- and CO2, Mn(II) is able to facilitate Fenton-type reactions.
Alanine↗