Tài liệu Báo cáo khoa học: Changes in rat liver mitochondria with aging Lon protease-like activity and N e-carboxymethyllysine accumulation in the matrix doc

Changes in rat liver mitochondria with aging Lon protease-like activity and N e -carboxymethyllysine accumulation in the matrix Hilaire Bakala 1 , Evelyne Delaval 1 , Maud Hamelin 1 , Jeanne Bismuth 1 , Caroline Borot-Laloi 1 , Bruno Corman 2 and Bertrand Friguet 1 1 Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris7-Denis Diderot, Paris, France; 2 Service de Biologie Cellulaire, Commissariat a ` l’Energie Atomique/Saclay, Gif-sur-Yvette, France Aging is accompanied by a gradual deterioration of cell functions. Mitochondrial dysfunction and accumulation of protein damage have been proposed to contribute to this process. The present study was carried out to examine the effects of aging in mitochondrial matrix isolated from rat liver. The activity of Lon protease, an enzyme implicated in the degradation of abnormal matrix proteins, was meas- ured and the accumulation of oxidation and glycoxidation (N e -carboxymethyllysine, CML) products was monitored using immunochemical assays. The function of isolated mitochondria was assessed by measuring respiratory chain activity. Mitochondria from aged (27 months) rats exhi- bited the same rate of oxygen consumption as those from adult (10 months) rats without any change in coupling efficiency. At the same time, the ATP-stimulated Lon protease activity, measured as fluorescent peptides released, markedly decreased from 10-month-old rats (1.15 ± 0.15 FUÆlgprotein )1 Æh )1 ) to 27-month-old-rats (0.59 ± 0.08 FUÆlgprotein )1 Æh )1 ). In parallel with this decrease in activity, oxidized proteins accumulated in the matrix upon aging while the CML-modified protein content assessed by ELISA significantly increased by 52% from 10 months (11.71 ± 0.61 pmol CMLÆlgprotein )1 ) to 27 months (17.81 ± 1.83 pmol CMLÆlgprotein )1 ). These results indicate that the accumulation of deleterious oxidized and carboxymethylated proteins in the matrix concomitant with loss of the Lon protease activity may affect the ability of aging mitochondria to respond to additional stress. Keywords: aging; mitochondria; matrix; Lon protease; carboxymethyllysine. A striking characteristic of normal aging in long-lived animals is the gradual decline in their physiological func- tions. This decline is associated with an increase in reactive oxygen species (ROS) production [1] and an accumulation of macromolecules damaged by post-translational nonenzy- matic modifications which alter the structure and function of tissue and cellular proteins [2–5]. Under oxidative stress, carbohydrates, lipids and proteins are the major targets of reactive oxygen species. Proteins can be damaged either directly or indirectly through the reactive carbonyl com- pounds derived from the oxidation of carbohydrates and lipids [6,7]. These carbonyl compounds react with protein amino-groups to give glycoxidation products such as N e -carboxymethyllysine (CML) [8]. This glycoxidation process modifies cell proteins and the cumulative effects lead to the tissue alterations and cell dysfunction typical of aging and diabetes [9,10]. Recent data also indicate that glycoxidative processes affect mitochondrial membrane phospholipids [11]. Mitochondria are in fact a major intracellular source of ROS during oxidative phosphoryla- tion and the increased production of ROS is implicated in the aging process [12–16]. Mitochondria are also the major targets of these ROS, which may damage mitochondrial proteins themselves, leading to dysfunction of these organelles [17,18]. Oxidative damage mainly concerns the activities of electron transport complexes of the inner mitochondrial membrane which are specifically modified during aging [19–21]. No attempt has yet been made to correlate the alterations in mitochondrial function with biochemical changes in the liver mitochondrial matrix of aging rats. Nevertheless, an age-related decrease in the expression of several genes involved in mitochondrial bioenergetics and mitochondrial biogenesis occurs in aging mice; the largest age-associated alteration affects the matrix enzyme Lon protease [22]. This ATP-stimulated protease is homologous to bacterial Lon protease [23] and has been found in several mammals including human tissues and cell lines [24–26]. It is responsible for the degradation of abnormal proteins and certain short-lived specific proteins [27,28]. In this study we have investigated the age-associated biochemical changes in mitochondrial matrix proteins from the rat liver. For this purpose we related the activity of Lon protease with the level of damaged proteins in the matrix, focusing on the accumulation of oxidized and CML-proteins as a marker of glycoxidative stress. Correspondence to H. Bakala, Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite ´ Paris7-Denis Diderot, T23-33 1 er e ´ tage CC 7128, 2 Place Jussieu, 75252 Paris, France. Fax: + 33 1 44 27 82 34; Tel.: + 33 1 44 27 82 35; E-mail: bakala@paris7.jussieu.fr Abbreviations: ROS, reactive oxygen species; AGE, advanced glycat- ion end products; CML, carboxymethyllysine; ABTS, 2,2¢-azinobis (3-ethylbenzo-6-thiazolinesulfonic acid); RCR, respiratory control ratio. (Received 22 January 2003, accepted 27 March 2003) Eur. J. Biochem. 270, 2295–2302 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03598.x Our results indicate that mitochondrial matrix proteins undergo oxidative and glycoxidative modifications. These damaged proteins accumulate with aging, in parallel with a large decrease in Lon protease activity. Materials and methods Animals Experiments were performed on male Wistar rats (WAG/ Rij) born and raised in the animal care facilities of the Commissariat a ` l’Energie Atomique (CEA Gif-sur-Yvette, France). This strain remains lean even when fed ad libitum and does not suffer from age-associated nephropathy [29]. The animals were fed a commercial diet (DO4; UAR, Villemoisson sur Orge, France) composed of 17% protein, 0.71% phosphorus, 0.78% calcium, 0.62% potassium, 0.27% sodium and 0.22% magnesium, with a total of 12.1 kJÆg )1 . Water was provided ad libitum. Cohorts were composed of adult and senescent animals (10 and 27 months old, respectively). All studies were conducted in accordance with the animal care policy of the National and European regulations. Chemicals Na(CN)BH 3 , glyoxylic acid, BSA (Fraction V) and HRP– conjugated anti-mouse IgG were purchased from Sigma. Anti-AGE mAb (clone no. 6D12) was from Trans Genic Inc. (Japan) and Oxyblot protein oxidation detection kit from Intergen. Chemical modification of BSA ( N e -carboxymethyllysine-BSA) Carboxymethylated bovine serum albumin (CML-BSA) was prepared according to Murata et al. [30]. Briefly, 100 mg BSA was incubated at 37 °Cfor24hwith0.15 M glyoxylic acid and 0.45 M Na(CN)BH 3 in 1 mL of 0.2 M sodium phosphate buffer (pH 7.4), and then extensively dialyzed against phosphate-buffered saline (NaCl/P i ). The CML content of the modified BSA was measured by amino acid analysis following hydrolysis of the modified protein in 6 M HCl, 0.2% phenol (Laboratoire de Micro- sequenc¸ age des Prote ´ ines, Institut Pasteur, Paris, France). There were 43.2 CML residues and 16.1 Lys residues in BSA-modified protein, while native BSA contained 57.9 Lys in a total of 692 residues. As the expected value in the primary sequence is 59 Lys, the error in the CML–adduct rate can be estimated to be as low as 2%. The CML content was expressed as 0.644 nmol CML per lg BSA, and this solution was used as the standard in a competitive ELISA. Isolation of mitochondria A 10% tissue homogenate was prepared in an ice-cold medium containing 220 m M mannitol, 70 m M sucrose, 2m M Hepes, 0.1 m M EDTA and 0.5% (w/v) BSA, pH 7.4. Nuclei and unbroken cells were pelleted by centrifugation for 10 min at 800 g and 0 °C. The super- natant was centrifuged at 8000 g for 10 min at 0 °C. The mitochondrial pellet was washed three times with the homogenization medium and used for polarographic measurements. For determination of matrix protease activity, mitochon- dria were suspended in 50 m M Tris/HCl buffer, pH 7.9, then disrupted by sonication (four times for 10 s). The resulting suspension was centrifuged at 15 000 g for 10 min and then at 100 000 g for 45 min. The supernatant (con- taining matrix protein) was stored at )80 °C for further determinations of protease activity and the level of carboxymethylated protein. Protein was assayed by the Bradford method. To estimate the contamination of mitochondrial prepar- ation with lysosomes, we used acid phosphatase activity as a marker. Measurements of mitochondrial respiration Oxygen consumption was measured polarographically with a Clark electrode in the sample, as described by Aprille and Asimakis [31] in a thermostatically controlled closed 2 mL chamber (30 °C). The rate of oxygen consumption was measured in the presence of 310 nmol ADP and 10 m M succinate or 5 m M glutamate and 5 m M malate (state 3) and when all the ADP has been consumed (state 4 or resting state). Oxygen-consumption rates are expressed as ng atoms of oxygen consumed per minute and per mg protein. The rate of oxygen consumption in state 3 and in state 4, respiratory control ratio (RCR) of oxygen consumptions in states 3 and 4 and the ADP/O ratio were calculated. Oxygen consumption in the presence of 40 l M of dinitrophenol (uncoupled state) was also checked. Enzymatic activities ATP-stimulated Lon protease activity was determined using casein-fluoresceine isothiocyanate (0.5 mgÆmL )1 ) as sub- strate. Casein was incubated with mitochondrial matrix extract (70 lgprotein)in70lL buffer (final concentration: 50 m M Tris/HCl, pH 7.9, 10 m M MgCl 2 , with or without 8m M ATP) for 1 h at 37 °C. The reaction was terminated by adding 30 lL of 40% trichloroacetic acid and 50 lLof 3% BSA. After centrifugation at 15 000 g for 30 min, 100 lLof2 M sodium borate was then added to 80 lLof supernatant. Fluorescence was measured with excitation/ emission wavelengths of 495/515 nm. Activity is expressed as fluorescence units per hour of incubation and per lg protein (FUÆlgprotein )1 Æh )1 ) corrected from the fluores- cence of casein alone, which represents around 25% of the measured fluorescence. Acid phosphatase activity was determined at each step of the isolation procedure using the acid phosphatase assay (Sigma kit), which measures the accumulation of p-nitrophenol from p-nitrophenyl phosphate disodium, at 410 nm. The reaction was stopped after 30 min incubation at room temperature. Determination of CML content in mitochondrial matrix proteins by competitive ELISA Each well of a 96-well microtiter plate (Nunc-immuno Plate, Nunc, Denmark) was coated with 100 lLCML-BSA (6.4 nmol CMLÆmL )1 )in50m M sodium carbonate buffer, 2296 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003 pH 9.6 by incubation overnight at 4 °C. The wells were washed three times with NaCl/P i containing 0.05% (v/v) Tween 20 (buffer A) and free binding sites were blocked by incubation for 1 h at room temperature with 100 lLNaCl/ P i containing 6% (w/v) skimmed milk. The wells were then washed with buffer A, 50 lL of competing antigen (test samples at 0.100 mgÆmL )1 or serial dilutions of standard CML-BSA from 0.64 m M to 128 m M ) was added, followed by 50 lL monoclonal antibody clone 6D12 (diluted 1 : 1000 in NaCl/P i ). The plate was incubated for 2 h at room temperature, washed and then incubated with 50 lL horseradish peroxidase-conjugated anti-mouse IgG (50 lL per well, second antibody diluted 1 : 1000) for 2 h at room temperature. The wells were washed, 100 lLofsubstrate solution [40 m M 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesul- fonic acid (ABTS) and 200 lL of 30% hydrogen peroxide in 20 mL acetate-phosphate buffer] were added per well and incubated for 30 min at 37 °C. The absorbance (A)was measured at 405 nm on a micro-ELISA plate reader (Spectra Rainbow, SLT Labinstruments, Austria). Results are expressed as the ratio B/B 0 , calculated as [experimental A minus background A (no antibody)]/[total A (no competitor) minus background A], vs. CML added as pmol CMLÆlgprotein )1 . Western blot analysis Western blot of CML proteins. Matrix protein samples (10 lg protein per lane) were electrophoresed on 10% (w/v) SDS/PAGE for 90 min at 100 V. One of two identical gels was stained with Coomassie blue to analyzed the pattern of matrix proteins. The proteins from the second gel were then transferred electrophoretically to a nitrocellulose membrane (Bio-Rad) for 1 h at 100 V. The membrane was saturated with 5% (w/v) skimmed milk in NaCl/P i /0.1% Tween 20 overnight at 4 °C, followed by four washes (10 min each) with NaCl/P i containing 0.2% (v/v) Tween 20 (wash buffer). The membrane was then incubated for 2 h at room temperature with anti-CML mAb clone 6D12 (diluted 1 : 1000 in NaCl/P i /0.1% Tween 20), washed four times with wash buffer, incubated for 1 h with anti-mouse IgG coupled to horseradish peroxidase (1 : 2500 dilution) and given a final wash. The proteins were revealed with an ECL reagent (Amersham-Pharmacia Biotech). Western blot of carbonylated proteins. Carbonylated proteins were analyzed using the oxyblot kit according to the manufacturer’s instructions (Oxyblot Detection, Inter- gen). Briefly, samples (10 lg protein per lane) were treated with 10 m M 2,4-dinitrophenolhydrazine in 2 M HCl, incu- bated at room temperature and neutralized. The derivatized proteins were separated by SDS/PAGE, transferred to a nitrocellulose membrane and treated as previous Western blotting (see below). The primary antibody used was against 2,4-dinitrophenol, and detection was performed using the ECL reagent. Western blot of Lon protease. A polyclonal antibody against Lon protease was raised in rabbit against a synthetic peptide corresponding to amino acids 208–221 of rat Lon. This antibody mainly recognized one protein band with an estimated molecular mass of 100 kDa corresponding to the molecularmassofLon. For Western blot analysis, we previously verified that signals were linear in the range from 10–60 lgoftotal protein. We used routinely 20 lg of mitochondrial matrix. Proteins were transferred onto nitrocellulose membrane (Bio-Rad), which was then incubated with Lon antibody (1 : 1000 dilution) for 1 h at room temperature and antigen were detected by chemiluminescence with Amersham’s ECL reagent. Electron microscopy Electron micrographs were taken of purified rat liver mitochondria. The mitochondrial pellet was washed with NaCl/P i , fixed by immersion in 2% paraformaldehyde/ 0.2% glutaraldehyde in NaCl/P i for 2 h at room tempera- ture, contrasted with 1% uranyl acetate. The resulting material was dehydrated and embedded in LRWhite. Ultrathin 60 nm sections were cut with a Leica ultramicro- tome, postfixed with 2% osmium tetroxide and examined in a Philips 400 electron microscope. Statistical analysis Results are presented as means ± SEM and differences between groups were assessed by means of Student’s unpaired t-test. Significance was set at P < 0.05. Results Age-related changes in the structure and respiratory function of isolated mitochondria As shown in Table 1, there was no difference in mito- chondrial oxygen consumptions between 10-month- and 27-month-old-rats, whatever the substrate used. The Table 1. Biochemical respiratory parameters in rat liver mitochondria from 10- and 27-month-old rats. Data represent mean values ± SEM (n, number of animals). Oxygen consumption rates were measured polarographically in the presence of 10 m M succinate or 5 m M glutamate plus 5m M malate. State 3 respiration was determined after adding 310 nmoles ADP. Glutamate (n ¼ 4) Succinate (n ¼ 7) 10 months 27 months 10 months 27 months State 4 (ng atom OÆmin )1 Æmg )1 ) 30.3 ± 0.3 26.5 ± 3.5 46.0 ± 3.5 47.1 ± 7.6 State 3 (ng atom OÆmin )1 Æmg )1 ) 120.8 ± 7.4 117.8 ± 9.4 189.7 ± 15.8 166.9 ± 14.1 RCR 3.98 ± 0.26 4.52 ± 0.32 4.15 ± 0.20 3.79 ± 0.36 P/O 2.85 ± 0.13 2.94 ± 0.13 2.05 ± 0.08 1.91 ± 0.06 Ó FEBS 2003 Protein alteration in mitochondrial matrix with aging (Eur. J. Biochem. 270) 2297 ADP/O ratios obtained with succinate or glutamate indicate no change in coupling efficiency. The yield of mitochondrial preparation as well as the respiratory control ratio (state 3/ state 4) did not change significantly during aging. Oxygen consumption rates were the same in the presence of 2,4- dinitrophenol or in the presence of ADP (state 3) at all ages considered. Electron micrography showed few alterations of crests and of mitochondrial membranes leading to some disrupted organelles (Fig. 1). Mitochondria preparations were free of contamination and both sets exhibited similar purity. ATP-stimulated protease activity in aging rats Results obtained by use of acid phosphatase determination in rat preparations indicated a very low lysosomal contami- nation (4% of mitochondrial matrix vs. total liver homo- genate). The activity of ATP-stimulated protease was determined in the presence or absence of ATP (Fig. 2A). Protease activity in the absence of ATP was lower in aging rats than in adults. Activity decreased by 51% between 10-month- old and 27-month-old rats (1.15 ± 0.15 and 0.59 ± 0.08 FUÆlgprotein )1 Æh )1 , respectively). Whatever the age, addition of ATP stimulated the degradation of the substrate casein about 2.5-fold but activity still decreases in an age- dependent manner. No significant difference could be detected in the level of Lon protein from the mitochondrial matrix of 10-month- and 27-month-old rats, as shown by Western blot analysis (Fig. 2B,C). Fig. 1. Assessment of the purity of mitochondrial preparations. Electron micrograph of isolated liver mitochondria from (A) 10-month-old and (B) 27-month-old rats. All the recognizable organelles are mito- chondria (arrow) showing different degrees of matrix density (star) and dilatation of the cristae (arrowhead). Disrupted mitochondria are visible (double arrow). Magnification, 30 000. Fig. 2. Lon protease quantification and activity in liver mitochondrial matrix from 10- and 27-month-old rats. Activitywasdeterminedinthe absence of ATP or in the presence of 8 m M ATP (A, white and grey columns respectively). Matrix proteins (20 lg) were subjected to Western blotting using a polyclonal antibody against Lon protease (B) and quantified by densitometric scanning (C), results being expressed in arbitrary units. Values are the mean ± SEM for five independent determinations. The P-value for enzymatic activity was significant (**P < 0.01 for 10-month-old rats). 2298 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003 CML adduct content in mitochondrial matrix proteins We used the monoclonal antibody clone 6D12 in competi- tive ELISA to evaluate the CML-protein content (Fig. 3). The CML content increased significantly by 52% from 11.71 ± 0.61 pmol CMLÆlgprotein )1 (n ¼ 8) in 10- month-old rats to 17.81 ± 1.83 pmol CMLÆlgprotein )1 (n ¼ 9) in 27-month-old rats (P ¼ 0.007). These data indicate an age-associated accumulation of CML adducts in mitochondrial matrix. Western blotting of modified proteins We identified the major proteins in the mitochondrial matrix by performing SDS/PAGE separation and staining the gel with Coomassie blue or Western blotting. Analysis of the SDS/PAGE revealed a broad spread of proteins with apparent molecular mass ranging from 10–170 kDa (Fig. 4A). The samples from the two different ages exhibited a comparable pattern of bands, although two bands of 60 and 150 kDa strongly visible at 10 months (lane 1) were absent at 27 months, and an important band of 70 kDa (lane 2) emerged in this latter sample. Western blotting with mAb 6D12 was used to detect matrix proteins undergoing CML modification with aging. Proteins from all molecular masses were immunolabelled in samples of both ages (Fig. 4B). The 10-month-old matrix proteins (lane 1) contained 14 bands intensely stained over an apparent range of 10–170 kDa, with the most prominent band at 60 kDa (lane 1). In the 27-month-old preparations (lane 2) only eight main bands are stained, with two intense signals of 70 and 50 kDa, while the bands at 60 and 150 kDa vanished. With oxyblot (Fig. 4C), antibodies stained carbonylated proteins mainly in band ranges of 30–60 and 70–120 kDa at 10 months (lane 1). These protein bands became strongly stained at 27 months, particularly those with apparent molecular masses of 30, 55, 75 and 105 kDa (lane 2). These results strongly support the hypothesis that CML- proteins and oxidized proteins do occur selectively in the liver mitochondrial matrix and that their recruitment varies with aging. Discussion We used isolated rat liver mitochondria to analyse the matrix defects that occur with aging. Mitochondria, similar to the cytosol, contain a proteolytic system that controls the metabolic stability of mitochondrial proteins and ensures the elimination of damaged proteins [28]. This continuous Fig. 3. Determination of CML-protein content in liver mitochondrial matrix with aging. The CML content in the matrix proteins from 10- and 27-month-old rats was measured by competitive ELISA using mAb 6D12. Results are expressed as pmol CMLÆlgprotein )1 (n, number of animals). ***P ¼ 0.007 vs. 10-month-old rats. Fig. 4. Western blot analyses of liver mitochondrial matrix proteins from 10-month- and 27-month-old rats. Samples (10 lg) were subjected to SDS/ PAGE under reducing conditions. The gel was stained with Coomassie blue (A) or subjected to Western blotting using either 6D12 monoclonal antibody to detect CML-modified proteins (B) or oxyblot kit to reveal oxidized proteins (C). Samples from 10-month-old (lane 1) and from 27-month-old rats (lane 2). Standard molecular masses (lane 3). The arrow and arrowhead show the disappearance and appearance of bands, respectively. Ó FEBS 2003 Protein alteration in mitochondrial matrix with aging (Eur. J. Biochem. 270) 2299 protein turnover appears to be important for their main- tenance and function, and consequently for cell integrity. Lon protease plays a pivotal role because it is responsible for breaking down abnormal matrix proteins. In our study, we have shown a large decline in ATP- stimulated Lon protease activity in the mitochondrial matrix with aging. This activity in 27-month-old rats is only 51% of that of adult (10-months) rats. Such a decrease in matrix enzyme activity cannot result from the disruption sensitivity of mitochondria as we observed roughly the same pool of disrupted organelles in preparations from both old and adult rats, as well as the same level of respiratory chain activity in the two groups of animals. Recently, age-related alterations of the mitochondrial function have been reported in liver mitochondria from young (4 months) and old (30 months) rats and correlated to a significant decrease of the specific activity of Complex I [32]. In our study we observed no change in the respiratory chain activity between 10-month-old and 27-month-old rats whatever the substrate used. It would be interesting to compare the mitochondrial function between young and adult rats (4 months vs. 10 months) to determine whether the matrix defect could be the consequence of precocious impairment in the inner membrane. The decrease in enzymatic Lon protease activity may be the result of either a loss of efficiency in relation to damaged protein and/or a decrease in the amount of this protein in the matrix. We have not detected any significant modifica- tion in the amount of this protein in the matrix. We can conclude that the decrease in this enzymatic activity is the result of accumulation of damaged protein occuring with aging. We found chemically modified proteins even in the mitochondrial matrix of adult rats, the concentration of which increased significantly with aging. On the one hand we found evidence of the occurrence of oxidative protein modifications. This oxidation appeared to selectively recruit proteins and their immunological signals increased with aging. These findings are consistent with previous reports showing an increase in carbonyl proteins in mitochondria from different tissues of several animals with age, because of increased oxidative stress [19,33,34]. On the other hand, our study demonstrated for the first time that matrix proteins undergo carboxymethylation. The level of CML-modified protein increased with aging but affected particular proteins, suggesting that some matrix proteins are more susceptible to glycoxidative stress. Although the source of the CM moiety is not defined, several findings have shown that CML can originate from both glycoxidation and lipoperoxidation reactions [6,35,36]. Recent studies on cultured vascular endothelial cells revealed that hyperglyceamia caused overproduction of mitochondrial ROS, which in turn initiate intracellular advanced glycation end products (AGE) formation primar- ily, if not exclusively, by increasing the concentration of AGE-forming methylglyoxal [37–39]. In addition, the inner mitochondrial membranes have a high percentage of cardiolipin phospholipid, which contains a very high level of the polyunsaturated fatty acid linoleic acid [40,41]. They are prime target of ROS due to their location near to the site of ROS production [42] and could be the source of carbonyl adducts. Other recent reports have shown that there is intracellular lipid glycoxidation that affects the phospha- tidylethanolamine from membranes of these organelles [11]. In the present investigation, we detected CML proteins using the anti-AGE mAb 6D12, which recognizes CML-like structures, as well as carboxyethyllysine and several unidentified AGE epitopes [43]. The oxidized proteins that accumulated with aging in the range 95–120 kDa could include the Lon protease, with an apparent molecular mass of 100 kDa. The Lon protease may be structurally modified by oxidation, which in turn could reduce its activity. In support of our assertion, recent reports claiming that carbonylated matrix enzymes such as aconitase accumulate in the housefly mitochondria with age accompanied by loss in its activity [17,44]. Nevertheless, the modifications we observed did not affect the ATPase binding site of the enzyme, as ATP stimulation was the same for enzymes from all mitochondria, regardless of age. Whatever the origin of the glycoxidation product, this accumulation of CML- and oxidized proteins with aging occurs while the Lon protease-like activity markedly decreases, could reveal an imbalance between the relative rates of glycoxidation/oxidation and proteolysis in the mitochondrial matrix. Although the respiratory function of mitochondria is not compromised in spite of the extent of altered proteins, we can speculate that the increase in glycoxidative and oxidative alteration is relevant only if the damage is severe enough to have an impact on mitochond- rial function. In addition this threshold severity will depend on tissue susceptibility: recent studies conducted on rat heart have demonstrated altered oxidative metabolism of cardiac mitochondria with aging [45], but this dysfunction selec- tively affected a specific subcellular region of the senescent myocytes. In conclusion, we have shown here that mitochondrial matrix proteins undergo glycoxidative modification with aging. We pointed out CML-modification in addition to carbonylation and accumulation of these altered proteins. Besides these damaged proteins, we reported a large decrease in the activity of a matrix enzyme, the Lon protease, which normally plays a key role in maintaining mitochondrial integrity. All together, these age-dependent alterations may contribute to disadvantage aged mitochon- dria to respond to conditions of stress and compromise cell viability. Acknowledgements The financial support of the Fondation pour la Recherche Me ´ dicale and the French Ministry of Research is gratefully acknowledged. References 1. Youngman, L.D., Park, J.Y. & Ames, B.N. (1992) Protein oxi- dation associated with aging is reduced by dietary restriction of protein or calories. Proc. Natl Acad. Sci. USA 89, 9112–9116. 2. Giardino, I., Edelstein, D. & Brownlee, M. (1994) Nonenzymatic glycosylation in vitro and in bovine endothelial cells alters basic fibroblast growth factor activity. A model for intracellular glyco- sylation in diabetes. J. Clin. Invest. 94, 110–117. 3. Vlassara, H., Bucala, R. & Striker, L. (1994) Pathogenic effects of advanced glycosylation: biochemical, biologic, and clinical impli- cations for diabetes and aging. Laboratory Invest. 70, 138–151. 2300 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003 4. King, G.L., Shiba, T., Oliver, J., Inoguchi, T. & Bursell, S.E. (1994) Cellular and molecular abnormalities in the vascular endothelium of diabetes mellitus. Annu. Rev. Med. 45, 179–188. 5. Verbeke, P., Perichon, M., Borot-Laloi, C., Schaeverbeke, J. & Bakala, H. (1997) Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging. J. Histochem. Cytochem. 45, 1059–1068. 6. Requena, J.R., Fu, M.X., Ahmed, M.U., Jenkins, A.J., Lyons, T.J., Baynes, J.W. & Thorpe, S.R. (1997) Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein. Biochem. J. 322, 317–325. 7. Miyata,T.,Inagi,R.,Asahi,K.,Yamada,Y.,Horie,K.,Sakai, H., Uchida, K. & Kurokawa, K. (1998) Generation of protein carbonyls by glycoxidation and lipoxidation reactions with auto- xidation products of ascorbic acid and polyunsaturated fatty acids. FEBS Lett. 437, 24–28. 8. Motomiya, Y., Oyama, N., Iwamoto, H., Uchimura, T. & Mar- uyama, I. (1998) N-epsilon-(carboxymethyl) lysine in blood from maintenance hemodialysis patients may contribute to dialysis- related amyloidosis. Kidney Int. 54, 1357–1366. 9. Traverso, N., Menini, S., Cottalasso, D., Odetti, P., Marinari, U.M. & Pronzato, M.A. (1997) Mutual interaction between gly- cation and oxidation during non-enzymatic protein modification. Biochim. Biophys. Acta 1336, 409–418. 10. Kristal, B.S. & B.P. (1992) An emerging hypothesis: synergistic induction of aging by free radicals and Maillard reactions. J. Gerontol. 47, B107–B114. 11. Pamplona, R., Requena, J.R., Portero-Otin, M., Prat, J., Thorpe, S.R. & Bellmunt, M.J. (1998) Carboxymethylated phosphati- dylethanolamine in mitochondrial membranes of mammals- Evidence for intracellular lipid glycoxidation. Eur. J. Biochem. 255, 685–689. 12. Chance, B., Sies, H. & Boveris, A. (1979) Hydroperoxide meta- bolism in mammalian organs. Physiol. Rev. 59, 527–605. 13. Kalous, M. & Drahota, Z. (1996) The role of mitochondria in aging. Physiol. Res. 45, 351–359. 14. Turrens, Z. (1997) Superoxide production by the mitochondrial respiratory chain. Bioscience Report 17, 3–7. 15. Shigenaga, M.K., Hagen, T.M. & Ames, B.N. (1994) Oxidative damage and mitochondrial decay in aging. Proc.NatlAcad.Sci. USA 91, 10771–10778. 16. Sastre, J., Pallardo, F.V., Garcia de la Ascunsion, J. & Vina, J. (1999) Mitochondria, oxidative stress and aging. Free Rad. Res. 32, 189–198. 17. Yan, L.J., Levine, R.L. & Sohal, R.S. (1997) Oxidative damage during aging targets mitochondrial aconitase. Proc.NatlAcad. Sci. USA 94, 11168–11172. 18. Yan, L.J. & Sohal, R.S. (1998) Mitochondrial adenine nucleotide translocase is modified oxidatively during aging. Proc. Natl Acad. Sci. USA 95, 12896–12901. 19. Martinez, M., Hernandez, A.I., Martinez, N. & Ferrandiz, M.L. (1996) Age-related increase in oxidized proteins in mouse synaptic mitochondria. Brain Res. 731, 246–248. 20. Boffoli, D., Scacco, S.C., Vergari, R., Persio, M.T., Solarino, G., Laforgia, R. & Papa, S. (1996) Ageing is associated in females with a decline in the content and activity of the bc 1 complex in skeletal muscle mitochondria. Biochim. Biophys. Acta 1315, 66–72. 21. Wong, L.K. & Sohal, R.S. (2000) Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch. Biochem. Biophys. 373, 16–22. 22. Lee, C.K., Klopp, R.G., Weindruch, R. & Prolla, T.A. (1999) Gene expression profile of aging and its retardation by caloric restriction. Science 285, 1390–1393. 23. Wang,N.,Gottesman,S.,Willingaham,M.C.,Gottesman,M.& Maurizi, M.R. (1993) A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc.NatlAcad.Sci.USA90, 11247–11251. 24. Desautels, M. & Goldberg, A.L. (1982) Demonstration of an ATP-dependent, vanadate-sensitive endoprotease in the matrix of rat liver mitochondria. J. Biol. Chem. 257, 11673–11679. 25. Watabe,S.&Kimura,T.(1985)Adrenalcortexmitochondrial enzyme with ATP-dependent protease and protein-dependent ATPase activities. J. Biol. Chem. 260, 14498–14504. 26. Ishii, S., Watabe, S. & Hamada, H. (1992) ATP-dependent pro- tease in human placenta. Placenta 13, 343–347. 27. Marcillat, O., Zhang, O., Lin, S.W. & Davies, K.J.A. (1988) Mitochondria contain a proteolytic system which can recognize and degrade oxidatively-denaturated proteins. Biochem. J. 254, 677–683. 28. Bota, D.A. & Davies, K.J. (2002) Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nature Cell Biol. 4, 674–680. 29. Gray, J.E., Van Zwieten, M.J. & Hollander, C.F. (1982) Early light microscopic changes of chronic progressive nephrosis in several strains of aging laboratory rats. J. Gerontol. 37, 142–150. 30. Murata,T.,Nagai,R.,Ishibashi,T.,Inomuta,H.,Ikeda,K.& Horiuchi, S. (1997) The relationship between accumulation of advanced glycation end products and expression of vascular endothelial growth factor in human diabetic retinas. Diabetologia 40, 764–769. 31. Aprille, J.R. & Asimakis, G.K. (1980) Postnatal development of rat liver mitochondria; state 3 respiration, adenine nucleotide translocase activity and the net accumulation of adenine nucleo- tide. Arch. Biochem. Biophys. 201, 564–575. 32. Ventura, B., Genova, M.L., Bovina, C., Formiggini, G. & Lenaz, G. (2002) Contol of oxidative phosphorylation by Complex I in rat liver mitochondria: implications for aging. Biochim. Biophys. Acta 1553, 249–260. 33. Sohal, R.S. & Dubay, A. (1994) Mitochondrial oxidative damage, hydrogen peroxide release, and aging. Free Radic. Biol. Med. 16, 621–626. 34. Nagai, M., Takahashi, R. & Goto, S. (2000) Dietary restriction initiated late in life can reduce mitochondrial protein carbonyls in rat livers: Western blot studies. Biogerontology 1, 321–328. 35. Fu, M.X., Requena, J.R., Jenkins, A.J., Lyons, T.J., Baynes, J.W. & Thorpe, S.R. (1996) The advanced glycation end product, N-(carboxymethyl) lysine, is a product of both lipid peroxidation and glycoxidation reactions. J. Biol. Chem. 271, 9982–9986. 36.Miyata,T.,Fu,M.X.,Kurokawa,K.,VanYperselede Strihou, C., Thorpe, S.R. & Baynes, J.W. (1998) Autoxidation products of both carbohydrates and lipids are increased in uremic plasma: is there oxidative stress in uremia? Kidney Int. 54, 1290– 1295. 37. Thornalley, P.J. (1990) The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life. Biochem. J. 269, 1–11. 38. Shinohara, M., Thornalley, P.J., Giardino, I., Beisswenger, P., Thorpe, S.R., Onorato, J. & Brownlee, M. (1998) Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis. J. Clin. Invest. 101, 1142–1147. 39. Nishikawa, T., Du Edelstein, D.X.L., Yamagishi, S., Matsumura, T.,Kaneda,Y.,Yorek,M.A.,Beebe,D.,Oates,P.J.,Hammes, H.P., Giardino, I. & Brownlee, M. (2000) Normalizing mito- chondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 404, 787–789. 40. Colbeau, A., Nachbaur, J. & Vignais, P.M. (1971) Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249, 462–492. Ó FEBS 2003 Protein alteration in mitochondrial matrix with aging (Eur. J. Biochem. 270) 2301 41. Daum, G. (1985) Lipids of mitochondria. Biochim. Biophys. Acta 822, 1–42. 42. Paradies, G., Petrosillo, G., Pistolese, M. & Ruggiero, F.M. (2000) The effect of reactive oxygen species generated from the mito- chondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart sub- mitochondrial particles. FEBS Lett. 466, 323–326. 43. Ikeda, K., Higashi, T., Sano, H., Jinnouchi, Y., Yoshida, M., Araki, T., Ueda, S. & Hurochi, S. (1996) N (epsilon) -(carboxy- methyl) lysine protein adduct is a major immunological epitope in proteins modified with advanced glycation end products of the Maillard reaction. Biochemistry 35, 8075–8083. 44. Das, N., Levine, R.L., Orr, W.C. & Sohal, R.S. (2001) Selectivity of protein oxidative damage during aging in Drosophila melano- gaster. Biochem. J. 15, 209–216. 45. Fannin, S.W., Lesnefsky, E.J., Slabe, T.J., Hassan, M.O. & Hoppel, C.L. (1999) Aging selectively decreases oxidative capacity in rat heart interfibrillar mitochondria. Arch. Biochem. Biophys. 372, 399–407. 2302 H. Bakala et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . proteins even in the mitochondrial matrix of adult rats, the concentration of which increased significantly with aging. On the one hand we found evidence. Determination of CML-protein content in liver mitochondrial matrix with aging. The CML content in the matrix proteins from 10- and 27-month-old rats was measured