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% f
Trang 1Changes in rat liver mitochondria with aging
Hilaire Bakala1, Evelyne Delaval1, Maud Hamelin1, Jeanne Bismuth1, Caroline Borot-Laloi1,
Bruno Corman2and Bertrand Friguet1
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
(Ne-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 FUÆlg protein)1Æh)1) to 27-month-old-rats (0.59 ± 0.08 FUÆlg protein)1Æh)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 CMLÆlg protein)1) to 27 months (17.81 ± 1.83 pmol CMLÆlg protein)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
Ne-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 producphosphoryla-tion 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)
Trang 2Our 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)BH3, 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
(Ne-carboxymethyllysine-BSA)
Carboxymethylated bovine serum albumin (CML-BSA)
was prepared according to Murata et al [30] Briefly,
100 mg BSA was incubated at 37C for 24 h with 0.15M
glyoxylic acid and 0.45M Na(CN)BH3 in 1 mL of 0.2M
sodium phosphate buffer (pH 7.4), and then extensively
dialyzed against phosphate-buffered saline (NaCl/Pi)
The CML content of the modified BSA was measured by
amino acid analysis following hydrolysis of the modified
protein in 6MHCl, 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 mM mannitol, 70 mM sucrose,
2 mM Hepes, 0.1 mM 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 0C The
super-natant was centrifuged at 8000 g for 10 min at 0C 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 mM 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 (30C) The rate of oxygen consumption was measured in the presence of 310 nmol ADP and 10 mM
succinate or 5 mMglutamate and 5 mMmalate (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 lM 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 lg protein) in 70 lL buffer (final concentration:
50 mM Tris/HCl, pH 7.9, 10 mMMgCl2, with or without
8 mMATP) for 1 h at 37C The reaction was terminated
by adding 30 lL of 40% trichloroacetic acid and 50 lL of 3% BSA After centrifugation at 15 000 g for 30 min,
100 lL of 2Msodium borate was then added to 80 lL of 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 (FUÆlg protein)1Æh)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 lL CML-BSA (6.4 nmol CMLÆmL)1) in 50 m sodium carbonate buffer,
Trang 3pH 9.6 by incubation overnight at 4C The wells were
washed three times with NaCl/Pi 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 lL NaCl/
Picontaining 6% (w/v) skimmed milk The wells were then
washed with buffer A, 50 lL of competing antigen (test
samples at 0.100 mgÆmL)1or serial dilutions of standard
CML-BSA from 0.64 mMto 128 mM) was added, followed
by 50 lL monoclonal antibody clone 6D12 (diluted
1 : 1000 in NaCl/Pi) 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 lL of substrate
solution [40 mM
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 37C 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/B0, calculated as [experimental
A minus background A (no antibody)]/[total A (no
competitor) minus background A], vs CML added as pmol
CMLÆlg protein)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/Pi/0.1% Tween 20
overnight at 4C, followed by four washes (10 min each)
with NaCl/Picontaining 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/Pi/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 mM2,4-dinitrophenolhydrazine in 2MHCl, 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 molecular mass of Lon
For Western blot analysis, we previously verified that signals were linear in the range from 10–60 lg of total 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/Pi, fixed by immersion in 2% paraformaldehyde/ 0.2% glutaraldehyde in NaCl/Pifor 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
5 m 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)1Æmg)1) 30.3 ± 0.3 26.5 ± 3.5 46.0 ± 3.5 47.1 ± 7.6 State 3 (ng atom OÆmin)1Æmg)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
Trang 4ADP/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-10-month-old rats (1.15 ± 0.15 and 0.59 ±
0.08 FUÆlg protein)1Æh)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 Activity was determined in the 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).
Trang 5CML 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 CMLÆlg protein)1 (n¼ 8) in
10-month-old rats to 17.81 ± 1.83 pmol CMLÆlg protein)1
(n¼ 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 CML-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 CMLÆlg protein)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.
Trang 6protein 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.
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