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INTRODUCTION |
Glycation and glycoxidation products are formed by the binding of
sugars or of aldehyde/ketone adducts to the accessible free 
-NH2 group of the basic amino acid constituents of a
protein (1, 2). The end result of this process is the formation of
several forms of advanced glycation/glycoxidation end products (AGE)1 that alter the
structure and function of proteins. Glycation and glycoxidation are
well known to occur in the extracellular compartment during aging and
in pathologies such as diabetes and Alzheimer's disease (3-6). In the
cytosol, the glycation process is more complex due to the fact that
glycation agents can be generated not only by the catabolism of sugars
but also by lipid, amino acid, as well as ascorbate metabolism (2, 7).
N-Carboxymethyl-lysine (CML) and fluorescent AGE, such as
pentosidine, are two forms of AGE known to accumulate in cells during
aging (6, 8). CML brings additional negative charge to the glycated
protein, whereas pentosidine and cyclic AGE formation leads to
intra/intermolecular cross-links. In contrast to the complex network of
antioxidant defenses that limit the deleterious effects of oxidation on
macromolecules (9), the cellular defenses against other
post-translational damage such as glycation have not been extensively
studied (10, 11). Indeed, no specific proteolytic pathway has been
shown to degrade cytosolic AGE. Because these structures are present in
aggregates of cross-linked proteins called AGE-pigment-like fluorophores, mainly accumulating in lysosomes during aging (8, 12,
13), a defect in the lysosomal degradation pathway has been suggested
(6, 14, 15). In addition, AGE have been shown to be resistant to
ATP-dependent proteolysis (16), and proteasome activity is
impaired in kidney and liver of rats during diabetes, a condition that
favors glycated protein formation (17).
The proteasome is the main cytosolic proteolytic complex responsible
for the degradation of damaged protein and general protein turnover
(18, 19). The 20 S proteasome, which is ubiquitin and ATP-independent,
is involved in 70-80% of the selective recognition and degradation of
the mildly oxidized proteins in the cytosol (19, 20). The 26 S
proteasome, formed upon association of the 19 S regulatory complex
with the 20 S catalytic core, has also been proposed to play a role in
oxidized protein degradation in ubiquitin and ATP-dependent
pathways (21, 22). An age-related decline of the peptidase activities
of both the 20 S and 26 S proteasome has been reported in cells and
tissues during aging in rat (23-25) or in human (26-29). Therefore,
the reported decrease of the 20 S proteasomal activity may explain, at
least in part, the accumulation of highly oxidized and aggregated
proteins in the cytosol of cells observed during aging (30). Finally,
it has been demonstrated that 4-hydroxy-2-nonenal (HNE) cross-linked proteins, as well as lipofuscin/ceroid fluorescent pigments, act as
inhibitors of the proteasome (31-33) which may also contribute to the
accumulation of these damaged proteins in aged cells.
In this study, the fate of the proteasome has been evaluated after
treatment of human dermal fibroblasts with the carbonyl compound
glyoxal under conditions that promote the formation of CML-modified
proteins (34, 35). The status of the cytosolic enzyme
glucose-6-phosphate dehydrogenase (G6PDH) has also been investigated
because it is the first enzyme in the pentose phosphate pathway that
catalyzes the formation of NADPH, important for the maintenance of
intracellular redox balance (36-38). Furthermore, it has been
demonstrated previously that G6PDH activity is decreased with age (39),
is affected by oxidation, and that the oxidized enzyme is susceptible
to proteolysis by the 20 S proteasome in vitro (40).
Therefore, the susceptibility of CML- and fluorescent AGE-modified
G6PDH to degradation by the 20 S proteasome has been investigated. In
contrast to the oxidized protein, both the CML- and fluorescent
AGE-modified enzymes were resistant to degradation by the 20 S
proteasome. To explain the observed resistance of glycated protein to
proteolysis by the 20 S proteasome, we have investigated several
structural parameters of CML- and fluorescent AGE-G6PDH as compared
with the oxidized and native forms by using biophysical characterization.
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EXPERIMENTAL PROCEDURES |
Chemicals and Antibodies--
G6PDH from Leuconostoc
mesenteroides was purchased from Worthington. Casein fluorescein
isothiocyanate (38 µg of FITC/mg of protein) was purchased from
Sigma. If not specified otherwise, all other chemicals were purchased
from Sigma and were of highest analytical grade available.
Rabbit anti-CML polyclonal antibody raised against CML-modified bovine
serum albumin was provided by Dr. H. Bakala (Université Denis
Diderot, Paris 7). Rabbit immune sera raised against 20 S rat
proteasome were obtained from Assay Research (Silver Spring, MD) as
described previously (23).
Quantification and Isolation of CML-modified Proteins from
Fibroblasts--
Primary cultures of human dermal fibroblasts were
obtained from M. Moreau (Inserm U505, Université Pierre et Marie
Curie, Paris 6) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine,
100 units of penicillin, and 100 µg/ml streptomycin at 37 °C and
5% CO2. Two days after seeding, cells were treated with
0.25 mM glyoxal for 1 week. The medium was changed every 2 days. Cells remaining viable during incubation were determined by their
ability to exclude trypan blue, and the viability of untreated and
glyoxal-treated cells was about the same (more than 99%). Cells were
collected, suspended in 10 mM Hepes, pH 8, supplemented
with 50 mM NaCl, 500 mM sucrose, 1 mM EDTA, 0.2% (v/v) Triton X-100, 5 mM
2-mercaptoethanol and sonicated 5 times for 5 s at 4 °C.
Cellular debris and organelles were removed from the crude extract by
centrifugation for 1 h at 10,000 × g at 4 °C.
A competitive enzyme-linked immunosorbent assay was performed as
described previously (27) to measure the amount of CML-modified protein
in glyoxal-treated fibroblasts.
Purification of CML-modified proteins was achieved using a
CNBr-activated Sepharose column coupled with polyclonal anti-CML antibodies. Crude cellular extracts were loaded on the column previously equilibrated with PBS, pH 7.4, and then washed with the same
buffer. The CML-modified proteins were eluted with 0.1 M
glycine, pH 2.8, and the fractions were immediately neutralized with 2 M Tris-HCl, pH 8. The eluted proteins were subjected to SDS-PAGE on a 12% gel and electrotransferred onto a nitrocellulose membrane. The blot was then processed utilizing polyclonal
anti-CML antibody (1/1000) and anti-ubiquitin antibody (1/500) (Dako,
Trappes, France).
Preparation of the Different Modified Forms of
G6PDH--
CML-modified G6PDH was prepared by reductive methylation of
G6PDH (41). Briefly, 6 mg of G6PDH was incubated with 45 mM glyoxylic acid and 150 mM sodium cyanoborohydride for
24 h at 37 °C in 0.1 M sodium phosphate buffer, pH
7.8. Fluorescent AGE-modified G6PDH was prepared using the procedure of
Grandhee and Monnier (42) with minor modifications. G6PDH (6 mg) was
incubated with 100 mM ribose and 1 mM
diethylenetriaminepentaacetic acid in 0.1 M sodium
phosphate buffer, pH 9.0. After 1 week of incubation at 37 °C,
samples were diluted 10 times with PBS, pH 7.4, and dialyzed twice for
24 h against the same buffer. Total cyclic fluorescent AGE were
characterized by fluorescence analysis (excitation, 370 nm; emission,
440 nm) in a Kontron SFM 25 spectrofluorimeter (Saint-Quentin en
Yvelines, France), and pentosidine content was measured using the
method from Odetti et al. (43). Protein concentration was
determined by the Lowry method (Bio-Rad), and the samples were stored
at 
20 °C. Metal-catalyzed oxidation of the enzyme was achieved as
described previously (32). Prior to use, the various forms of G6PDH
were chromatographed through a PD-10 column (Amersham Pharmacia
Biotech) equilibrated with PBS, pH 7.4. The eluted fractions were
concentrated by using a Centricon-30 system (Millipore, Bedford, MA).
Measurement of G6PDH Activity--
The enzymatic reaction was
performed at 25 °C by incubating 25, 50, or 75 ng of the different
forms of G6PDH diluted in 1 ml of 50 mM Hepes, pH 7.8, 100 mM KCl in the presence of 10 mM NADP+ and 100 mM glucose 6-phosphate. G6PDH
activity was determined by following the appearance of NADPH
spectrophotometrically at 340 nm for 5 min.
G6PDH activity in crude cellular extracts was determined by the same
method using 250 µg of protein. Assaying G6PDH activity in biological
samples is complicated by the presence of endogenous 6-phosphogluconate
dehydrogenase, the second enzyme of the pentose phosphate pathway that
also produces NADPH. Therefore, the activity of G6PDH was determined
after inactivation of 6-phosphogluconate dehydrogenase by maleimide at
a final concentration of 5 mM (44).
20 S Proteasome: Purification, Peptidase Activities,
and Western Blot Analysis--
20 S proteasome was purified from
human placenta according to the procedure described previously (40)
with minor modifications (45). Peptidase activities of the proteasome
were assayed using fluorogenic peptides,
succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (LLVY-AMC) for the
chymotrypsin-like activity,
N-t-butyloxycarbonyl-Leu-Ser-Thr-Arg-amidomethylcoumarin (LSTR-AMC) for the trypsin-like activity, and
N-benzyloxycarbonyl-Leu-Leu-Glu-
-naphthylamide (LLE-NA)
for the peptidylglutamyl-peptide hydrolase activity as described
previously (27) with minor modifications. The mixture, containing 20 µg of crude homogenate total protein in 25 mM Tris-HCl, pH 7.5, was incubated at 37 °C with the appropriate peptide
substrate (LLVY-AMC at 25 µM or LLE-NA at 150 µM or LSTR-AMC at 40 µM) in a final volume
of 200 µl. Enzymatic kinetics were conducted in a
temperature-controlled microplate fluorimetric reader (Fluostar Galaxy,
bMG, Stuttgart, Germany). Excitation/emission wavelengths were 350/440
nm and 340/410 nm for aminomethylcoumarin and -naphthylamine, respectively. Proteasome activities were determined as the difference between total activity and the remaining activity of the crude extract
in the presence of 20 µM proteasome inhibitor
N-Cbz-Leu-Leu-leucinal (MG132). For inactivation
experiments, 2 µg of purified proteasome were incubated with various
concentrations of glyoxal for 30 min at room temperature. After
preincubation, proteasome peptidases activities were determined as
described above. The proteasome amount was estimated by Western blot
analysis of cytosolic extracts. 20 µg of total proteins were
separated on a 12% SDS-PAGE. Immunoblot experiments were then
performed using anti-20 S proteasome polyclonal antibody (1/2500).
Proteolytic Degradation of the Various Forms of G6PDH and
FITC-Casein--
Proteolysis of the various forms of G6PDH at
different concentrations was achieved in the presence of 0.3 µM of purified 20 S proteasome at 37 °C in PBS, pH
7.4. At different times, a 20-µl aliquot was removed and submitted to
a 10% (w/v) trichloroacetic acid precipitation. The mixture was
centrifuged at 4 °C at 15,000 × g for 30 min, and
the supernatant containing the released peptides was neutralized by
adding 100 µl of 2 M potassium borate, pH 10. The amount
of amino groups was determined after addition of fluorescamine (3 mg/ml
in acetone) by measuring the emission intensity of the fluorescent
derivatives (excitation, 375 nm; emission, 475 nm). Proteolysis of
FITC-casein (50 µg/ml) was performed with 0.3 µM proteasome at 37 °C in the absence or presence of 18 µM of the various forms of G6PDH. At different times, a
20-µl aliquot was removed and processed as described above. The
proteolysis rate was estimated by measuring the emission intensities of
FITC fluorescent peptides at 515 nm (excitation at 495 nm).
8-Anilino-1-naphthalenesulfonic Acid (ANSA) Binding--
The
various forms of G6PDH (at 0.4 µM) were incubated with
100 µM ANSA in 50 mM Hepes, pH 7.8, and 100 mM KCl at 37 °C for 30 min. The fluorescence emission
spectrum from 430 to 600 nm (excitation at 370 nm) was recorded for
ANSA alone and for ANSA in the presence of the different forms of G6PDH
in a Kontron SFM 25 spectrofluorimeter (Saint-Quentin en Yvelines,
France). Fluorescence increase occurring upon binding of ANSA to
protein was obtained after subtraction of the emission spectrum of ANSA alone.
Circular Dichroism Spectra--
Native and modified forms of the
protein were diluted to a final concentration of 1 mg/ml in 10 mM potassium phosphate buffer, pH 7.4, and dialyzed against
the same buffer overnight at 4 °C. CD spectra were recorded in a
Jobin-Yvon CD6 spectropolarimeter (Longjumeau, France) at 20 °C,
using 1-cm or 0.1-mm path length cells for the near (250-320 nm) and
far (190-260 nm) UV analysis, respectively. Each spectrum results from
averaging five successive individual scans with a 0.5-nm step and an
integration time of 2 s per step. Buffer contribution was
eliminated by subtraction of the corresponding spectrum acquired under
the same conditions.
Thermal Inactivation--
0.02 µM native, oxidized
or CML-modified G6PDH was incubated at 45 °C in 50 mM
Hepes, pH 7.8, 100 mM KCl. At the indicated times, 50 µl
of the mixture was added to 900 µl of 50 mM Hepes, pH
7.8, 100 mM KCl, at 25 °C to terminate the thermal
inactivation, and the residual activity was measured as described above.
Urea Denaturation--
Denaturation was performed by incubating
0.3 µM of the different forms of G6PDH in 20 mM potassium phosphate, pH 7.2, with increasing
concentration of urea (from 0 to 8 M). The mixture was
gently stirred for 3 h at 20 °C. At each urea concentration, the fluorescence emission spectrum (300-450 nm, excitation 290 nm) of
the samples was determined. The native enzyme fraction was obtained
from the ratio F Fd/Fn Fd and plotted as a function of urea concentration.
F is the fluorescence at 320 nm of the sample at a given
concentration of urea; Fn and Fd are the
fluorescence intensities (320 nm) of the sample at 0 M and
8 M urea, respectively.
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RESULTS |
Protein Modification and Proteasome Activity in Glyoxal-treated
Cells--
The effects of intracellular glycation on proteasome
peptidase activities were analyzed in primary cultures of human dermal fibroblasts treated for 1 week with 0.25 mM glyoxal. These
conditions induced the formation of CML-modified proteins without
affecting cell viability. Indeed, using an enzyme-linked immunosorbent
assay with anti-CML polyclonal antibody, intracellular CML-protein
adducts exhibited a 5-fold increase in the treated cells as compared
with the control cells (data not shown). The three main proteasome peptidase activities, chymotrypsin-like, trypsin-like, and
peptidylglutamyl-peptide hydrolase activities, were monitored in
cellular extracts of treated and untreated fibroblasts. As shown in
Fig. 1A, all three activities decreased significantly in the glyoxal-treated cells as compared with
the control cells. To determine whether the observed decline in the
proteasome peptidase activities was due to a decreased proteasome
content, the amount of proteasome was estimated by Western blot
analysis of cellular extracts, using an anti-proteasome polyclonal
antibody (Fig. 1A). No change in proteasome content was
detected in treated cells indicating that the loss of proteasome activity is likely due to an alteration of proteasome function. To
determine whether the observed decline of proteasome activity could be
explained by direct inactivation of the 20 S proteasome by glyoxal,
peptidase activities were assayed after incubation of purified
proteasome with various concentrations of this compound. The results
presented in Fig. 1B indicate that all three proteasome peptidase activities were inhibited with 0.1 mM glyoxal and
to a higher extent with increasing concentrations.
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Fig. 1.
Effect of glyoxal on proteasome
function. A, proteasome peptidase activities and
content in glyoxal-treated cells. Primary culture of human dermal
fibroblasts was treated with 0.25 mM glyoxal for 1 week.
The chymotrypsin-like (black bars), peptidylglutamyl-peptide
hydrolase (striped bars), and trypsin-like (white
bars) activities were assayed in cytosolic extracts either in
treated cells (G) or in control cells (C) using
fluorogenic peptide substrates, LLVY-AMC at 25 µM, LLE-NA
at 150 µM, and LSTR-AMC at 40 µM,
respectively. The measurement of peptidase activities in the untreated
cells (C) was taken as 100%. The error bars
represent the S.E. of the mean of three independent experiments.
p values (t test) for glyoxal-treated cells
versus control are as follows: *, p  0.01, and  , p 0.002. Soluble proteins (20 µg) from
glyoxal-treated or untreated fibroblasts were subjected to SDS-PAGE on
a 12% gel and electrotransferred onto a nitrocellulose membrane. The
blot was processed with anti-proteasome polyclonal antibodies.
B, effect of glyoxal on proteasome peptidase activities
in vitro. Proteasome purified from human placenta was
incubated for 30 min with glyoxal at indicated concentrations.
Chymotrypsin-like (black bars), peptidylglutamyl-peptide
hydrolase (striped bars), and trypsin-like (white
bars) activities were determined using fluorogenic peptide
substrates LLVY-AMC at 25 µM, LLE-NA at 150 µM, LSTR-AMC at 40 µM, respectively. The
results are expressed as percentage of the activities of the proteasome
incubated without glyoxal.
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To examine whether CML-modified proteins are substrates for ubiquitin
conjugation, an immunopurification of the crude extracts from
glyoxal-treated and control cells was achieved on an anti-CML polyclonal antibody affinity column. After elution, the purified proteins were submitted to Western blot analysis using anti-CML and
anti-ubiquitin antibodies. As shown in Fig.
2, the major bands corresponding to
CML-modified proteins are also ubiquitinated, indicating these proteins
may be marked for degradation by the 26 S proteasome. Among cytosolic
enzymes that may be targets for glyoxal-induced modification, the fate
of G6PDH was investigated because this enzyme has an essential lysine
residue in its active site. Moreover, it plays a critical role in
maintenance of intracellular redox status, and this enzyme is a well
characterized model for the effects of various forms of modifications
on susceptibility to proteasome degradation. We observed a 60% loss in
G6PDH activity in glyoxal-treated cells defining this enzyme as a
target for glyoxal-induced inactivation and also as a good model for
further investigation of glycated protein degradation by the
proteasome.
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Fig. 2.
Ubiquitination of CML-modified proteins in
glyoxal-treated cells. Cellular proteins from glyoxal-treated
cells were immunopurified on an anti-CML affinity column. The eluted
proteins were subjected to SDS-PAGE on a 12% gel, electrotransferred
onto a nitrocellulose membrane, and immunoblotted with anti-CML and
anti-ubiquitin antibodies. The blot depicted is representative of three
separate experiments.
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Characterization of the Various Modified Forms of
G6PDH--
Because CML-modified and fluorescent AGE-modified proteins
are known to accumulate with age, these glycated forms of G6PDH were
prepared and used to assess their susceptibility to proteolysis by the
20 S proteasome in vitro as described under "Experimental Procedures." Oxidatively modified G6PDH was also prepared to be used
as a positive control. The formation of CML on protein was revealed by
immunoblotting experiments using anti-CML antibodies. Amino acid
analysis indicates a loss of 4 lysines in the CML-modified G6PDH and a
loss of 9 lysines plus 4 arginines in the fluorescent AGE-modified form
(data not shown). Pentosidine content (2.25 nmol/mg of enzyme) was
measured by high performance liquid chromatography analysis, and the
presence of other cyclic AGE structures was detected by
spectrofluorometry. No intermolecular cross-links were detected after
SDS-PAGE indicating that this modified G6PDH exhibits only pentosidine
or other cyclic AGE cross-links within the same polypeptide (data not
shown). The activities of both oxidatively modified and CML-modified
G6PDH declined 50%, whereas the fluorescent AGE-modified form was
completely inactivated.
Proteolysis of Native and Various Modified Forms of G6PDH by the
Proteasome--
The rates of proteolysis for the native and modified
forms of G6PDH were determined by monitoring the appearance of small peptides containing free amino groups generated after incubation of the
various forms of G6PDH with purified 20 S proteasome (Fig. 3A). This method has been
validated previously for native and modified G6PDH because it has been
established that the loss of up to 13 lysines (upon modification by
HNE) per molecule of G6PDH did not affect the values of the rates of
proteolysis (31). As demonstrated previously, treatment of the enzyme
with metal-catalyzed oxidation resulted in an increase in
susceptibility to proteasome-mediated degradation (40). In contrast,
the CML-modified protein was degraded to the same extent as the native
protein, whereas the fluorescent AGE-modified form was more resistant
to proteolysis than the native form. Kinetic parameters for degradation
of the various forms of G6PDH (Table I)
were calculated from direct linear plots (not shown) according to the
representation of Eisenthal and Cornish-Bowden (46). The apparent
KM for the CML-modified G6PDH was almost the same as
for the native enzyme (41.8 and 42.7 µM, respectively),
whereas fluorescent AGE-modified and oxidized G6PDH exhibited a lower
KM (13 and 8 µM, respectively). The
kcat value was about the same for the native,
the CML-modified, and the oxidized G6PDH, whereas this turnover number
was decreased for the fluorescent AGE-modified G6PDH. To determine
whether these modified proteins behave as inhibitors of the proteasome,
competition experiments using FITC-casein as substrate were performed
in the presence of 18 µM of the various modified forms of
G6PDH. As a substrate of the proteasome, the oxidatively modified
protein was competing to almost 50% for the degradation of casein,
whereas the CML- and the fluorescent AGE-modified protein did not
exhibit an enhanced inhibitory effect as compared with the native
enzyme (Fig. 3B).
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Fig. 3.
Effect of G6PDH modifications on
proteasome-mediated proteolysis. A, proteolysis of native
and modified forms of G6PDH (at 18 µM) by the proteasome
was performed and monitored as described under "Experimental
Procedures." The rates of proteolysis are presented as percentage of
the value obtained for native G6PDH (bars represent the
mean ± S.E. of three independent experiments). B,
proteolysis of FITC-casein by the proteasome in the presence of various
forms of G6PDH. Proteolysis of FITC-casein was determined in the
presence of native and modified G6PDH. The rate of FITC-casein
degradation alone was set as 100%. The bars represent the
S.E. of the mean for three independent experiments.
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Table I
Kinetic parameters for degradation of G6PDH substrates by the 20 S
proteasome
Data represent mean values ± S.E. obtained from three separate
experiments.
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Binding of the Hydrophobic Fluorescent Probe ANSA to the Modified
Forms of G6PDH--
Because glycated proteins build up both as a
function of disease and aging, it was important to investigate
potential reasons why these proteins are not recognized and degraded by
the proteasome. Hydrophobic surface exposure has been proposed to be
the recognition signal for 20 S proteasome degradation. To determine
whether the glycated forms of G6PDH expose hydrophobic regions at their
surface, we have monitored the binding of the hydrophobic fluorescent
probe ANSA which exhibits an increase and a shift in fluorescence when associated with surface-exposed hydrophobic sequences (Fig.
4). As opposed to free radical oxidation
of protein which results in increased ANSA binding, no binding was
observed with the CML- and fluorescent AGE-modified G6PDH. Oxidative
modification of G6PDH has also been shown to promote dissociation of
the dimeric enzyme that may explain hydrophobic surfaces exposure at
the monomer-monomer interface (40). As expected, no dissociation of the
dimer was observed for both the native and the glycated forms of the
enzyme using gel filtration on a Superose 6 column (Amersham Pharmacia Biotech) (data not shown).
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Fig. 4.
Spectrofluorimetric analysis of ANSA binding
to the various forms of G6PDH. Emission spectra (excitation 370 nm) of ANSA (100 µM) in the presence of various forms of
G6PDH (0.4 µM) were performed as described under
"Experimental Procedures." The increase in fluorescence intensity
resulting from the binding of ANSA to the enzyme was determined by
subtracting the emission spectrum of ANSA from that of ANSA in the
presence of the different forms of enzyme. From top to
bottom: oxidized, native, and fluorescent AGE- and
CML-G6PDH.
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Structural Properties of the Modified Forms of
G6PDH--
Structural changes that may occur upon G6PDH modification
were analyzed by far- and near-UV CD for secondary and tertiary structural alterations, respectively. The CD spectra of oxidized, fluorescent AGE- and CML-modified were nearly identical in the far-UV
region, indicating no change in secondary structure (Fig. 5A). The slight change
observed for the oxidized protein at 190 nm is not accompanied by a
change at 222 nm. It is therefore unlikely that any significant
modification of the helical content of the protein has occurred upon
oxidation. However, the oxidized enzyme exhibited a decreased signal
intensity in the near-UV region, at 280 and 287 nm (Fig.
5B), suggesting alterations in tertiary structure. In
contrast, the spectrum of the CML- and the fluorescent AGE-modified
proteins remained close to the native form, indicating no major
modification of their tertiary structure. The spectral deviations
observed for the fluorescent AGE-modified enzyme at wavelengths greater
than 290 nm can be explained by the absorbance properties of the AGE
adducts. To assess potential changes in the structural stability of the
modified forms of G6PDH, thermal denaturation experiments were
performed at 45 °C as depicted in Fig.
6A. Because the thermal
denaturation was followed by monitoring the enzymatic activity, the
fluorescent AGE-modified protein could not be assayed. Interestingly,
the CML-modified G6PDH was found to be even more heat-resistant than
the native protein, whereas oxidized protein exhibited enhanced
thermolability. The conformational stability of the native and the
various modified forms of G6PDH was assayed by equilibrium denaturation
experiments in the presence of increasing concentrations of urea. As
shown in Fig. 6B, glycated forms of G6PDH appeared more
structurally stable than native enzyme, whereas the oxidized form of
the enzyme was far less stable. Moreover, the shape of the denaturation
curves obtained for both CML- and fluorescent AGE-modified G6PDH is
indicative of a heterogenous population of modified enzymes with
different conformational stabilities.
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Fig. 5.
CD spectral analysis of the different
forms of G6PDH. A, far-UV CD spectra were recorded as
described under "Experimental Procedures." From top to
bottom: fluorescent AGE- ( ), CML- ( ), native ( ), and
oxidized ( ) G6PDH. B, near-UV CD spectra were recorded as
described under "Experimental Procedures." From top to
bottom at 287 nm: oxidized (), CML-(), fluorescent
AGE-(), and native () G6PDH.
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Fig. 6.
Thermal and conformational stabilities of the
different forms of G6PDH. A, heat inactivation of the
different forms of G6PDH. Native, oxidized, or CML-modified G6PDH (0.02 µM) was incubated at 45 °C for the indicated times.
Heat inactivation was terminated, and the residual G6PDH activity was
determined as described under "Experimental Procedures."  ,
CML-modified G6PDH;  , native G6PDH;  , oxidized G6PDH. The
points represent the means, and the bars the S.E.
of the mean for three independent experiments. B,
urea-induced denaturation of the various forms of G6PDH. Denaturation
was performed by incubating 0.3 µM of the different forms
of G6PDH with an increased amount of urea (from 0 to 8 M)
and was followed by monitoring the fluorescence emission spectrum of
the samples (300-450 nm, excitation 290 nm). The folded enzyme
fraction was determined at 320 nm and plotted as a function of urea
concentration as described under "Experimental Procedures." ,
native G6PDH; , oxidized G6PDH; , CML-modified G6PDH;
+, fluorescent AGE-modified G6PDH.
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DISCUSSION |
Modifications of protein by glycation/glycoxidation, as evidenced
by the presence of CML, pentosidine, and vesperlysine adducts on
proteins, are formed during the progression of various diseases and as
a result of normal aging (4-6, 47). The present study was
undertaken to determine whether the glycation process and the
subsequent accumulation of glycated proteins affect the proteasomal system, which is responsible for intracellular degradation of ubiquitinated and oxidized proteins (18, 19). Indeed, proteasome function has been reported to be impaired during human aging as a
result of both the decreases of its intrinsic activity and protein content (27, 30). Contributing factors may include the occurrence of
structural modifications (28) or the inhibitory effects of cross-linked
HNE-modified protein and lipofuscin (31-33). Protein glycation was
induced by treatment of human dermal fibroblasts with glyoxal, a
carbonyl compound, under conditions known to promote the formation of
CML-modified proteins in cultured rat sensory neurons (35) and fetal
lung cells (15). In this latter study, the proteolytic activity as well
as the expression of lysosomal cathepsin D were shown to decrease,
indicating that AGE formation and/or subsequent oxidative stress can
alter the lysosomal protein degradation pathway. In the present study,
we provide evidence that the proteasomal system is impaired in
glyoxal-treated cells. Chymotrypsin-like, trypsin-like, and
peptidylglutamyl-peptide hydrolase activities of the proteasome are
significantly decreased, although no change in proteasome content could
be detected (Fig. 1A). In vitro incubation of
purified 20 S proteasome with glyoxal indicated that glyoxal can
inactivate all three proteasome peptidase activities (Fig.
1B). This finding raises the possibility that the proteasome
is a target for glyoxal-mediated inactivation in vivo.
However, the sensitivity of proteasome peptidase activities to glyoxal
appears to be slightly different in glyoxal-treated cells as compared
with that of 20 S proteasome in vitro; the
peptidylglutamyl-peptide hydrolase activity is the most sensitive to
inhibition in vivo and the least sensitive in
vitro. This apparent discrepancy can be explained by the fact that
within cells the proteasome, one of the protein targets for glyoxal, is
also present as a complex with the 19 S regulatory particle to form
the 26 S proteasome. Moreover, cellular AGE-modified proteins have
been shown to promote oxidative stress (48-50), and the proteasome
peptidylglutamyl-peptide hydrolase activity is known to be readily
inactivated by oxidants (23). In addition, an inhibitory effect of
glycated proteins, as reported previously for HNE cross-linked proteins
and lipofuscin (32, 33), cannot be excluded.
We also found that G6PDH in glyoxal-treated cells exhibits a dramatic
decrease in activity. This inactivation could be due to the formation
of CML on the active site lysine residue. This would be expected to
result in a decline in reduced glutathione regeneration and subsequent
alterations in redox control modulation. This, in turn, would affect
the activity of GSH- and NADPH-dependent enzymes involved
in the detoxification of reactive carbonyls, main precursors of AGE
(11). The drop of G6PDH activity may also explain, at least in part,
the increase in intracellular oxidative stress associated with AGE
formation (49, 50). Interestingly, G6PDH activity, shown to be
sensitive to oxidative stress (36-38), decreased with age (39).
G6PDH is a target for glyoxal-mediated inactivation that has been
extensively studied with respect to the effects of various post-translational modifications on susceptibility to degradation by
the 20 S proteasome (40, 51). Therefore, the susceptibility of
glycated G6PDH to degradation by the 20 S proteasome was investigated in vitro. Two forms of glycated G6PDH, non-fluorescent,
minimally modified by CML and highly modified by fluorescent AGE with
intramolecular cross-links such as pentosidine, were assayed for the
degradation by the 20 S purified proteasome. As opposed to oxidatively
modified G6PDH which is a good substrate of the 20 S proteasome, these two forms of glycated proteins are no more susceptible to proteolysis by the proteasome than the native enzyme (Fig. 3A). In fact,
as shown in Table I, almost similar kcat and
KM values were obtained for CML-modified and native
G6PDH. Due to a lower kcat, fluorescent
AGE-modified G6PDH appeared more resistant than the native enzyme,
despite a lower KM. Finally, in contrast to oxidized
G6PDH, both native, CML-, and fluorescent AGE-modified G6PDH exhibited
about the same specificity constant
kcat/KM. These findings
provide one explanation for the observed resistance of glycated
proteins to proteolysis. The formation of intramolecular cross-links
generated by cyclic fluorescent adducts, such as pentosidine, may
explain this resistance toward proteasome degradation, as already
observed with HNE cross-linked G6PDH (31, 32, 40).
Oxidized protein degradation by the 20 S proteasome has been studied
extensively by different groups (20, 52-55). Mild oxidation leads to
an increased proteolytic susceptibility, whereas highly oxidized
protein becomes resistant to degradation by the 20 S proteasome. It
has been shown that mildly oxidized proteins exhibit decreased thermal
and thermodynamic stability (56, 57) and bind more efficiently the
hydrophobic fluorescent probe ANSA (40). Both the exposure of
hydrophobic surface on modified proteins and the loss of secondary
structure have been proposed to act as a recognition signal for binding
and degradation of the substrate protein by the 20 S proteasome (19,
54, 55). Because glycated proteins are poor substrates of the 20 S
proteasome, structural studies of the glycated forms of G6PDH aimed at
assessing their secondary and tertiary structure as well as their
conformational stability were performed. These structural properties
were then compared with those of the native and oxidized forms of the
enzyme. As opposed to oxidized G6PDH, hydrophobic amino acids are not exposed at the surface of glycated proteins, as shown in ANSA-binding experiments (Fig. 4). Moreover, the secondary as well as the tertiary structures of these proteins, as monitored by far- and near-UV CD,
respectively (Fig. 5, A and B), were nearly
identical to those of the native counterpart. CML-modified G6PDH is
more thermostable than the native enzyme (Fig. 6A), and both
CML- and fluorescent AGE-modified G6PDH exhibit an increased resistance
toward urea denaturation (Fig. 6B). Furthermore, the shape
of the transition curves indicates that both CML- and fluorescent
AGE-modified enzymes represent a heterogeneous population of modified
proteins. Indeed, depending on the nature and the number of
intramolecular cross-links (pentosidine and versperlysine), the
fluorescent AGE-modified proteins may have different conformational
stabilities. Additional negative charges brought by the
carboxymethylation reaction may stabilize the protein conformation as
well. Taken together, these findings show that, as opposed to oxidative
modification that promotes an increased flexibility and decreased
conformational stability of the protein, glycation of G6PDH induces an
increased structural stability that correlates with the observed
resistance of these modified proteins to degradation by the 20 S proteasome.
We have shown that CML-modified proteins in glyoxal-treated cells are
also ubiquitinated (Fig. 2). Interestingly, HNE-modified proteins
generated upon oxidative stress were also found to be ubiquitinated
(58). These observations raise the possibility that the ubiquitin-26 S
proteasome pathway is implicated in the removal of these modified
proteins. However, our in vitro results suggest that even if
ubiquitinated chains are recognized by the 19 S complex, glycated
proteins, as well as HNE-modified proteins, are resistant to
degradation by the 20 S catalytic core of the proteasome. Future
studies will address whether ubiquitination of CML-modified proteins is
a marking step for their degradation by the 26 S proteasome. On the
other hand, ubiquitination of glycated proteins, if not a 26 S
proteasome-targeting signal, may also be a signal for their targeting
to other pathways such as protein degradation by the lysosomal pathway
(59, 60).
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel./Fax:
33-1-44-27-82-34; E-mail: bfriguet@paris7.jussieu.fr.
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