Protein modification and replicative senescenceof WI-38 human embryonic fibroblasts
Emad K. Ahmed,1 Adelina Rogowska-Wrzesinska,2
Peter Roepstorff,2 Anne-Laure Bulteau1 and BertrandFriguet1
1Laboratoire de Biologie Cellulaire du Vieillissement, UR4,
Universite Pierre et Marie Curie-Paris 6, Paris, France2Department of Biochemistry and Molecular Biology, University of
Southern Denmark, Odense, Denmark
Summary
Oxidized proteins as well as proteins modified by the lipid
peroxidation product 4-hydroxy-2-nonenal (HNE) and by
glycation (AGE) have been shown to accumulate with
aging in vivo and during replicative senescence in vitro.
To better understand the mechanisms by which these
damaged proteins build up and potentially affect cellular
function during replicative senescence of WI-38 fibro-
blasts, proteins targeted by these modifications have
been identified using a bidimensional gel electrophoresis-
based proteomic approach coupled with immunodetec-
tion of HNE-, AGE-modified and carbonylated proteins.
Thirty-seven proteins targeted for either one of these
modifications were identified by mass spectrometry and
are involved in different cellular functions such as protein
quality control, energy metabolism and cytoskeleton.
Almost half of the identified proteins were found to be
mitochondrial, which reflects a preferential accumulation
of damaged proteins within the mitochondria during cel-
lular senescence. Accumulation of AGE-modified proteins
could be explained by the senescence-associated
decreased activity of glyoxalase-I, the major enzyme
involved in the detoxification of the glycating agents
methylglyoxal and glyoxal, in both cytosol and mitochon-
dria. This finding suggests a role of detoxification systems
in the age-related build-up of damaged proteins. More-
over, the oxidized protein repair system methionine sulf-
oxide reductase was more affected in the mitochondria
than in the cytosol during cellular senescence. Finally, in
contrast to the proteasome, the activity of which is
decreased in senescent fibroblasts, the mitochondrial
matrix ATP-stimulated Lon-like proteolytic activity is
increased in senescent cells but does not seem to be suffi-
cient to cope with the increased load of modified mito-
chondrial proteins.
Key words: protein oxidation; protein glycation; proteo-
mics; replicative senescence; WI-38 fibroblasts; protein
maintenance.
Introduction
In the early 1960s, the concept that primary cells isolated from
mammalian tissues can undergo only a finite number of divisions
when grown in culture was established by Hayflick and Moor-
head (Hayflick & Moorhead, 1961; Hayflick, 1965). Once cul-
tured, cells loose their replicative potential they are termed
senescent and viewed as aged cells. Cellular senescence has
been demonstrated to play an important role in tumor suppres-
sion (Campisi, 2005), while replicative senescence in vitro has
been shown to reflect at least some features of aging in vivo.
Hence, senescent cells represent a valid model for studying
mammalian cellular aging, and it is generally believed that
understanding the mechanisms of cellular aging will provide
insight into organismal aging (Rohme, 1981). Moreover, evi-
dence has been provided for the occurrence of senescent cells in
aged tissues such as human skin, rat kidney and in different
mouse organs (Dimri et al., 1995; Melk et al., 2003; Wang
et al., 2009), while the presence of senescent cells has been
associated with a variety of age-related diseases such as athero-
sclerosis, osteoarthritis, or chronic obstructive pulmonary dis-
ease (Minamino et al., 2002; Yudoh et al., 2005; Tsuji et al.,
2006). Although telomere shortening has been described as the
underlying cause of cellular senescence (Bodnar et al., 1998),
other events such as expression of oncogenes and a variety of
stimuli including oxidative stress have also been recognized as
potent inducers of cellular senescence (Serrano & Blasco, 2001;
Toussaint et al., 2002; Passos & Von Zglinicki, 2006).
Accumulation of altered proteins both within cells or extracell-
ulary is a common feature of aging. Intracellular proteins are car-
rying carbonyl groups during aging (Stadtman, 1992, 2002),
and many studies have shown that oxidized proteins are build-
ing up during the normal nonpathological aging of cells and
organisms. These studies were performed in flies, rats, human
tissues (Carney et al., 1991; Smith et al., 1991; Stadtman,
1992; Heinecke et al., 1993; Agarwal & Sohal, 1994; Chao
et al., 1997), human fibroblasts (Sitte et al., 2000; Chondrogi-
anni et al., 2003), and human keratinocytes (Petropoulos et al.,
2000). Intracellular build-up of damaged protein with age
results, at least in part, from the increase in reactive oxygen spe-
cies (ROS) and other toxic compounds coming from both cellular
metabolism and external factors, but failure of protein mainte-
nance (i.e. degradation and repair) is also a major contributor to
Correspondence
Bertrand Friguet, Laboratoire de Biologie Cellulaire du Vieillissement, UR4,
Universite Pierre et Marie Curie-Paris 6, case courrier 256, 7 quai Saint
Bernard, 75252 Paris cedex 05, France. Tel.: +33 (0)1 44 27 82 34, fax:
+33 (0)1 44 27 82 34; e-mail: [email protected]
Accepted for publication 14 January 2010
252 ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
Aging Cell (2010) 9, pp252–272 Doi: 10.1111/j.1474-9726.2010.00555.x
the age-associated accumulation of damaged proteins (Friguet,
2006). An inevitable side-product of oxidative metabolism is the
production of ROS, which can damage lipids, nucleic acids, and
proteins (Berlett & Stadtman, 1997). Within proteins, ROS can
attack virtually any amino acid, among which arginine, lysine,
threonine, and proline were shown to be oxidized to carbonyl
derivatives. Carbonyl groups can also be brought into proteins
through conjugation of histidine, lysine and cysteine residues
with lipid peroxidation products. For instance, both protein
carbonylation and modification by aldehydes of cornified enve-
lope proteins have been evidenced in skin stratum corneum and
the extent of these modifications was found higher in areas that
are continuously exposed to sunlight-induced oxidative stress
(Hirao & Takahashi, 2005). Indeed, ROS can attack lipids causing
the generation of reactive aldehydes such as 4-hydroxynonenal
(HNE). HNE is generated from the peroxidation of polyunsatu-
rated fatty acids of membrane lipids and is considered as a
potent mediator of cellular damage as a result of its relative solu-
bility and stability, which enable it to react with proteins within
and outside membranes. HNE can covalently bind to the side-
chains of cysteine, lysine, and histidine through a nucleophilic
attack to form Michael adducts (Uchida, 2003). Such HNE-modi-
fied proteins were found to accumulate upon serial passaging of
human keratinocytes (Petropoulos et al., 2000) and in the retina
of aged rats (Kapphahn et al., 2006). In the latter study, HNE-
modified proteins (e.g. triosephosphate isomerase, a enolase,
Hsc70, and bB2 crystallin) were involved in metabolism, chaper-
one function, and fatty acid transport. Proteins can also be mod-
ified through the reaction of arginine and lysine amino groups
with reducing sugars or reactive dicarbonyl compounds, such as
glyoxal and methylglyoxal, based on the Maillard reaction. This
reaction is named glycation or nonenzymatic glycosylation
(Jeanmaire et al., 2001). Glycation, which leads to the formation
of early and advanced glycation end products (AGE), is consid-
ered as one of the major cause of spontaneous damage to cellu-
lar and extracellular proteins (Thornalley et al., 2003). Formation
of AGE on proteins is found in many tissues and is thought to
contribute to a variety of age-associated diseases (Reddy &
Beyaz, 2006). Interestingly, Hsc 70 has been recently shown to
be AGE-modified in senescent human dermal fibroblasts
(Unterluggauer et al., 2009). Finally, ROS can oxidize cysteine as
well as methionine residues within proteins to generate disul-
fides and cysteic acids or methionine sulfoxides, respectively. In
contrast with irreversible oxidative damage, certain oxidation
products of cysteine and methionine can be repaired by thiore-
doxin ⁄ thioredoxin reductase or glutaredoxin ⁄ glutathione ⁄ gluta-
thione reductase and methionine sulfoxide reductase,
respectively (Petropoulos & Friguet, 2006). Nonrepairable pro-
tein damage can be removed by proteasome and lysosome,
however, proteasome (Bulteau et al., 2000; Sitte et al., 2000;
Chondrogianni et al., 2003), lysosome (Massey et al., 2006) and
methionine sulfoxide reductase (Picot et al., 2004; Petropoulos
& Friguet, 2005) activities were shown to decline with age and
during replicative senescence indicating their involvement in the
accumulation of modified proteins during the aging process.
Although an increased load of modified proteins has been
clearly associated with cellular aging, in most cases the target
proteins have not been identified. Identification of these pro-
teins would be expected to give some insights into the mecha-
nisms by which these damaged proteins could affect cellular
function. Moreover, identification of such modified proteins
may also help understanding how these damaged proteins are
building up in senescent cells. In a previous study, we had pro-
vided evidence for the accumulation of oxidized proteins, as well
as proteins modified by glycation and conjugation with the lipid
peroxidation product HNE, in senescent human embryonic fibro-
blasts WI-38 (Ahmed et al., 2007). Therefore, the current study
was aimed at identifying target proteins subjected to such modi-
fications in the course of replicative senescence of WI-38 fibro-
blasts.
Results
Evaluation of the senescent state and the
accumulation of modified proteins in WI-38
fibroblasts
WI-38 human embryonic fibroblasts were grown as described in
Experimental procedures and the proliferation curve (Fig. 1A)
showed a linear rate of cell division from the beginning of the
cell culture until reaching the Hayflick-limit after about 45 popu-
lation doublings (PD). Two stages of cell life were investigated:
young (PD < 25) and senescent (PD > 42). As expected, the
number of cells that showed positive staining for the senes-
cence-associated b-galactosidase (Fig. 1B) was higher in senes-
cent cells (90%) in comparison with young ones (10%). In
addition, when entering replicative senescence, WI-38 cells
exhibited morphological changes as they became larger and
assumed irregular shapes. Another biochemical marker used to
assess the senescent state of cultured cells was the proteasome
activity. As shown in Fig. 1C,D, two proteasome peptidase activ-
ities, chymotrypsin-like and peptidyl glutamyl-peptide hydrolase
(CT-L and PGPH) were monitored and found to be significantly
decreased (more than 50% reduction in CT-L activity and about
25% in PGPH activity) in senescent cells compared to young
ones. To further identify HNE- and AGE-modified proteins that
were previously reported to accumulate during WI-38 replicative
senescence (Ahmed et al., 2007), HNE and AGE protein adducts
were immunodetected after 2D gel electrophoresis separation
of cellular protein lysates. For each sample, two gels were run in
parallel, one gel was stained with silver nitrate to visualize pro-
tein spots (Fig. 2A). The second gel was transferred onto a nitro-
cellulose membrane for western blotting using polyclonal
antibodies specific for either HNE or AGE protein adducts. In the
first 2D gel electrophoresis analyses, IPG strips with broad linear
pH range 3–10 were used and several spots were visualized on
the immunoblots, which are increasingly modified by HNE (15
spots) and AGE (14 spots) in senescent WI-38 fibroblasts com-
pared to young ones (Fig. 2B,C). Because of the close proximity
of the spots within this pH gradient, their resolution was not
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
253
good enough for them to be correctly matched and excised
from their respective silver-stained gels for their identification by
mass spectrometry. However, because the majority of these
spots were falling in the pH range between 4 and 9, young and
senescent WI-38 protein lysates were further analyzed using
three narrow-range strips covering pH between 4.5 and 9.
Identification and characterization of HNE-modified,
AGE-modified, and carbonylated proteins in
senescent WI-38 fibroblasts
Three narrow-range IPG strips supplied from GE Healthcare for
the following pH gradients (4.5–5.5), (5.5–6.7), and (6–9) were
used. As mentioned earlier, two gels were prepared in parallel
for each sample, one for visualization of proteins and subse-
quent identification by MS analysis and the other for blotting
against either HNE or AGE protein adducts polyclonal antibod-
ies. The results for HNE-modified proteins are presented in
Figs 3B, 4B, and 5B; and the results for AGE-modified proteins
are presented in Figs 3C and 4C. Proteins modified with car-
bonylation in the course of WI-38 replicative senescence were
also analyzed (Fig. 6) using nonlinear pH gradient (3–10 NL) IPG
strips, which permits a better protein separation at the central
region between pH 5 and 7. Protein spots on gels and blots were
digitized using UMAX UTA-100 scanner and then analyzed
using IMAGE MASTER 2D software (GE Healthcare, Saclay, France).
Protein spots were quantified as % volume (% V) in pixels,
which is a normalized value that remains relatively independent
of irrelevant variations between images. Because of variations in
protein expression levels in senescent cells, for each spot a rela-
tive modification index (RMI) was calculated, which is the ratio
of quantification value of protein spot on membrane to its corre-
sponding value on gel. To normalize the RMI values of the differ-
ent spots in their respective gels, protein spots exhibiting RMI
ratio (RMIsenescent ⁄ RMIyoung) consistently higher than 1.5 were
considered as increasingly modified (Tables 1, 2, and 3). The
basis for choosing a RMI ratio higher than 1.5 as minimum level
for significant increases comes from previous knowledge on the
maximum overall age-related increase in oxidatively modified
proteins that falls in the range between 1.5 and 2 (Levine,
2002). Moreover, a similar range for the maximum amplitude
was also observed for carbonylated, AGE- and HNE-modified
proteins in senescent fibroblasts compared to young ones
(Ahmed et al., 2007).
Modified protein spots over all pH gradients used were
excised from their respective silver-stained gels, subjected to
tryptic digestion and analyzed by MALDI-TOF MS. All the pro-
teins listed in Tables 1, 2, and 3 have been assigned a significant
Mascot score based on the probability that the observed match
is a random event and protein scores greater than 56 are signifi-
cant (P < 0.05). Proteins that were identified as targets for
either one of the three modifications are found to fall into differ-
ent categories: cytoskeleton and cytoskeleton-associated pro-
teins, molecular chaperones, respiratory chain complexes and
(A)
(C)
(B)
(D)
Fig. 1 Assessment of the senescent state of WI-38 fibroblasts. (A) Proliferation curve of WI-38 human embryonic fibroblasts. The two crosses mark the limits for
population doublings and age of the culture of the two stages under study (young and senescent). (B) Early-passage cells (< 25 PD) were defined as young because
only 10% were positive for SA-b-Gal staining. Late-passage cells (> 42 PD) were defined as senescent cells because more than 90% of the cells were positive for
SA-b-Gal staining. As shown on the light microscopy images of representative cells, senescence is associated with distinct morphological changes. Senescent cells
become larger, assume irregular shape with diffuse, thin cytoplasm, and show the characteristic SA-b-Gal staining. (C and D) Proteasome activities CT-L and PGPH
were measured as described in Experimental procedures for young and senescent WI-38 cells. Both activities were found to be significantly decreased (**P < 0.01)
in senescent cells.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
254
energy metabolism, protein degradation, protein biosynthesis
and mRNA processing, amino acids metabolism, mitochondrial
protein import, annexins, aldehyde metabolism, general catabo-
lism, and few proteins with unknown cellular function (Tables 1,
2 and 3). The subcellular location of the modified proteins indi-
cated that the modified proteins are found in four major frac-
tions: mitochondria (44%), cytosol (28%), endoplasmic
reticulum (11%), and cytoskeleton (8%) with the highest per-
centage for the mitochondria, thus reflecting the higher level of
protein damage inside this organelle especially when taking into
account that the mitochondrial proteins represent only 10% of
total cellular protein lysates (data not shown). Some identified
proteins were migrated in multiple spots with nearly the same
molecular weight but different pI values as vimentin (6 spots,
Fig. 3), actin (3 spots, Fig. 3), and mitochondrial inner mem-
brane protein (2 spots, Fig. 4). Such variants from individual pro-
teins are known as charge trains and result from post-
translational modifications that alter the intrinsic charge of the
protein. Examples of such modifications include deamidation,
phosphorylation, and HNE modification (Colvis & Garland,
2002; Ueda et al., 2002). Fragments of some proteins were also
found to be modified with HNE like those for Hsc70 where 3
spots with mass between 25 and 37 kDa were identified as
Hsc70 with IPG strip (4.5–5.6).
The cytoskeleton protein vimentin is a preferred
target of the different types of modification in
senescent WI-38 fibroblasts
Vimentin has been identified among the proteins modified
with HNE (spots 3-8, Fig. 3), AGE (spots 5 & 6, Fig. 3) and
also with carbonylation (spot 41, Fig. 6) in senescent fibro-
blasts where it shows a much larger modification yield for
all three modifications when compared to young cells. Inter-
estingly, the upregulation of this protein has been suggested
to play a role in determining senescence morphology in late-
passage fibroblasts (Nishio et al., 2001), and it has been
recently reported that vimentin is a specific target in skin
glycation during in vivo aging (Kueper et al., 2007). Taken
together with our results, these findings prompted us to
Fig. 2 2D gel-based immunodetection of HNE- and AGE-modified proteins in WI-38 cell lysates after IEF on linear pH gradient 3–10. (A) Silver-stained 2D patterns
of proteins (300 lg) from young and senescent cells separated using IPG strips with linear pH gradient 3–10 in the first dimension. More than 600 spots were
detected using IMAGE MASTER 2D Software. (B) Modification of proteins by HNE was detected by Western blot analysis using an anti-HNE polyclonal antibody. (C)
Protein glycation was detected by Western blot analysis using an anti-AGE polyclonal antibody. Several proteins were increasingly modified by HNE and AGE in
senescent WI-38 fibroblasts.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
255
further analyze the modification of vimentin by HNE and
AGE. Using an anti-vimentin antibody for western blotting, a
higher amount of vimentin was observed in senescent cells
when compared to young cells (Fig. 7A). In addition, vimen-
tin in senescent samples is running as multiple bands that
may originate from post-translational modifications or prote-
olytic cleavage. To further demonstrate that vimentin is tar-
geted for modification by HNE and AGE, vimentin was
immunoprecipitated in lysates from young and senescent
fibroblasts and then blotted with anti-HNE or anti-AGE anti-
bodies and a matching-position was observed for the HNE
and AGE signals with one of the vimentin bands in both
young and senescent cell lysates (Fig. 7B). To further investi-
gate whether HNE or AGE modification is targeting free
vimentin subunits or vimentin within either native or frag-
mented intermediate filament (IF) polymers, intermediate fila-
ments were fractionated into two pelletable fractions: IF
polymers (F1), fragmented IF (F2) and one nonpelletable
vimentin subunit fraction (F3). The three fractions were ana-
lyzed by western blot using anti-HNE, anti-AGE, and anti-
vimentin antibodies. The vimentin band in native polymer
(F1) showed a weak signal with HNE and AGE in both
young and senescent cells, while both fragmented polymers
(F2) and free vimentin subunits (F3) showed a stronger HNE-
and AGE-signal with one of the vimentin-derived fragments
that were already more abundant in senescent cells when
compared to young ones (Fig. 7C). Finally and to test
whether the vimentin network shows an overlapping with
HNE-modified proteins inside the cell, an immunofluores-
cence study was performed using anti-vimentin and anti-HNE
antibodies as described in Experimental procedures. Analysis
of the immunofluorescence microscopy reveals colocalization
of the two fluorescent signals in both young and senescent
cells. The observation that vimentin colocalize with HNE
although the polymeric form of vimentin is only slightly
modified in fractionation study could be because of a basal
level of vimentin modification by HNE or because of another
protein that colocalizes with vimentin filaments and is also
modified by HNE. In addition, the colocalization is more pro-
nounced in senescent cells at the edges of the vimentin net-
work where the intermediate filaments are less organized
and do not reach the periphery of cells (Fig. 7D).
Fig. 3 2D gel-based immunodetection of HNE- and AGE-modified proteins in WI-38 cell lysates after IEF on pH gradient 4.5–5.5. Protein lysates (500 lg) from
young and senescent WI-38 fibroblasts were separated by 2D gel electrophoresis using the IPG strips of pH 4.5-5.5 and then silver stained (A). Gels were also
blotted onto nitrocellulose membrane and hybridized with anti-HNE (B) and anti-AGE (C) antibodies as described under Experimental procedures. The blots were
then matched with their respective gels using IMAGE MASTER 2D software and proteins ‘spots 1–14’ that were increasingly modified with HNE or AGE in senescent
cells were picked for identification by MALDI-TOF MS.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
256
Impairment of glyoxal- and aldehyde-detoxification
systems (GLO-I and GST) in senescent WI-38
fibroblasts
Because almost half of the modified proteins that were identi-
fied are of mitochondrial origin, which may reflect the preferen-
tial targeting of modifications for proteins within the
mitochondria during senescence, the status of glyoxalase-I
(GLO-I) and glutathione-S-transferase (GST) was monitored in
the mitochondrial and cytosolic fractions of young and senes-
cent cells. GLO-I is part of the glyoxalase system, present in the
cytosol and the mitochondria of all cells, where it provides an
enzymatic defense against glyoxal and methylglyoxal-induced
glycation (Thornalley, 2003), while GST is a major enzyme
involved in the detoxification of HNE (Zimniak, 2008). For this
purpose, mitochondrial and cytosolic fractions were prepared
from young and senescent cells as described in Experimental
procedures. Aconitase was used as a mitochondrial marker and
was absent in the cytosolic fractions (Fig. 8B). Analysis of the
activity of GLO-I in cytosolic and mitochondrial fractions showed
a 40 to 50% decrease in both fractions with no change in its
expression levels in senescent cells (Fig. 8A,B), a finding which
may explain the observed accumulation of glycated proteins in
senescent cells. On the contrary, the activity level of GST did not
significantly change in the cytosol but showed a slight but signif-
icant decrease in the mitochondria of senescent fibroblasts com-
pared to young ones (Fig. 8C).
Fate of methionine sulfoxide reductase (Msr) and
mitochondrial ATP-stimulated proteolytic activity in
senescent WI-38 fibroblasts
Because protein degradation and repair, also referred as protein
maintenance, play an important role in the elimination of altered
proteins, the oxidized protein repair system methionine sulfox-
ide reductase (Msr) and the mitochondrial ATP-stimulated prote-
olytic activity, which has been implicated in the degradation of
oxidatively modified proteins, were investigated. The Msr system
is responsible for catalyzing the reduction of protein-bound
methionine sulfoxides to methionine, and thus prevents their
conversion and further accumulation as irreversibly oxidized
forms. It consists of two distinct enzyme families, MsrA that is
Fig. 4 2D gel-based immunodetection of HNE- and AGE-modified proteins in WI-38 cell lysates after IEF on pH gradient 5.5–6.7. Protein lysates (500 lg) from
young and senescent WI-38 fibroblasts were separated by 2D gel electrophoresis using the IPG strips of pH 5.5–.7 and then silver stained (A). Gels were also
blotted onto nitrocellulose membrane and hybridized with anti-HNE (B) and anti-AGE (C) antibodies as described under Experimental procedures. The blots were
then matched with their respective gels using IMAGE MASTER 2D software and proteins ‘spots 15–36’ that were increasingly modified with HNE or AGE in senescent
cells were picked up for identification by MALDI-TOF MS.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
257
specific for the S-form of methionine sulfoxide and MsrB for the
R-form. Figure 9 shows a 62% decrease in Msr activity in the
mitochondrial fraction compared to a lower 22% decrease in
the cytosol. In addition, the decrease in Msr activity inside the
mitochondria is coupled with a decreased MsrA content (30%),
while no change of MsrA content was observed in the cytosol.
This reflects more deterioration of the Msr system in the mito-
chondria than in the cytosol of senescent fibroblasts.
On the other hand, mammalian mitochondrial matrix contains
two known ATP-stimulated proteolytic systems, Lon and ClpXP
(Van Dyck & Langer, 1999; Kaser & Langer, 2000). Each of these
proteases is believed to contribute to the degradation of
unfolded and damaged proteins. In addition, Lon protease has
been implicated in the removal of oxidized proteins (Bota et al.,
2002). Proteolytic activity was measured as the rate of fluores-
cein isothiocyanate (FITC)-casein degradation in the presence of
8 mM ATP. Interestingly, a 2.5-fold increase in ATP-dependent
proteolytic activity was monitored in senescent mitochondrial
fraction compared to young ones (Fig. 10A). However, Lon pro-
tease did not show a significant increase in its protein level,
while a 20% increase in ClpP was observed in mitochondria
from senescent cells (Fig. 10B). To determine whether Lon is
involved in this increase in proteolytic activity, the same assay
was performed after 30 min incubation of mitochondrial frac-
tions with the potent Lon-inhibitor peptidyl boronate MG262 at
10 lM. About 50% inhibition of the ATP-dependent proteolytic
activity was monitored in mitochondrial extracts from young
cells compared to about 40% in mitochondrial extracts from
senescent cells (Fig. 10C), which represents the contribution of
Lon protease activity to the total proteolytic activity in young
and senescent mitochondria and corresponds to a two-fold
increase in Lon protease activity in mitochondria from senescent
cells.
Discussion
Protein oxidative damage is believed to contribute to cellular
aging because many studies have pointed out the accumula-
tion of oxidatively damaged proteins during aging (Yan et al.,
1997; Yan & Sohal, 1998) and age-related diseases (Levine,
2002; Dalle-Donne et al., 2003). Because we had previously
reported an increase in proteins modified with HNE, AGE, and
Fig. 5 2D gel-based immunodetection of HNE-modified proteins in WI-38 cell lysates after IEF on pH gradient 6–9. Protein lysates (500 lg) from young and
senescent WI-38 fibroblasts were separated by 2D gel electrophoresis using the IPG strips of pH 6–9 and then silver stained (A). Gels were also blotted onto
nitrocellulose membrane and hybridized with anti-HNE antibody (B) as described under Experimental procedures. The blots were then matched with their
respective gels using IMAGE MASTER 2D software and proteins ‘spots 37–39’ that were increasingly modified with HNE in senescent cells were picked for
identification by MALDI-TOF MS.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
258
carbonylation in senescent WI-38 human embryonic fibroblasts
cells (Ahmed et al., 2007), proteins targeted by these modifi-
cations have been identified using a 2D gel electrophoresis-
based proteomic approach coupled with immunodetection of
HNE-, AGE-modified, and carbonylated proteins. The aim of
this study was to better understand the mechanisms by which
these damaged proteins are building up and potentially affect
cellular function during replicative senescence of WI-38 cells.
Indeed, the damaging effects associated with oxidation, conju-
gation with HNE, and formation of AGE (Bassi et al., 2002;
Cao et al., 2003; Kaplan et al., 2007; Bigl et al., 2008) high-
light the importance of identifying proteins that are targets for
these modifications. Despite the limitations of 2D gel electro-
phoresis, it is still considered as one of the best technique for
separation of a complex mixture of soluble proteins. However,
membrane proteins are usually missed and the resolution in
the first dimension is limited by the selection of the pH range.
In this study, either nonlinear pH-range IPG strips 3–10 or a
selection of narrow pH-range IPG strips were used to provide a
better protein separation.
The cytoskeletal proteins vimentin, actin, and tubulin were
found among the proteins identified as HNE-modified. Cyto-
skeletal proteins are classical targets for ROS-mediated oxida-
tive damage inside mammalian cells. For example, actin was
shown to be a target for protein carbonylation in many mam-
malian cells including fibroblasts under oxidative stress (Banan
et al., 2001; Dalle-Donne et al., 2001). In addition, HNE has
been shown to modify the cytoskeletal proteins vimentin,
actin, and tubulin (Montine et al., 1996; Mattson et al., 1997)
and disrupts microtubules via Michael addition and causes
tubulin modification (Neely et al., 1999). Vimentin has been
further analyzed because it was found carbonylated, modified
with HNE and AGE in senescent WI-38 fibroblasts. In fact,
many structural changes of the intermediate filament protein
vimentin have been associated with cellular senescence. For
example, vimentin filaments form thick, long, dense bundles in
senescent cells in comparison with irregular and small fur-like
networks in young or early-passage fibroblasts (Wang, 1985).
Furthermore, while over-expression of vimentin in young fibro-
blasts induces the appearance of a senescent phenotype
(A) (B)
(A) (B)
Fig. 6 2D gel-based immunodetection of carbonylated proteins in WI-38 cell lysates after IEF on a nonlinear pH gradient 3–10. Protein lysates (500 lg) from
young and senescent WI-38 fibroblasts were separated by 2D gel electrophoresis using the IPG strips of pH 3–10 NL. Gels were silver stained (A) or blotted onto
nitrocellulose membrane and hybridized with anti-carbonyl-DNP adduct antibodies as described in Experimental procedures. The blots were then matched with
their respective gels using IMAGE MASTER 2D software and proteins ‘spots 40-49’ that were increasingly carbonylated (B) in senescent cells were picked up for
identification by MALDI-TOF MS.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
259
(Nishio et al., 2001), vimentin knockout in primary embryonic
fibroblasts causes an accelerated rate of replicative senescence
(Tolstonog et al., 2001), a finding that suggests a protective
function of vimentin against damage that induces telomere
shortening-independent senescence (Hwang et al., 2009).
More vimentin bands or spots are visualized in 1D or 2D gels,
respectively, for senescent cells compared to young ones. Such
finding may be because of modifications of the protein by
phosphorylation or proteolytic cleavage by calpains and casp-
ases, which have previously been documented (Perides et al.,
1987; Byun et al., 2001; Eriksson et al., 2004). Vimentin has
also been identified as a major target of modification by carbo-
xymethyllysine (CML) in human dermal fibroblasts during aging
in vivo (Kueper et al., 2007). Both modifications of vimentin by
HNE and AGE were confirmed by assaying anti-HNE and anti-
AGE reactivity on immunoprecipitated vimentin (Fig. 7B). HNE
and AGE modifications of vimentin-derived fragments were
evidenced in both fragmented intermediate filaments and
vimentin monomers (Fig. 7C). Interestingly, a band with a
molecular weight lower than native vimentin, which may cor-
respond to a proteolytic fragment, was mostly targeted for
both modifications (Fig. 7C). Finally, colocalization of vimentin
and HNE was monitored in young and senescent fibroblasts
(Fig. 7D). In senescent cells, colocalization was found at the
edges of the vimentin network where the intermediate fila-
ments are less organized and do not reach the periphery of
cells (Fig. 7D). In fact, vimentin modification with HNE has pre-
viously been reported in cultured rat neonatal cardiomyocytes
after exposure to HNE and confirmed by indirect immunofluo-
rescent localization (VanWinkle et al., 1994). Taken together,
our results indicate that, in addition to AGE adducts formation,
the intermediate filament protein vimentin is a target for modi-
fication by HNE that may therefore contribute to the alteration
of vimentin function in senescent WI-38 fibroblasts.
Some modified proteins were found to have a chaperone
function such as Hsc70, calreticulin, endoplasmic reticulum pro-
(A)
(C)
(B)
Fig. 7 Characterization of HNE-modified vimentin. (A) Western blot detection of vimentin in the total cell extract of young and senescent WI-38 cells. (B) For
immunoprecipitation of vimentin, young and senescent cells were lysed as described under Experimental procedures, and the blots were hybridized with either
anti-vimentin, anti-HNE or anti-AGE antibodies and vimentin bands were found to correspond to bands modified with HNE and AGE. (C) Fractionation of the
intermediate filament (IF) pool into IF polymers (1), mixture of fragmented IF and soluble subunits (2), and soluble subunits (3). Fractions were denatured and
analyzed by SDS–PAGE as described in Experimental procedures and then immunoblotted with anti-vimentin, anti-HNE, or anti-AGE antibodies. Vimentin bands
were detected on HNE and AGE blots using a separate experiment. A lane of sample protein was splitted into two halves, one half was probed with either anti-
HNE or anti-AGE antibodies and the other half was probed with anti-vimentin antibody (data not shown). Then the two halves were realigned and the main band
that comatched with the vimentin bands is the one indicated by an arrow. Vimentin-derived fragments and vimentin monomers (fractions 2 and 3) were more
abundant in senescent fractions and were showing more HNE- and AGE modifications. (D) Immunofluorescence showing colocalization of vimentin with HNE
signals. Colocalization is more evident in the periphery of the senescent cell where vimentin net is disorganized and filaments are retracted and do not reach the
cell membrane.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
260
tein ERp29, T-complex 1 subunit zeta, and elongation factor Tu.
Hsc70 has been previously identified as one of the targets for
modification by HNE in retina of aged rats (Kapphahn et al.,
2006). In addition, it was identified as target for AGE modifica-
tion in human senescent dermal fibroblasts (Unterluggauer
et al., 2009). Calreticulin and Hsc70 also showed increased oxi-
dative modification in HL60 cells under oxidative stress (Magi
et al., 2004), while elongation factor Tu is modified by HNE after
treatment of plant mitochondria with hydrogen peroxide, anti-
mycin A, or menadione (Winger et al., 2007). One interesting
finding is that, in addition to full length Hsc70 (spot 1, Fig. 3B),
truncated forms of Hsc70 were found modified with HNE (spots
12, 13 and 14, Fig. 3B), which may suggest an interplay
between HNE modification and protein turnover. In fact, the
presence of modified cytosolic, mitochondrial, and endoplasmic
reticulum chaperones in senescent cells supports the idea of a
defective protein quality control during cellular aging. Indeed,
another system getting deteriorated with aging is the protea-
some, which is the major intracellular proteolytic system impli-
cated in the removal of abnormal and oxidized proteins. The
age-related decrease in proteasomal activity has been attributed
to decreased proteasome cellular content but also to accumula-
tion of endogenous proteasome inhibitors and occurrence of
oxidative and glycoxidative modifications on proteasome subun-
its (Carrard et al., 2003; Ishii et al., 2005; Farout et al., 2006;
Gonzalez-Dosal et al., 2006). Because the 20S proteasome a2
subunit was identified as increasingly modified by carbonylation
(Table 3), this modification could contribute, together with the
previously documented decrease in proteasome subunits
expression, to the decreased proteasomal activity observed in
senescent WI-38 fibroblasts (Fig. 1C,D).
Senescent cells and cells from old donors have been shown
to exhibit an increased glycolytic activity with subsequent accu-
mulation of lactate (Prahl et al., 2008; Unterluggauer et al.,
2008). At the same time, an age-associated decrease in the
mitochondrial capability of ATP regeneration has been
observed (Dierick et al., 2002), most likely because of an
increased production of ROS and subsequent accumulation of
oxidative damage in the mitochondrial membrane, proteins,
and DNA (Jacobs, 2003). In senescent WI-38 fibroblasts, we
identified iron–sulfur subunit of complex I and subunit a of
ATP synthase as HNE-modified, subunit 1 of complex III as car-
bonylated, and FAD subunit of complex II as AGE-modified
(Tables 1, 2 and 3). Interestingly, iron-sulfur subunit of com-
plex I and FAD subunit of complex II were previously found to
be HNE-modified in kidney mitochondria of aged rat (Choksi
et al., 2007). In addition, HNE has been previously found to
inhibit the activity of many respiratory chain complexes in vitro
(Chen et al., 1998, 2001; Picklo et al., 1999; Isom et al.,
2004; Lashin et al., 2006) and mitochondrial respiration is also
affected by incubation of mitochondria with the AGE inducer
methylglyoxal (Rosca et al., 2002). Among the modified pro-
teins, we also found the enzymes malate dehydrogenase,
2-oxoglutarate dehydrogenase E1 component, glycerol-3-
phosphate dehydrogenase, glycerol kinase, and glutaminase
(Tables 1, 2 and 3). E1 component of pyruvate dehydrogenase
enzyme complex, identical to oxoglutarate dehydrogenase
enzyme, was also found among HNE-modified proteins in
plant mitochondria (Winger et al., 2007). These findings sug-
gest that modification of proteins responsible for energy
metabolism may participate in the impairment of mitochon-
drial function observed in senescent cells.
Aged cells suffer from deterioration in the redox homeostasis
exerted by the antioxidant machinery including NADPH (Finkel &
Holbrook, 2000). One of the reasons for decreased NADPH is
the decreased content and ⁄ or activity of glucose-6-phosphate
dehydrogenase (G6PDH), a main producer for NADPH through
the pentose phosphate pathway (Schwartz & Pashko, 2004).
Interestingly, G6PDH was identified among the AGE-modified
proteins (spot 34) in senescent WI-38 and such modification
may also participate to its inactivation in senescent cells. In addi-
tion, proteins involved in aldehyde metabolism such as mito-
chondrial aldehyde dehydrogenase 2 (ALDH2) and delta-
1-pyrroline carboxylate dehydrogenase (ALDH4A1) are found to
be HNE-modified. These proteins are involved in acetaldehyde
and glutamate semialdehyde detoxification, respectively. Modi-
fication of these enzymes with HNE may have an impact on their
function and subsequently increase the load of reactive alde-
hydes in senescent cells. Proteins that are playing an important
role in metabolic homeostasis inside mitochondria were also
found among modified proteins: isovaleryl-CoA-dehydroge-
nase, ETHE1 protein, 3-hydroxyacyl-CoA dehydrogenase, orni-
thine aminotransferase, and succinyl-CoA:3-ketoacid-coenzyme
A transferase (Tables 1, 2 and 3). Moreover, other proteins of
different cellular functions as neutral a glucosidase AB, mito-
chondrial-processing peptidase, filamin, tryptophanyl-tRNA syn-
thetase, and annexin A5 were also identified together with two
proteins with no exact cellular function, mitochondrial inner
membrane protein (mitofilin), and uncharacterized protein C19
(Tables 1, 2 and 3).
Accumulation of damaged proteins may be because of
increased modification, decreased elimination through degrada-
tion, and repair or the combination of both mechanisms. To fur-
ther investigate the pathways that are acting upstream of
protein modification, hence regulating the steady-state level of
modified proteins, the activity of representative enzymes
involved in either detoxification of reactive aldehydes (glutathi-
one-S-transferase or GST) or dicarbonyl glycating compounds as
glyoxal and methylglyoxal (glyoxalase-I or GLO-I) was quantified.
These activities were measured in the cytosolic and mitochon-
drial fractions of young and senescent fibroblasts because the
main part of the modified proteins identified in this study is
located in the mitochondria. Among detoxification reactions,
removal of reactive electrophiles such as HNE by GST-catalyzed
conjugation is representing a major longevity assurance mecha-
nism (Zimniak, 2008). Various data from the literature have
shown that conjugation of aldehydes with glutathione repre-
sents up to 60% of the metabolism of endogenous aldehydes
formed in cells of different tissues (Esterbauer et al., 1985). GST
demonstrates the highest affinity toward 4-hydroxyalkenals and
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
261
Tab
le1
HN
E-m
odifi
edpro
tein
sin
WI-38
fibro
bla
sts
Prote
in
spot
no
a
Iden
tified
pro
tein
nam
e
Iden
tified
pro
tein
funct
ion
Swis
s-Pr
ot
acce
ssio
nno
b
Prote
in
mas
sc
Mas
cot
score
d
Sequen
ce
cove
rage
(%)e
No.
of
mat
ched
pep
tides
f
No.
of
sequen
ced
pep
tides
gIP
Gst
rip
iRM
Ira
tio
j
1H
eat
shock
cognat
e71
kDa
pro
tein
Chap
erone
HSP
7C
_HU
MA
N70
898
87
16
10
14.5
–5.5
h
2Tu
bulin
bet
ach
ain
Cyt
osk
elet
alTB
B5_H
UM
AN
49
671
119
14
11
44.5
–5.5
h
3V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
516
29
18
84.5
–5.5
h
4V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
458
25
17
64.5
–5.5
h
5V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
599
33
20
74.5
–5.5
7
6V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
627
20
23
94.5
–5.5
2.2
7V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
640
25
24
11
4.5
–5.5
1
8V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
416
21
19
84.5
–5.5
3.3
9A
ctin
,cy
topla
smic
1C
ytosk
elet
alA
CTB
_HU
MA
N41
737
127
36
72
4.5
–5.5
5.2
10
Act
in,
cyto
pla
smic
1C
ytosk
elet
alA
CTB
_HU
MA
N41
737
460
35
18
64.5
–5.5
2.6
11
Act
in,
cyto
pla
smic
1C
ytosk
elet
alA
CTB
_HU
MA
N41
737
652
36
20
74.5
–5.5
9.7
12
Hea
tsh
ock
cognat
e71
kDa
pro
tein
Chap
erone
HSP
7C
_HU
MA
N70
898
300
25
16
54.5
–5.5
h
13
Hea
tsh
ock
cognat
e71
kDa
pro
tein
Chap
erone
HSP
7C
_HU
MA
N70
898
272
28
17
54.5
–5.5
h
14
Hea
tsh
ock
cognat
e71
kDa
pro
tein
Chap
erone
HSP
7C
_HU
MA
N70
898
279
28
17
44.5
–5.5
2.4
15
Mitoch
ondrial
inner
mem
bra
ne
pro
tein
No
exac
tfu
nct
ion
IMM
T_H
UM
AN
83
626
88
77
15.5
–6.7
33
16
Neu
tral
alpha-
glu
cosi
das
eA
Bpre
curs
or
Gly
copro
tein
bio
synth
esis
GA
NA
B_H
UM
AN
106
807
217
12
16
45.5
–6.7
3.8
17
Mitoch
ondrial
inner
mem
bra
ne
pro
tein
No
exac
tfu
nct
ion
IMM
T_H
UM
AN
83
626
328
18
17
35.5
–6.7
7.7
18
2-O
xoglu
tara
tedeh
ydro
gen
ase
E1co
mponen
t,
mitoch
ondrial
pre
curs
or
Ener
gy
met
abolis
mO
DO
1_H
UM
AN
113
403
284
11
18
55.5
–6.7
1.6
19
Gly
cero
l-3-p
hosp
hat
edeh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
Ener
gy
met
abolis
mG
PDM
_HU
MA
N80
783
112
14
13
25.5
–6.7
1.1
20
Het
erogen
eous
nucl
ear
ribonucl
eopro
tein
mRN
Apro
cess
ing
HN
RH
1_H
UM
AN
49
198
79
63
15.5
–6.7
1.1
20
Mitoch
ondrial
-pro
cess
ing
pep
tidas
esu
bunit
alpha,
mitoch
ondrial
pre
curs
or
Prote
inim
port
MPP
A_H
UM
AN
58
216
66
96
12
5.5
–6.7
1.1
21
Ald
ehyd
edeh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
Ald
ehyd
edet
oxi
fica
tion
ALD
H2_H
UM
AN
56
346
193
27
16
25.5
–6.7
1.9
22
NA
DH
deh
ydro
gen
ase
[ubiq
uin
one]
iron-s
ulfur
pro
tein
2,
mitoch
ondrial
pre
curs
or
Res
pirat
ory
com
ple
xN
DU
S2_H
UM
AN
52
512
75
15
10
35.5
–6.7
2.8
23
Ald
ehyd
edeh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
[N-t
erm
inal
frag
men
t]
Ald
ehyd
edet
oxi
fica
tion
ALD
H2_H
UM
AN
56
346
67
13
73
5.5
–6.7
1.5
24
Unch
arac
terize
dpro
tein
C19orf
10
pre
curs
or
Cel
lpro
lifer
atio
nC
S010_H
UM
AN
18
783
333
32
11
55.5
–6.7
1.5
37
Del
ta-1
-pyr
rolin
e-5
-car
boxy
late
deh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
Am
ino
acid
met
abolis
mA
L4A
1_H
UM
AN
61
681
78
12
93
6.0
–9.0
4.2
38
Isova
lery
l-C
oA
deh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
Am
ino
acid
met
abolis
mIV
D_H
UM
AN
46
290
121
77
36.0
–9.0
1.9
39
ATP
synth
ase
subunit
alpha,
mitoch
ondrial
pre
curs
or
resp
irat
ory
com
ple
xA
TPA
_HU
MA
N59
714
204
18
12
36.0
–9.0
1.5
Spots
of
inte
rest
wer
eid
entified
by
MS
asdes
crib
edin
Exper
imen
talpro
cedure
s.Pr
ote
insp
ot
no
(a)
refe
rto
num
ber
edsp
ots
on
Figs
3,
4,
and
5.
For
each
spot,
diffe
rent
par
amet
ers
clar
ifyi
ng
pro
tein
iden
tifica
tion
by
MS
are
indic
ated
[(ac
cess
ion
num
ber
(b),
mas
s(c
),m
asco
tsc
ore
(d),
%se
quen
ceco
vera
ge
(e),
no
of
mat
ched
pep
tides
(f),
and
no
of
sequen
ced
pep
tides
(g)]
.IP
Gst
rip
(i)re
fers
toth
efirs
tdim
ensi
on
pH
gra
die
nt
use
dfo
rth
e2D
gel
elec
trophore
sis.
(j)RM
Ira
tio
repre
sents
the
Rel
ativ
eM
odifi
cation
Index
Rat
ioan
d(h
)m
eans
that
the
modifi
edpro
tein
was
only
det
ecte
din
senes
cent
sam
ple
s.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
262
Tab
le2
AG
E-m
odifi
edpro
tein
sin
WI-38
fibro
bla
sts
Prote
in
spot
no
aId
entified
pro
tein
nam
e
Iden
tified
pro
tein
funct
ion
Swis
s-Pr
ot
acce
ssio
nno
b
Prote
in
mas
sc
Mas
cot
score
d
Sequen
ce
cove
rage
(%)e
No
of
mat
ched
pep
tides
f
No
of
sequen
ced
pep
tides
gIP
GSt
rip
i
RM
I
ratio
j
3V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
516
29
18
84.5
–5.5
h
4V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
458
25
17
64.5
–5.5
1
5V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
599
33
20
74.5
–5.5
4
6V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
627
20
23
94.5
–5.5
1
7V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
640
25
24
11
4.5
–5.5
h
8V
imen
tin
Cyt
osk
elet
alV
IME_
HU
MA
N53
652
416
21
19
84.5
–5.5
h
15
Mitoch
ondrial
inner
mem
bra
ne
pro
tein
No
exac
tfu
nct
ion
IMM
T_H
UM
AN
83
626
88
77
15.5
–6.7
37
17
Mitoch
ondrial
inner
mem
bra
ne
pro
tein
No
exac
tfu
nct
ion
IMM
T_H
UM
AN
83
626
328
18
17
35.5
–6.7
h
25
Fila
min
-A–
Hom
osa
pie
ns
(Hum
an)
[C-t
erm
inal
frag
men
t]
Cyt
osk
elet
al-
bin
din
gFL
NA
_HU
MA
N280
564
60
35
25.5
–6.7
h
26
T-co
mple
xpro
tein
1su
bunit
zeta
Chap
erone
TCPZ
_HU
MA
N57
988
339
19
11
35.5
–6.7
4.3
27
Succ
inat
edeh
ydro
gen
ase
[ubiq
uin
one]
flav
opro
tein
subunit,
mitoch
ondrial
pre
curs
or
Res
pirat
ory
com
ple
xD
HSA
_HU
MA
N72
645
153
11
73
5.5
–6.7
h
28
Glu
tam
inas
eki
dney
isofo
rm,
mitoch
ondrial
pre
curs
or
Ener
gy
met
abolis
mG
LSK
_HU
MA
N73
414
119
12
72
5.5
–6.7
1.1
28
Gly
cero
lki
nas
eEn
ergy
met
abolis
mG
LPK
_HU
MA
N57
452
100
11
62
5.5
–6.7
1.1
29
Succ
inyl
-CoA
:3-k
etoac
id-c
oenzy
me
Atr
ansf
eras
e1,
mitoch
ondrial
pre
curs
or
Ket
one
bodie
sca
tabolis
mSC
OT_
HU
MA
N56
122
160
19
11
25.5
–6.7
h
30
Tryp
tophan
yl-t
RN
Asy
nth
etas
e,cy
topla
smic
Prote
inbio
synth
esis
SYW
C_H
UM
AN
53
132
82
17
72
5.5
–6.7
h
31
Mitoch
ondrial
-pro
cess
ing
pep
tidas
esu
bunit
bet
a,
mitoch
ondrial
pre
curs
or
Prote
inim
port
MPP
B_H
UM
AN
54
331
137
97
25.5
–6.7
1.2
32
Orn
ithin
eam
inotr
ansf
eras
e,m
itoch
ondrial
pre
curs
or
Am
ino
acid
met
abolis
mO
AT_
HU
MA
N48
504
87
18
10
35.5
–6.7
1.8
33
26S
pro
teas
om
enon-A
TPas
ere
gula
tory
subunit
11
Prote
indeg
radat
ion
PSD
11_H
UM
AN
47
434
116
24
10
15.5
–6.7
1
34
Glu
cose
-6-p
hosp
hat
e1-d
ehyd
rogen
ase
NA
DPH
pro
duct
ion
G6PD
_HU
MA
N59
219
106
18
14
25.5
–6.7
1.7
35
Dih
ydro
lipoyl
deh
ydro
gen
ase,
mitoch
ondrial
pre
curs
or
Ener
gy
met
abolis
mD
LDH
_HU
MA
N54
116
104
78
25.5
–6.7
h
36
Elongat
ion
fact
or
Tu,
mitoch
ondrial
pre
curs
or
Chap
erone
EFTU
_HU
MA
N49
510
360
26
14
55.5
–6.7
2
Spots
of
inte
rest
wer
eid
entified
by
MS
asdes
crib
edin
Exper
imen
tal
pro
cedure
s.Pr
ote
insp
ot
no
(a)
refe
rto
num
ber
edsp
ots
on
Figs
3an
d4.
For
each
spot,
diffe
rent
par
amet
ers
clar
ifyi
ng
pro
tein
iden
tifica
tion
by
MS
are
indic
ated
[(ac
cess
ion
num
ber
(b),
mas
s(c
),m
asco
tsc
ore
(d),
%se
quen
ceco
vera
ge
(e),
no
of
mat
ched
pep
tides
(f),
and
no.
of
sequen
ced
pep
tides
(g)]
.IP
Gst
rip
(i)re
fers
toth
efirs
tdim
ensi
on
pH
gra
die
nt
use
d
for
the
2D
gel
elec
trophore
sis.
(j)RM
Ira
tio
repre
sents
the
Rel
ativ
eM
odifi
cation
Index
Rat
ioan
d(h
)m
eans
that
the
modifi
edpro
tein
was
only
det
ecte
din
senes
cent
sam
ple
s.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
263
Table 3 Carbonylated proteins in WI-38 fibroblasts
Protein
spot no.a Identified protein name
Identified protein
function
Swiss-Prot
accession nob
Protein
massc
Mascot
scored
Sequence
coverage (%)e
No. of
matched
peptidesf
No of
sequenced
peptidesg
IPG
Stripi
RMI
ratioj
40 Calreticulin precursor Chaperone CALR_HUMAN 48 112 434 32 18 3 3–10 NL 2
41 Vimentin Cytoskeletal VIME_HUMAN 53 652 Vim 3B4
Ab blot
3–10 NL > 3
42 Cytochrome b-c1 complex
subunit 1, mitochondrial
precursor
Respiratory
complex
QCR1_HUMAN 52 612 61 21 9 1 3–10 NL 2
43 Annexin A5 Blood
coagulation
ANXA5_HUMAN 35 914 104 25 10 1 3–10 NL 6.5
44 Endoplasmic
reticulum protein
ERp29 precursor
Chaperone ERP29_HUMAN 28 975 183 24 9 2 3–10 NL h
45 ETHE1 protein,
mitochondrial
precursor
Mitochonrial
homeostasis
ETHE1_HUMAN 27 855 137 17 11 3 3–10 NL h
46 Proteasome subunit alpha
type-2
Protein
degradation
PSA2_HUMAN 25 882 96 20 4 1 3–10 NL h
47 3-Hydroxyacyl-CoA
dehydrogenase
type-2
tRNA
processing
HCD2_HUMAN 26 906 274 22 9 3 3–10 NL 1.7
48 Elongation factor Tu,
mitochondrial
precursor
Chaperone EFTU_HUMAN 49 510 69 9 5 2 3–10 NL h
49 Malate dehydrogenase,
mitochondrial precursor
Energy
metabolism
MDHM_HUMAN 35 509 101 17 6 2 3–10 NL 6.8
Spots of interest were identified by MS as described in Experimental procedures. Protein spot no (a) refer to numbered spots on Fig. 6. For each spot,
different parameters clarifying protein identification by MS are indicated [(accession number (b), mass (c), mascot score (d), % sequence coverage (e), no. of
matched peptides (f) and no of sequenced peptides (g)]. IPG strip (i) refers to the first dimension pH gradient used for the 2D gel electrophoresis. (j) RMI
ratio represents the Relative Modification Index Ratio and (h) means that the modified protein was only detected in senescent samples.
(A)
(C)
(B)
Fig. 8 Activity of the detoxification enzymes glyoxalase-I and glutathione-S-transferase in young and senescent WI-38 fibroblasts. Glyoxalase-I and glutathione-
S-transferase activities were assayed in the cytosolic and mitochondrial fraction of young and senescent WI-38 cells as described in the Experimental procedures. A
significant decrease in the activity of glyoxalase-I (**P < 0.01) was detected (A), while its protein expression level was not significantly changed in both cytosolic
and mitochondrial fractions (B). Western blot detection of aconitase in mitochondrial and cytosolic fractions indicated a good mitochondrial isolation with nearly
no mitochondrial leakage into the cytosol (B). No change in glutathione-S-transferase activity was detected in the cytosolic fraction, while a small but significant
decrease (*P < 0.05) was observed in the mitochondrial one (C).
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
264
the resistance of Chinese hamster fibroblasts to HNE toxicity is
related to the activity of GST (Spitz et al., 1991). A small but
significant decrease in the activity of mitochondrial GST was
observed in senescent fibroblasts compared to no change in the
cytosolic fraction (Fig. 8C). This decrease in GST activity inside
mitochondria may contribute to the accumulation of HNE and
may promote its reaction with mitochondrial proteins. On the
other hand, accumulation of glycated proteins in senescent
WI-38 fibroblasts seems to be related to the observed decrease
in activity of the GLO-I for both mitochondrial and cytosolic frac-
tions (Fig. 8A,B). A similar finding has been recently reported in
aged C. elegans, and it was proposed that the observed
decrease in GLO-I activity with age promotes further accumula-
tion of methylglyoxal-derived protein adducts (Morcos et al.,
2008). Indeed, GLO-I is the major enzyme involved in the detoxi-
fication of the dicarbonyl glycating agents glyoxal and methyl-
glyoxal in physiological systems (Westwood et al., 1997), and it
has been recently shown that overexpression of GLO-I protects
mitochondrial proteins from methylglyoxal modification and
prolongs C. elegans life span (Morcos et al., 2008).
Concerning the elimination of damaged proteins, the activity
of the oxidized protein repair system methionine sulfoxide
reductase (Msr) was also determined in the cytosolic and mito-
chondrial fractions of young and senescent fibroblasts. Msr
enzymes, MsrA, and MsrB are catalyzing the reduction of the S
and R forms of methionine sulfoxide, respectively, into methio-
nine within proteins (Moskovitz et al., 2000; Weissbach et al.,
2002). It has been previously reported that Msr activity is
decreased in aged rat organs (Petropoulos et al., 2001) and
senescent fibroblasts (Picot et al., 2004), and it has been pro-
posed that this decline in Msr activity participate to the age-
associated accumulation of oxidized proteins (Petropoulos &
Friguet, 2006). We have found that the decrease in Msr activity
is more prominent in the mitochondrial fraction of senescent
WI-38 fibroblasts (38% residual activity) than in the cytosolic
one (78% residual activity) (Fig. 9A,B). In addition, mitochondria
from senescent cells showed a significant decrease in the MsrA
protein level, which was not found in the cytosol (Fig. 9C),
reflecting the higher impairment of this system within the mito-
chondria. Degradation of damaged protein also represents an
important mechanism of protein maintenance insuring cellular
homeostasis and survival (Morimoto & Cuervo, 2009). Inside the
mitochondria, irreversibly oxidized proteins are targeted to deg-
radation by proteolytic systems such as the Lon protease (Ngo &
Davies, 2007). Lon protease expression decreases with age in
skeletal muscle (Bota et al., 2002), and it has been pointed out
that the age-related impairment of Lon protease is likely organ-
specific (Delaval et al., 2004). Interestingly, an increased ATP-
stimulated proteolytic activity was detected in the senescent
WI-38 fibroblasts mitochondrial fraction compared to young
ones without increased expression of Lon and only a 20%
increase for ClpP, another ATP-stimulated protease in the mito-
chondrial matrix (Fig. 10A,B). This increased activity can be
explained, at least in part, by activation of the Lon protease
because a twofold increase in Lon protease activity was
observed in senescent mitochondrial fraction when compared
to young one (Fig. 10C). In addition to ClpXP, mitochondrial
membrane-associated proteases of the AAA+ family could be
also involved in the age-associated increase in ATP-stimulated
proteolytic activity.
In conclusion, proteins increasingly modified by HNE, AGE, or
carbonylation during replicative senescence and identified in this
study represent a restricted set of proteins that fall in different
functional categories, the most represented being protein qual-
ity control, energy metabolism, cytoskeleton, and aldehyde
detoxification. Because impairment of these systems has been
previously documented in senescent cells, the reported protein
modifications may therefore be implicated in their age-associ-
ated functional decline. Interestingly, almost half of the modi-
fied proteins that were identified are located in the
(A) (B)
(C)
Fig. 9 Activity of the oxidized protein repair system methionine sulfoxide reductase in young and senescent WI-38 fibroblasts. Msr total activity and expression of
MsrA protein were detected in both cytoplasmic and mitochondrial fractions of young and senescent WI-38 fibroblasts. A less important but significant decrease
(*P < 0.05) in Msr activity was detected in the cytosolic fraction (A) compared to a more pronounced decrease (**P < 0.01) in the mitochondrial one (B). The
decrease in activity is accompanied with a decrease in MsrA protein expression level in mitochondria (30%) but not in cytosol. As expected, the cytosolic isoform of
MsrA is shorter than the mitochondrial isoform (Vougier et al., 2003) (C).
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
265
mitochondria, which indicate the occurrence of higher protein
damage inside this organelle. In senescent cells, GST, the major
enzyme involved in the removal of reactive aldehydes such as
HNE, was only slightly affected in the mitochondria, while GLO-
I, the major enzyme involved in the detoxification of the dicar-
bonyl glycating agents glyoxal and methylglyoxal, was found to
be impaired in both mitochondria and cytosol, pointing out a
potential role of detoxification systems in the age-related build-
up of damaged proteins. Moreover, the senescence-associated
decreased activity of the oxidized protein repair enzymes Msr,
which was more important in the mitochondria, suggests that
this decline in Msr activity contribute to accumulation of oxi-
dized proteins. Finally, in contrast to the proteasome, the activity
of which is decreased in senescent fibroblasts, the mitochondrial
matrix ATP-stimulated proteolytic activity is increased in senes-
cent cells. This increased activity, which does not seem to be suf-
ficient to cope with the increased load of modified
mitochondrial proteins, can be explained, at least in part, by the
activation of the Lon protease through a mechanism that needs
to be further elucidated and that might be related to increased
oxidative events within the mitochondria.
Experimental procedures
Chemicals and reagents
IPG strips (pH: 3–10 L, 3–10 NL, 4.5–5.5, 5.5–6.7, 6–9), IPG buf-
fer (pH: 3–10 L, 3–10 NL, 4.5–5.5, 5.5–6.7, 6–11), Destreak
rehydration solution, nitrocellulose membranes, and chemilumi-
nescence kit ECL+ were purchased from GE Healthcare (Saclay,
France). Bradford protein assay reagent kit and chemicals for
SDS ⁄ PAGE were purchased from BioRad (Marnes La Coquette,
France). Protein OxyBlot kit was purchased from Millipore (Saint-
Quentin en Yvelines, France). Mitochondrial isolation kit for
mammalian cells was purchased from Perbio (Brebieres, France).
Polyclonal anti-AGE antibody has been characterized as previ-
ously described (Verbeke et al. 1997; Poggioli et al., 2002), and
it recognizes a variety of AGE products such as CML and CEL.
Polyclonal anti-HNE recognize HNE-adducts with lysine, cyste-
ine, and histidine amino acids within proteins and were raised as
originally described (Szweda et al., 2000). Anti-MsrA and anti-
aconitase antibodies have been previously described (Petropou-
los et al., 2001; Bulteau et al., 2003). Anti-vimentin V9 and 3B4
antibodies are from Abcam (Paris, France), anti-glyoxalase-I and
anti-GAPDH are from Santa-Cruz Biotechnology (Santa Cruz,
CA, USA). Human anti-Lon antibodies were raised as previously
described (Bulteau et al., 2007), while anti-ClpP antibodies were
raised against the peptide SAMERDYMSPMEAQE of the ClpP
protein. All other chemicals were of analytical grade and
obtained from Sigma–Aldrich (Saint-Quentin Fallavier, France).
Cell line and culture conditions
Human embryonic fibroblasts WI-38 were grown in Dulbecco’s
minimal essential medium (1 g L)1 glucose) supplemented with
10% fetal calf serum, 100 U mL)1 penicillin, 100 lg mL)1
streptomycin, and 2 mM L-glutamine. Cultures were kept in an
incubator at 37 �C and 5% CO2-containing atmosphere. Sub-
(A)
(B)
(C)
Fig. 10 ATP-stimulated proteolytic activity in the mitochondria of young and senescent WI-38 fibroblasts. (A) The ATP-stimulated degradation of FITC-casein
substrate was assayed as described in Experimental procedures and found to be 2.5-fold higher in senescent WI-38 mitochondrial extracts compared to young
ones. The expression level of Lon protease did not significantly change but a 20% increase was observed for ClpP in senescent WI-38 cells compared to young
ones (B). The effect of the Lon protease inhibitor MG262 at 10 lM on the total ATP-stimulated proteolytic activity was tested and a 50% & 40% inhibition for Lon
protease activity was obtained in young and senescent cells, respectively, indicating that part of the increased ATP-stimulated degradation of FITC-casein in
senescent cells is because of the two-fold activation of the Lon protease (C).
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
266
confluent cultures were obtained by seeding 5 · 105 cells ⁄ 75
cm2 culture flasks until they entered senescence after about 45
cumulative population doublings (CPD). In all experimental pro-
cedures described in the following paragraphs, early-passage
(young at CPD < 25) and late-passage (senescent at CPD > 42)
WI-38 cultures were used.
b-galactosidase staining
Staining for b-galactosidase (SA-b-gal) activity was performed as
described by Dimri et al. (1995). Briefly, WI-38 cells were
washed with PBS, fixed in 0.2% glutaraldehyde ⁄ 2% formalde-
hyde for 5 min at room temperature, and washed again with
PBS. Cells were then stained at 37 �C (in the absence of CO2)
with fresh SA-b-gal staining solution (150 mM NaCl, 2 mM
MgCl2, 5 mM potassium ferricyanide, 5 mM potassium ferrocya-
nide, and 40 mM citric acid ⁄ sodium phosphate, pH 6.0) contain-
ing 1 mg mL)1 5-bromo-4-chloro-3-indolyl-b-D-galactoside
(X-gal). Staining was maximal after 15–20 h of incubation. Four
hundred cells were counted each time, and the number of posi-
tive cells was calculated as a percentage of the whole cells.
Proteasome peptidase activities
Peptidase activities of the proteasome were assayed using fluor-
ogenic peptides, succinyl-Leu-Leu-Val-Tyr-aminomethylcouma-
rin (LLVY-AMC) for the chymotrypsin-like activity and
N-benzyloxycarbonyl-Leu-Leu-Glu-naphthylamide (LLE-NA) for
the peptidylglutamyl-peptide hydrolase activity as previously
described (Bulteau et al., 2000). The mixture containing 20 lg
of protein lysate in 25 mM Tris–HCl, pH 7.5, was incubated at
37 �C with the appropriate peptide substrate (LLVY-AMC at
12.5 lM or LLE-NA at 150 lM) in a final volume of 200 lL. Enzy-
matic kinetics were conducted in a temperature-controlled
microplate fluorimetric reader (Fluostar Galaxy, bMG, Stuttgart,
Germany). Excitation ⁄ emission wavelengths were 350 ⁄ 440 and
340 ⁄ 410 nm for aminomethylcoumarin and b-naphthylamine,
respectively. Proteasome activities were determined as the dif-
ference between total activity and the remaining activity of the
crude lysate in the presence of 20 lM of proteasome inhibitor
N-Cbz-Leu-Leu-Leucinal (MG132).
Two-dimensional gel electrophoresis
Sample preparation
Young and senescent WI-38 fibroblasts were washed once with
PBS, scrapped into 5 mL PBS, pelleted by centrifugation for
5 min at 1000 g, and then kept at )20 �C until analysis. Cell
pellets were suspended in lysis buffer (2% CHAPS, 0.1 M DTT in
50 mM Tris–HCl, pH 7.4, and complete protease inhibitor cock-
tail) and incubated 10 min over ice with occasional vortexing.
Cells were lysed by sonication for 4 · 5 s using a Branson Soni-
fier 150 followed by incubation with benzonase (500 U mL)1)
for 90 min at 4 �C with agitation. Cell lysates were obtained
after centrifugation at 15 000 g for 10 min at 4 �C and protein
concentration was determined by the method of Bradford.
Isoelectrofocusing (IEF)
For IPG strips 3–10 L and 3-10 NL (13 cm), aliquots of 150–
300 lg of proteins were diluted in 250 lL rehydration solution
(7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethyl-
ammonio]-1-propanesulfonate (CHAPS), 65 mM DTT, 2% IPG
buffer (v ⁄ v). The protein samples were loaded into the IPG
strips by in-gel rehydration at room temperature overnight. IEF
was performed using a Multiphor II Electrophoresis unit and an
EPS 3500 XL power supply (GE Healthcare, Saclay, France) at
20 �C using the following electrical profile: 3500 V, 4 h;
750 V, 0.5 h; 1500 V, 0.5 h; 2500 V, 16 h; 3500 V, 2 h (total
49.4 kVh). For IPG strips 4.5–5.5 and 5.5–6.7 (18 cm), aliquots
of 250–600 lg of proteins were diluted in 350 lL of rehydra-
tion solution and loaded into IPG strips by in-gel rehydration.
IEF was performed using the following electrical profile: 0–
50 V, 1 min; 50 V, 1 h; 50–1000 V, 3 h; 1000–3500 V, 3 h;
3500 V, 19 h (total 74.9 kVh). For IPG strips pH 6–9 (18 cm),
aliquots of 250–600 lg of proteins were diluted in 350 lL of
Destreak rehydration solution supplemented with 1% Pharma-
lyte pH 6-11 and were loaded into rehydrated IPG (Destreak
rehydration solution supplemented with 1% Pharmalyte pH 6–
11) using bridge electrode strips (Sabounchi-Schutt et al.,
2000). The following electrical profile protocol was used: 0–
1000 V, 2.5 h; 1000–3500 V, 2.5 h; 3500 V, 21 h (total
80.4 kVh). After IEF, the IPG strips were stored at –80 �C until
used in the second dimension.
SDS–PAGE
IPG strips were equilibrated for 15 min in 10 mL equilibration
buffer (6 M urea, 2% SDS, 30% glycerol, 1% DTT in 50 mM
Tris–HCl, pH 8.8) and then for 15 min in equilibration buffer
containing traces of bromophenol blue and 3% iodoacetamide
instead of DTT. The strips were placed on 12% polyacrylamide
gels according to Laemmli (1970) and electrophoresis was car-
ried out using the cooling Protean II system (BioRad, Marnes La
Coquette, France) at 95 V for 16–18 h.
Western blotting
The gels were electroblotted on a nitrocellulose membrane
for 3 h at a fixed voltage of 100 V. The blots were then
incubated for 2 h at room temperature in blocking buffer
(PBS supplemented with 1% BSA and 0.1% Tween 20).
Detection of HNE- and AGE-modified proteins was achieved
after overnight incubation at 4 �C with the rabbit polyclonal
antibody raised against HNE-modified KLH and AGE-modified
RNAse (1 ⁄ 2000 in blocking buffer). The membranes were
then washed three times in washing buffer (PBS supple-
mented with 0.1% Tween 20) and incubated with HRP-
linked secondary antibody (1 ⁄ 10 000 in blocking buffer) 1 h
at room temperature. After being washed 3 times in wash-
ing buffer, HNE- and AGE-modified proteins were finally
revealed by the chemiluminescence kit ECL+.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
267
In-strip derivatization of protein-bound carbonyls and 2D-
OxyBlot
After IEF, the IPG strips 3–10 NL were incubated at room tem-
perature in 10 mL of 10 mM DNPH ⁄ 2 M HCl for 20 min with
agitation. Strips were neutralized with 2 M Tris-base containing
30% glycerol for 10 min, and this step is repeated one time for
30 min. Equilibration of strips is made as described earlier in
SDS–PAGE. After 2D electrophoresis, the proteins are blotted
onto a nitrocellulose membrane as described earlier in western
blotting and detection of carbonyl groups was performed with
the OxyBlot oxidized protein detection kit according to the man-
ufacturer’s protocol.
Protein staining and image analysis
The 2D separated protein spots were visualized with a modified
silver staining method that is compatible with MS ⁄ MS (Merril
et al., 1981). The silver-stained gels and films were digitized
with UMAX UTA-100 scanner (GE Healthcare, Saclay, France).
The spot detection and quantification were carried out with the
IMAGE MASTER 2D software (GE Healthcare), and the data were
expressed as spot % volume in pixels.
In-gel digestion, mass spectrometry, and database
searches
Spots ⁄ bands of interest were manually excised from gels and
washed with deionized water followed by two washes with
100% acetonitrile for 15 and 2 min. The gel plugs were dehy-
drated in a vacuum centrifuge and rehydrated with a solution of
2% trypsin (Promega Inc., Madison, WI, USA) in 50 mM
NH4HCO3, at 4�C. After 20 min, the excess of trypsin solution
was removed and 30 lL of 50 mM NH4HCO3 were added and
digestion proceeded at 37�C overnight, followed by storage at
)20�C until use. Peptides desalting was performed on custom-
made reverse-phase microcolumns, prepared with R2 resin
(Perseptive Biosystems Inc., Framingham, MA, USA) as described
elsewhere (Gobom et al., 1999). Peptide solution, obtained
from digestion of each spot, was loaded onto the microcolumn,
followed by washing with 10 lL of 1% trifluoroacetic acid
(TFA). Bound peptides were eluted with 0.8 lL of matrix solu-
tion (5 lg lL)1 of a-cyano-4-hydrocynnamic acid in 70% aceto-
nitrile and 0.1% TFA) directly onto the matrix-assisted laser
desorption ionization (MALDI) target plate. Peptide mass spectra
were acquired in positive reflector mode on a 4800 Plus MALDI
TOF ⁄ TOF� Analyzer (Applied Biosystems, Foster City, CA, USA)
using 20 kV of acceleration voltage. Each spectrum was
obtained with a total of 800 laser shots and was externally cali-
brated using peptides derived by tryptic digestion of ß-lacto-
globulin. Tandem mass spectra were acquired using the same
instrument in MS ⁄ MS positive mode. From the raw data output,
peak lists were generated by Data Explorer (Applied Biosystems,
Foster City, CA, USA). MS and MS ⁄ MS peak lists were combined
into search files and used to search SwissProt databases using
the Mascot search engine (Matrix Science Ltd, London, UK).
Search parameters were as follows: Database: SwissProt 55;
Taxonomy: all entries or mammalian; Enzyme: trypsin; Allow up
to 1 missed cleavage; Fixed modifications: none; Variable modi-
fications: methionine oxidation; Peptide mass tolerance:
70 ppm; and Fragment mass tolerance: 500 ppm.
Vimentin immunoprecipitation and fractionation
Vimentin was immunoprecipitated as previously described
(Kumar et al., 2007). Young and senescent fibroblasts were
washed with phosphate-buffered saline (PBS) and pelleted by
scrapping into PBS. Cell pellets were lysed in 25 mM HEPES
buffer, pH 7.4 (containing 150 mM NaCl, 5 mM b-glycerophos-
phate, 1 mM sodium orthovanadate, 5 mM sodium pyrophos-
phate, 5 mM EDTA, 5 mM EGTA, 0.9% Triton X-100 and 0.1%
NonIdet P40, and 1 complete minitablet of protease inhibitors
per 50 mL) for 15 min at 4�C with agitation. The supernatant
was recovered after centrifugation at 16 200 g for 15 min at
4�C. For immunoprecipitation, 500 lg of the above-mentioned
supernatant protein was incubated with about 200 lg of vimen-
tin antibody [Vim 3B4] overnight at 4�C on rotating platform,
then for 2–3 h with 50 lL of protein G-sepharoseTM beads.
After centrifugation, beads were washed three times with lysis
buffer. Bound proteins were eluted with SDS sample buffer
under nonreducing conditions, resolved by SDS–PAGE, and
transferred onto nitrocellulose membranes. Nitrocellulose mem-
branes were incubated at room temperature for 1 h in blocking
buffer containing PBS with 0.1% Tween (PBS-T) and 1% bovine
serum albumin, followed by incubation with the anti-vimentin,
anti-HNE, or anti-AGE antibodies in the blocking buffer. After
being washed three times for 10 min each with PBS-T, the mem-
brane was incubated with horseradish peroxidase-linked anti-
mouse or anti-rabbit IgG antibodies followed by washing with
PBS-T. Immunoreactive bands were visualized with ECL+ detec-
tion kit. Vimentin fractionation was performed as described by
Goldman (Eriksson et al., 2004). Cell pellets were lysed for
10 min over ice in a buffer containing 1% Triton X-100,
120 mM NaCl, 10 mM sodium pyrophosphate supplied with
complete protease inhibitors. Lysates were then centrifuged for
5 min at 10 000 g, and the pellet (fraction 1) and part of the
supernatant proteins (fraction 2) were denatured by boiling in
sample loading buffer. The remaining part of the supernatant
was centrifuged at 100 000 g for 75 min, and the supernatant
proteins (fraction 3) were denatured by boiling in sample buffer.
Fraction 1 corresponds to IF polymers, fraction 2 to a mixture of
fragmented IF and solubilized subunits and fraction 3 to soluble
subunits. After resolving by SDS–PAGE and blotting onto mem-
branes, immunodetection was performed as described earlier
and bands were revealed with the ECL+ kit.
Immunofluorescence staining
Cells were rinsed with PBS and fixed with cold methanol for
10 min at room temperature. After blocking for 15 min with
5% fetal calf serum (FCS) in PBS supplemented with 0.1% Triton
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
268
X-100, cells were incubated for 1.5 h at room temperature with
primary monoclonal anti-vimentin clone V9 (1 ⁄ 800) or poly-
clonal anti-HNE (1 ⁄ 50). At the end of incubation, the cells were
rinsed five times with PBS and incubated for 1 h at dark with
1% FCS solution containing fluorescent-labeled secondary anti-
bodies (AlexaFluor 488 goat anti-rabbit IgG and sheep anti-
mouse CY3) at dilutions 1 ⁄ 600 and 1 ⁄ 800, respectively. After
several washings with PBS, fluorescence images were recorded
on a fluorescence microscope (Carl Zeiss Axioskop 40, Oberko-
chen, Germany).
Isolation of mitochondria
Young and senescent WI-38 fibroblasts cultures were washed
once with PBS, trypsinized with 0.25% trypsin (1 ⁄ 1 in PBS),
washed again in PBS, and pelleted by centrifugation. Cell pellets
were suspended in 5 volumes homogenization buffer (0.3 M
mannitol, 0.2 M EDTA, 10 mM HEPES, pH 7.4), homogenized
over ice with Dounce glass homogenizer. Whole cells, cell deb-
ris, and nuclei were removed by centrifugation at 1000 g. The
supernatant was further centrifuged at 10 000 g, and the
resulting supernatant was saved as the cytosolic fraction, while
the resulting pellets were washed once with the cold isolation
buffer and resuspended in a small volume of the buffer. Protein
concentration was assessed with the Bradford method. Mito-
chondrial and cytosolic samples were denatured with Laemmli
sample buffer under reducing conditions for 5 min at 100�C.
Proteins were then resolved on a 12% SDS ⁄ PAGE gels and elec-
trotransferred onto a Hybond nitrocellulose membrane. Nitro-
cellulose membrane-immobilized proteins were analyzed using
antibodies specific to glyoxalase-I, GAPDH, Lon, ClpP, and MsrA
at dilutions 1 ⁄ 300, 1 ⁄ 300, 1 ⁄ 400, 1 ⁄ 400, and 1 ⁄ 4000, respec-
tively. Blots were developed with ECL+ kit. Films were scanned
and the amount of signal was quantified by densitometric analy-
sis using IMAGEMASTER 1D software.
Glyoxalase-I, glutathion S-transferase, and
methionine sulfoxide reductase assays
The glyoxalase-I assay was performed by monitoring the
increase in absorbance at 240 nm because of the formation of
S-D-lactoylglutathione for 2 min at 25 �C (Oray & Norton,
1982). The standard assay mixture contained 7.9 mM methylgly-
oxal, 1 mM glutathione, 14.6 mM magnesium sulfate, and
182 mM Imidazole ⁄ HCl, pH 7. Before initiating the reaction by
adding the sample (30 lg cytosolic or 75 lg mitochondrial frac-
tion) to the assay mixture, the mixture was allowed to stand for
4 min at 25�C to ensure the equilibration of hemithioacetal for-
mation. One unit of enzyme activity is the amount of enzyme
catalyzing the formation of 1 lmole of S-D-lactoylgllutathi-
one ⁄ min. GST activity was detected by the conventional method
(Habig & Jakoby, 1981). The typical reaction mixture in a volume
of 1 mL of 100 mM phosphate buffer pH 6.5, contained 1 mM
1-chloro-2,4-dinitrobenzene (CDNB), and 1 mM reduced gluta-
thione. The reaction was initiated by addition of the sample. The
reaction was monitored by following the rate of thioether for-
mation at 340 nm. One unit of enzyme activity was defined as
one micromole of thioether formed per min knowing that the
molar extinction coefficient of CDNB is 9.6 mM)1 cm)1. Total
Msr activity was measured in cytoplasmic and mitochondrial
fractions of young and senescent WI-38 fibroblasts using N-ace-
tyl-[3H]methionine R,S sulfoxide substrate as described earlier
(Brot et al., 1982).
Measurement of ATP-stimulated proteolytic activity
Proteolytic activity was assayed as the rate of FITC-casein degra-
dation (Delaval et al., 2004; Bulteau et al., 2007). Mitochondrial
lysates were diluted to 2.5 mg mL)1 in assay buffer containing
10 mM MgCOOCH3, 2.0 mM DTT, 150 mM NaCl, 0.05% Triton
X-100, 8.0 mM ATP, and 100 mM HEPES, pH 8, in the presence
or absence of 10 lM of the Lon protease inhibitor N-Cbz-Leu-
Leu-Leu-B(OH)2 (MG262) (Frase et al., 2006). WI-38 mitochon-
dria were preincubated with MG262 for 30 min at 37 �C before
addition of FITC-casein. Proteolysis of FITC-casein (25 lg) was
then performed at 37 �C. At incubation times of 0-75 min, a
20-lL aliquot was removed, and the protein was precipitated
with 10% (w ⁄ v) TCA. The mixture was then centrifuged at
15 000 g for 30 min at 4 �C. The supernatant containing the
peptide fragments was neutralized upon addition of 500 lL of
saturated potassium borate at pH 9.2. The level of FITC-peptide
fragments was determined by spectrofluorometric analysis (exci-
tation at 495 nm; emission at 515 nm). Proteolytic activity was
linear for 75 min under the assay conditions.
Statistical analysis
All measurements were repeated a minimum of three times and
results are expressed as mean ± standard deviation (SD). Data
were tested for normality and statistical significance for the
comparison of two groups was performed using student’s t-test:
* P < 0.05; ** P < 0.01.
Acknowledgments
The authors thank Dr. D. Paulin and Dr. Z. Xue for their help and
advice for vimentin fractionation and immunofluorescence
microscopy. EKA is a research associate from Ain Shams Univer-
sity and recipient of a fellowship from the Egyptian Ministry of
Research. This work is supported by funds from MENRT and
European 6th Framework Program Grant Proteomage LSHM-
CT-518230.
Author contributions
EKA participated in the design and coordination of the study, car-
ried out most of the experiments and drafted the manuscript;
ARW participated in the design of the study and carried out all
mass spectrometry experiments; PR participated in the design
and coordination of the mass spectrometry-based proteomic
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
269
studies; ALB participated in the design and coordination of the
mitochondrial functional analyses; BF conceived the study, partic-
ipated in its design and coordination and drafted the manuscript.
References
Agarwal S, Sohal RS (1994) Aging and proteolysis of oxidized proteins.
Arch. Biochem. Biophys. 309, 24–28.
Ahmed EK, Picot CR, Bulteau AL, Friguet B (2007) Protein oxidative
modifications and replicative senescence of WI-38 human embryonic
fibroblasts. Ann. N Y Acad. Sci. 1119, 88–96.
Banan A, Fitzpatrick L, Zhang Y, Keshavarzian A (2001) OPC-com-
pounds prevent oxidant-induced carbonylation and depolymerization
of the F-actin cytoskeleton and intestinal barrier hyperpermeability.
Free Radic. Biol. Med. 30, 287–298.
Bassi AM, Ledda S, Valentini S, De Pascale MC, Rossi S, Odetti P, Cottalasso
D (2002) Damaging effects of advanced glycation end-products in the
murine macrophage cell line J774A.1. Toxicol In Vitro. 16, 339–347.
Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease,
and oxidative stress. J. Biol. Chem. 272, 20313–20316.
Bigl K, Gaunitz F, Schmitt A, Rothemund S, Schliebs R, Munch G,
Arendt T (2008) Cytotoxicity of advanced glycation endproducts in
human micro- and astroglial cell lines depends on the degree of
protein glycation. J Neural Transm. 115, 1545–1556.
Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Har-
ley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-
span by introduction of telomerase into normal human cells. Science
279, 349–352.
Bota DA, Van Remmen H, Davies KJ (2002) Modulation of Lon prote-
ase activity and aconitase turnover during aging and oxidative stress.
FEBS Lett. 532, 103–106.
Brot N, Werth J, Koster D, Weissbach H (1982) Reduction of N-acetyl
methionine sulfoxide: a simple assay for peptide methionine sulfox-
ide reductase. Anal. Biochem. 122, 291–294.
Bulteau AL, Petropoulos I, Friguet B (2000) Age-related alterations of
proteasome structure and function in aging epidermis. Exp. Geron-
tol. 35, 767–777.
Bulteau AL, Ikeda-Saito M, Szweda LI (2003) Redox-dependent modu-
lation of aconitase activity in intact mitochondria. Biochemistry 42,
14846–14855.
Bulteau AL, Dancis A, Gareil M, Montagne JJ, Camadro JM, Lesuisse E
(2007) Oxidative stress and protease dysfunction in the yeast model
of Friedreich ataxia. Free Radic. Biol. Med. 42, 1561–1570.
Byun Y, Chen F, Chang R, Trivedi M, Green KJ, Cryns VL (2001) Cas-
pase cleavage of vimentin disrupts intermediate filaments and pro-
motes apoptosis. Cell Death Differ. 8, 443–450.
Campisi J (2005) Suppressing cancer: the importance of being senes-
cent. Science 309, 886–887.
Cao Z, Hardej D, Trombetta LD, Li Y (2003) The role of chemically
induced glutathione and glutathione S-transferase in protecting
against 4-hydroxy-2-nonenal-mediated cytotoxicity in vascular
smooth muscle cells. Cardiovasc. Toxicol. 3, 165–177.
Carney JM, Starke-Reed PE, Oliver CN, Landum RW, Cheng MS, Wu
JF, Floyd RA (1991) Reversal of age-related increase in brain protein
oxidation, decrease in enzyme activity, and loss in temporal and spa-
tial memory by chronic administration of the spin-trapping com-
pound N-tert-butyl-alpha-phenylnitrone. Proc. Natl Acad. Sci. USA
88, 3633–3636.
Carrard G, Dieu M, Raes M, Toussaint O, Friguet B (2003) Impact of
ageing on proteasome structure and function in human lympho-
cytes. Int. J. Biochem. Cell Biol. 35, 728–739.
Chao CC, Ma YS, Stadtman ER (1997) Modification of protein surface
hydrophobicity and methionine oxidation by oxidative systems. Proc.
Natl Acad. Sci. USA 94, 2969–2974.
Chen J, Schenker S, Frosto TA, Henderson GI (1998) Inhibition of cyto-
chrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE
adduct formation with the enzyme subunits. Biochim. Biophys. Acta
1380, 336–344.
Chen J, Henderson GI, Freeman GL (2001) Role of 4-hydroxynonenal
in modification of cytochrome c oxidase in ischemia ⁄ reperfused rat
heart. J. Mol. Cell. Cardiol. 33, 1919–1927.
Choksi KB, Nuss JE, Boylston WH, Rabek JP, Papaconstantinou J
(2007) Age-related increases in oxidatively damaged proteins of
mouse kidney mitochondrial electron transport chain complexes.
Free Radic. Biol. Med. 43, 1423–1438.
Chondrogianni N, Stratford FL, Trougakos IP, Friguet B, Rivett AJ, Go-
nos ES (2003) Central role of the proteasome in senescence and
survival of human fibroblasts: induction of a senescence-like pheno-
type upon its inhibition and resistance to stress upon its activation.
J. Biol. Chem. 278, 28026–28037.
Colvis C, Garland D (2002) Posttranslational modification of human
alphaA-crystallin: correlation with electrophoretic migration. Arch.
Biochem. Biophys. 397, 319–323.
Dalle-Donne I, Rossi R, Milzani A, Di Simplicio P, Colombo R (2001)
The actin cytoskeleton response to oxidants: from small heat shock
protein phosphorylation to changes in the redox state of actin itself.
Free Radic. Biol. Med. 31, 1624–1632.
Dalle-Donne I, Giustarini D, Colombo R, Rossi R, Milzani A (2003) Pro-
tein carbonylation in human diseases. Trends Mol Med. 9, 169–176.
Delaval E, Perichon M, Friguet B (2004) Age-related impairment of
mitochondrial matrix aconitase and ATP-stimulated protease in rat
liver and heart. Eur. J. Biochem. 271, 4559–4564.
Dierick JF, Kalume DE, Wenders F, Salmon M, Dieu M, Raes M, Roe-
pstorff P, Toussaint O (2002) Identification of 30 protein species
involved in replicative senescence and stress-induced premature
senescence. FEBS Lett. 531, 499–504.
Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano
EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke M, Campisi J
(1995) A biomarker that identifies senescent human cells in culture
and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92, 9363–9367.
Eriksson JE, He T, Trejo-Skalli AV, Harmala-Brasken AS, Hellman J,
Chou YH, Goldman RD (2004) Specific in vivo phosphorylation sites
determine the assembly dynamics of vimentin intermediate fila-
ments. J. Cell Sci. 117, 919–932.
Esterbauer H, Zollner H, Lang J (1985) Metabolism of the lipid peroxi-
dation product 4-hydroxynonenal by isolated hepatocytes and by
liver cytosolic fractions. Biochem. J. 228, 363–373.
Farout L, Mary J, Vinh J, Szweda LI, Friguet B (2006) Inactivation of
the proteasome by 4-hydroxy-2-nonenal is site specific and depen-
dant on 20S proteasome subtypes. Arch. Biochem. Biophys. 453,
135–142.
Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology
of ageing. Nature 408, 239–247.
Frase H, Hudak J, Lee I (2006) Identification of the proteasome inhibitor
MG262 as a potent ATP-dependent inhibitor of the Salmonella enterica
serovar Typhimurium Lon protease. Biochemistry 45, 8264–8274.
Friguet B (2006) Oxidized protein degradation and repair in ageing
and oxidative stress. FEBS Lett. 580, 2910–2916.
Gobom J, Nordhoff E, Mirgorodskaya E, Ekman R, Roepstorff P (1999)
Sample purification and preparation technique based on nano-scale
reversed-phase columns for the sensitive analysis of complex peptide
mixtures by matrix-assisted laser desorption ⁄ ionization mass spec-
trometry. J. Mass Spectrom. 34, 105–116.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
270
Gonzalez-Dosal R, Sorensen MD, Clark BF, Rattan SI, Kristensen P
(2006) Phage-displayed antibodies for the detection of glycated pro-
teasome in aging cells. Ann. N Y Acad. Sci. 1067, 474–478.
Habig WH, Jakoby WB (1981) Assays for differentiation of glutathione
S-transferases. Methods Enzymol. 77, 398–405.
Hayflick L (1965) The Limited In Vitro Lifetime Of Human Diploid Cell
Strains. Exp. Cell Res. 37, 614–636.
Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid
cell strains. Exp. Cell Res. 25, 585–621.
Heinecke JW, Li W, Daehnke HL 3rd, Goldstein JA (1993) Dityrosine, a
specific marker of oxidation, is synthesized by the myeloperoxidase-
hydrogen peroxide system of human neutrophils and macrophages.
J. Biol. Chem. 268, 4069–4077.
Hirao T, Takahashi M (2005) Carbonylation of cornified envelopes in
the stratum corneum. FEBS Lett. 579, 6870–6874.
Hwang ES, Yoon G, Kang HT (2009) A comparative analysis of the cell
biology of senescence and aging. Cell. Mol. Life Sci. 66, 2503–
2524.
Ishii T, Sakurai T, Usami H, Uchida K (2005) Oxidative modification of
proteasome: identification of an oxidation-sensitive subunit in 26 S
proteasome. Biochemistry 44, 13893–13901.
Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar V
(2004) Modification of Cytochrome c by 4-hydroxy- 2-nonenal: evi-
dence for histidine, lysine, and arginine-aldehyde adducts. J Am Soc
Mass Spectrom. 15, 1136–1147.
Jacobs HT (2003) The mitochondrial theory of aging: dead or alive?
Aging Cell 2, 11–17.
Jeanmaire C, Danoux L, Pauly G (2001) Glycation during human der-
mal intrinsic and actinic ageing: an in vivo and in vitro model study.
Br. J. Dermatol. 145, 10–18.
Kaplan P, Tatarkova Z, Racay P, Lehotsky J, Pavlikova M, Dobrota D
(2007) Oxidative modifications of cardiac mitochondria and inhibi-
tion of cytochrome c oxidase activity by 4-hydroxynonenal. Redox
Rep. 12, 211–218.
Kapphahn RJ, Giwa BM, Berg KM, Roehrich H, Feng X, Olsen TW, Fer-
rington DA (2006) Retinal proteins modified by 4-hydroxynonenal:
identification of molecular targets. Exp. Eye Res. 83, 165–175.
Kaser M, Langer T (2000) Protein degradation in mitochondria. Semin.
Cell Dev. Biol. 11, 181–190.
Kueper T, Grune T, Prahl S, Lenz H, Welge V, Biernoth T, Vogt Y,
Muhr GM, Gaemlich A, Jung T, Boemke G, Elsasser HP, Wittern KP,
Wenck H, Stab F, Blatt T (2007) Vimentin is the specific target in
skin glycation. Structural prerequisites, functional consequences,
and role in skin aging. J. Biol. Chem. 282, 23427–23436.
Kumar N, Robidoux J, Daniel KW, Guzman G, Floering LM, Collins S
(2007) Requirement of vimentin filament assembly for beta3-adren-
ergic receptor activation of ERK MAP kinase and lipolysis. J. Biol.
Chem. 282, 9244–9250.
Laemmli UK (1970) Cleavage of structural proteins during the assem-
bly of the head of bacteriophage T4. Nature 227, 680–685.
Lashin OM, Szweda PA, Szweda LI, Romani AM (2006) Decreased
complex II respiration and HNE-modified SDH subunit in diabetic
heart. Free Radic. Biol. Med. 40, 886–896.
Levine RL (2002) Carbonyl modified proteins in cellular regulation,
aging, and disease. Free Radic. Biol. Med. 32, 790–796.
Magi B, Ettorre A, Liberatori S, Bini L, Andreassi M, Frosali S, Neri P,
Pallini V, Di Stefano A (2004) Selectivity of protein carbonylation in
the apoptotic response to oxidative stress associated with photody-
namic therapy: a cell biochemical and proteomic investigation. Cell
Death Differ. 11, 842–852.
Massey AC, Kiffin R, Cuervo AM (2006) Autophagic defects in aging:
looking for an ‘‘emergency exit’’? Cell Cycle 5, 1292–1296.
Mattson MP, Fu W, Waeg G, Uchida K (1997) 4-Hydroxynonenal, a
product of lipid peroxidation, inhibits dephosphorylation of the
microtubule-associated protein tau. Neuroreport 8, 2275–2281.
Melk A, Kittikowit W, Sandhu I, Halloran KM, Grimm P, Schmidt BM,
Halloran PF (2003) Cell senescence in rat kidneys in vivo increases
with growth and age despite lack of telomere shortening. Kidney
Int. 63, 2134–2143.
Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive
stain for proteins in polyacrylamide gels shows regional variation in
cerebrospinal fluid proteins. Science 211, 1437–1438.
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I
(2002) Endothelial cell senescence in human atherosclerosis: role of
telomere in endothelial dysfunction. Circulation 105, 1541–1544.
Montine TJ, Amarnath V, Martin ME, Strittmatter WJ, Graham DG
(1996) E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskele-
tal proteins in P19 neuroglial cultures. Am. J. Pathol. 148, 89–93.
Morcos M, Du X, Pfisterer F, Hutter H, Sayed AA, Thornalley P, Ahmed
N, Baynes J, Thorpe S, Kukudov G, Schlotterer A, Bozorgmehr F, El
Baki RA, Stern D, Moehrlen F, Ibrahim Y, Oikonomou D, Hamann A,
Becker C, Zeier M, Schwenger V, Miftari N, Humpert P, Hammes HP,
Buechler M, Bierhaus A, Brownlee M, Nawroth PP (2008) Glyoxalase-
1 prevents mitochondrial protein modification and enhances lifespan
in Caenorhabditis elegans. Aging Cell 7, 260–269.
Morimoto RI, Cuervo AM (2009) Protein homeostasis and aging: tak-
ing care of proteins from the cradle to the grave. J. Gerontol. A
Biol. Sci. Med. Sci. 64, 167–170.
Moskovitz J, Poston JM, Berlett BS, Nosworthy NJ, Szczepanowski R,
Stadtman ER (2000) Identification and characterization of a putative
active site for peptide methionine sulfoxide reductase (MsrA) and its
substrate stereospecificity. J. Biol. Chem. 275, 14167–14172.
Neely MD, Sidell KR, Graham DG, Montine TJ (1999) The lipid peroxi-
dation product 4-hydroxynonenal inhibits neurite outgrowth, dis-
rupts neuronal microtubules, and modifies cellular tubulin. J.
Neurochem. 72, 2323–2333.
Ngo JK, Davies KJ (2007) Importance of the lon protease in mitochon-
drial maintenance and the significance of declining lon in aging.
Ann. N Y Acad. Sci. 1119, 78–87.
Nishio K, Inoue A, Qiao S, Kondo H, Mimura A (2001) Senescence and
cytoskeleton: overproduction of vimentin induces senescent-like mor-
phology in human fibroblasts. Histochem. Cell Biol. 116, 321–327.
Oray B, Norton SJ (1982) Glyoxalase I from mouse liver. Methods Enz-
ymol. 90 Pt E, 542–546.
Passos JF, Von Zglinicki T (2006) Oxygen free radicals in cell senes-
cence: are they signal transducers? Free Radic. Res. 40, 1277–1283.
Perides G, Kuhn S, Scherbarth A, Traub P (1987) Probing of the struc-
tural stability of vimentin and desmin-type intermediate filaments
with Ca2 + -activated proteinase, thrombin and lysine-specific endo-
proteinase Lys-C. Eur. J. Cell Biol. 43, 450–458.
Petropoulos I, Friguet B (2005) Protein maintenance in aging and repli-
cative senescence: a role for the peptide methionine sulfoxide
reductases. Biochim. Biophys. Acta 1703, 261–266.
Petropoulos I, Friguet B (2006) Maintenance of proteins and aging:
the role of oxidized protein repair. Free Radic. Res. 40, 1269–
1276.
Petropoulos I, Conconi M, Wang X, Hoenel B, Bregegere F, Milner Y,
Friguet B (2000) Increase of oxidatively modified protein is associ-
ated with a decrease of proteasome activity and content in aging
epidermal cells. J. Gerontol. A Biol. Sci. Med. Sci. 55, B220–B227.
Petropoulos I, Mary J, Perichon M, Friguet B (2001) Rat peptide methi-
onine sulphoxide reductase: cloning of the cDNA, and down-regula-
tion of gene expression and enzyme activity during aging. Biochem.
J. 355, 819–825.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
271
Picklo MJ, Amarnath V, McIntyre JO, Graham DG, Montine TJ (1999)
4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at
multiple sites. J. Neurochem. 72, 1617–1624.
Picot CR, Perichon M, Cintrat JC, Friguet B, Petropoulos I (2004) The
peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1),
are downregulated during replicative senescence of human WI-38
fibroblasts. FEBS Lett. 558, 74–78.
Poggioli S, Bakala H, Friguet B (2002) Age-related increase of protein
glycation in peripheral blood lymphocytes is restricted to preferential
target proteins. Exp. Gerontol. 37, 1207–1215.
Prahl S, Kueper T, Biernoth T, Wohrmann Y, Munster A, Furstenau M,
Schmidt M, Schulze C, Wittern KP, Wenck H, Muhr GM, Blatt T
(2008) Aging skin is functionally anaerobic: importance of coenzyme
Q10 for anti aging skin care. Biofactors 32, 245–255.
Reddy VP, Beyaz A (2006) Inhibitors of the Maillard reaction and AGE
breakers as therapeutics for multiple diseases. Drug Discov Today
11, 646–654.
Rohme D (1981) Evidence for a relationship between longevity of
mammalian species and life spans of normal fibroblasts in vitro
and erythrocytes in vivo. Proc. Natl Acad. Sci. USA 78, 5009–
5013.
Rosca MG, Monnier VM, Szweda LI, Weiss MF (2002) Alterations in
renal mitochondrial respiration in response to the reactive
oxoaldehyde methylglyoxal. Am J Physiol Renal Physiol. 283, F52–
F59.
Sabounchi-Schutt F, Astrom J, Olsson I, Eklund A, Grunewald J, Bjellq-
vist B (2000) An immobiline DryStrip application method enabling
high-capacity two-dimensional gel electrophoresis. Electrophoresis
21, 3649–3656.
Schwartz AG, Pashko LL (2004) Dehydroepiandrosterone, glucose-6-
phosphate dehydrogenase, and longevity. Ageing Res Rev. 3, 171–187.
Serrano M, Blasco MA (2001) Putting the stress on senescence. Curr.
Opin. Cell Biol. 13, 748–753.
Sitte N, Merker K, von Zglinicki T, Grune T (2000) Protein oxidation
and degradation during proliferative senescence of human MRC-5
fibroblasts. Free Radic. Biol. Med. 28, 701–708.
Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd
RA, Markesbery WR (1991) Excess brain protein oxidation and
enzyme dysfunction in normal aging and in Alzheimer disease. Proc.
Natl Acad. Sci. USA 88, 10540–10543.
Spitz DR, Sullivan SJ, Malcolm RR, Roberts RJ (1991) Glutathione
dependent metabolism and detoxification of 4-hydroxy-2-nonenal.
Free Radic. Biol. Med. 11, 415–423.
Stadtman ER (1992) Protein oxidation and aging. Science 257, 1220–
1224.
Stadtman ER (2002) Importance of individuality in oxidative stress and
aging. Free Radic. Biol. Med. 33, 597–604.
Szweda LI, Szweda PA, Holian A (2000) Detection of 4-hydroxy-2-
nonenol adducts following lipid peroxidation from ozone exposure.
Methods Enzymol. 319, 562–570.
Thornalley PJ (2003) Glyoxalase I – structure, function and a critical
role in the enzymatic defence against glycation. Biochem. Soc.
Trans. 31, 1343–1348.
Thornalley PJ, Battah S, Ahmed N, Karachalias N, Agalou S, Babaei-
Jadidi R, Dawnay A (2003) Quantitative screening of advanced gly-
cation endproducts in cellular and extracellular proteins by tandem
mass spectrometry. Biochem. J. 375, 581–592.
Tolstonog GV, Shoeman RL, Traub U, Traub P (2001) Role of the inter-
mediate filament protein vimentin in delaying senescence and in the
spontaneous immortalization of mouse embryo fibroblasts. DNA Cell
Biol. 20, 509–529.
Toussaint O, Royer V, Salmon M, Remacle J (2002) Stress-induced pre-
mature senescence and tissue ageing. Biochem. Pharmacol. 64,
1007–1009.
Tsuji T, Aoshiba K, Nagai A (2006) Alveolar cell senescence in patients
with pulmonary emphysema. Am. J. Respir. Crit. Care Med. 174,
886–893.
Uchida K (2003) 4-Hydroxy-2-nonenal: a product and mediator of oxi-
dative stress. Prog. Lipid Res. 42, 318–343.
Ueda Y, Duncan MK, David LL (2002) Lens proteomics: the accumula-
tion of crystallin modifications in the mouse lens with age. Invest.
Ophthalmol. Vis. Sci. 43, 205–215.
Unterluggauer H, Mazurek S, Lener B, Hutter E, Eigenbrodt E,
Zwerschke W, Jansen-Durr P (2008) Premature senescence of
human endothelial cells induced by inhibition of glutaminase. Bio-
gerontology 9, 247–259.
Unterluggauer H, Micutkova L, Lindner H, Sarg B, Hernebring M,
Nystrom T, Jansen-Durr P (2009) Identification of Hsc70 as target
for AGE modification in senescent human fibroblasts. Biogerontolo-
gy 10, 299–309.
Van Dyck L, Langer T (1999) ATP-dependent proteases controlling
mitochondrial function in the yeast Saccharomyces cerevisiae. Cell.
Mol. Life Sci. 56, 825–842.
VanWinkle WB, Snuggs M, Miller JC, Buja LM (1994) Cytoskeletal
alterations in cultured cardiomyocytes following exposure to the
lipid peroxidation product, 4-hydroxynonenal. Cell Motil. Cytoskele-
ton 28, 119–134.
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 Cyto-
chem 45, 1045–1068.
Wang E (1985) Are cross-bridging structures involved in the bundle
formation of intermediate filaments and the decrease in locomotion
that accompany cell aging? J. Cell Biol. 100, 1466–1473.
Wang C, Jurk D, Maddick M, Nelson G, Martin-Ruiz C, von Zglinicki T
(2009) DNA damage response and cellular senescence in tissues of
aging mice. Aging Cell 8, 311–323.
Weissbach H, Etienne F, Hoshi T, Heinemann SH, Lowther WT, Mat-
thews B, St John G, Nathan C, Brot N (2002) Peptide methionine
sulfoxide reductase: structure, mechanism of action, and biological
function. Arch. Biochem. Biophys. 397, 172–178.
Westwood ME, Argirov OK, Abordo EA, Thornalley PJ (1997) Methyl-
glyoxal-modified arginine residues – a signal for receptor-mediated
endocytosis and degradation of proteins by monocytic THP-1 cells.
Biochim. Biophys. Acta 1356, 84–94.
Winger AM, Taylor NL, Heazlewood JL, Day DA, Millar AH (2007) The
Cytotoxic lipid peroxidation product 4-hydroxy-2-nonenal covalently
modifies a selective range of proteins linked to respiratory function
in plant mitochondria. J. Biol. Chem. 282, 37436–37447.
Yan LJ, Sohal RS (1998) Mitochondrial adenine nucleotide translocase
is modified oxidatively during aging. Proc. Natl Acad. Sci. USA 95,
12896–12901.
Yan LJ, Levine RL, Sohal RS (1997) Oxidative damage during aging tar-
gets mitochondrial aconitase. Proc. Natl Acad. Sci. USA 94, 11168–
11172.
Yudoh K, Nguyen T, Nakamura H, Hongo-Masuko K, Kato T, Nishioka
K (2005) Potential involvement of oxidative stress in cartilage senes-
cence and development of osteoarthritis: oxidative stress induces
chondrocyte telomere instability and downregulation of chondrocyte
function. Arthritis Res Ther. 7, R380–R391.
Zimniak P (2008) Detoxification reactions: relevance to aging. Ageing
Res Rev. 7, 281–300.
Protein modification in senescent WI-38 fibroblasts, E. K. Ahmed et al.
ª 2010 The AuthorsJournal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2010
272
Top Related