Protein modification and replicative senescence of WI38 human embryonic fibroblasts

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.



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


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.



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.



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


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.



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.



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.



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.



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



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.



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

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ensi

on

pH

gra

die

nt

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for

the

2D

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(j)RM

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repre

sents

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odifi

cation

Index

Rat

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)m

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was

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det

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din

senes

cent

sam

ple

s.



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).



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).



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).



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+.



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



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



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.

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