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Mechanisms of Ageing and Development xxx (2014) xxx–xxx

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Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver

Elena Bellavista a,b,*, Morena Martucci b, Francesco Vasuri c, Aurelia Santoro b,Michele Mishto d,e, Alexander Kloss d, Elisa Capizzi c, Alessio Degiovanni c, Catia Lanzarini b,Daniel Remondini a,f, Alessandro Dazzi g, Sara Pellegrini g, Matteo Cescon g,Miriam Capri a,b, Stefano Salvioli a,b, Antonia D’Errico-Grigioni c, Burkhardt Dahlmann d,Gian Luca Grazi h, Claudio Franceschi a,b,i,j,k

a Interdepartmental Centre ‘‘L. Galvani’’ for Integrated Studies on Biophysics, Bioinformatics and Biocomplexity (CIG), Alma Mater Studiorum, University of

Bologna, 40126 Bologna, Italyb Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Alma Mater Studiorum, University of Bologna, 40126 Bologna, Italyc ‘‘F. Addarii’’ Institute of Oncology and Transplant Pathology at Department of Experimental, Diagnostic and Specialty Medicine (DIMES), S. Orsola-Malpighi

Hospital, 40138 Bologna, Italyd Institute of Biochemistry, Charite Universitaetsmedizin Berlin, 10117 Berlin, Germanye Centro Interdipartimentale di Ricerca sul Cancro ‘‘Giorgio Prodi’’ (CIRC), University of Bologna, 40126 Bologna, Italyf Department of Physics and Astronomy (DIFA) and INFN Sez. Bologna, Alma Mater Studiorum, University of Bologna, 40127 Bologna, Italyg Department of General Surgery and Organ Transplantation, S. Orsola-Malpighi Hospital, 40138 Bologna, Italyh Istituto Nazionale Tumori Regina Elena, 00144 Roma, Italyi IRCCS Institute of Neurological Sciences, 40139 Bologna, Italyj National Research Council of Italy, CNR, Institute for Organic Synthesis and Photoreactivity (ISOF), 40129 Bologna, Italyk National Research Council of Italy, CNR, Institute of Molecular Genetics, Unit of Bologna IOR, 40136 Italy

A R T I C L E I N F O

Article history:

Received 12 June 2014

Received in revised form 18 September 2014

Accepted 19 September 2014

Available online xxx

Keywords:

Aging

Human liver

Standard proteasome

Immunoproteasome

PA28ab regulator

Gender

A B S T R A C T

Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function

and duration of such transplanted livers are apparently comparable to those from young donors,

suggesting that, despite some morphological and structural age-related changes, no major functional

changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on

proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating

donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three

major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of

proteasomes composition showed an age-related increased of b5i/a4 ratio, suggesting a shift toward

proteasomes containing inducible subunits and a decreased content of PA28a subunit, mainly in the

cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from

aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect

the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are

emerging and they deserve further investigation owing to the different aging trajectories between man

and women. Finally, our data support the safe use of livers from old donors for transplantation.

� 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-

ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

* Corresponding author at: Interdepartmental Centre ‘‘L. Galvani’’ and Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum, University

of Bologna, 40126 Bologna, Italy. Tel.: +39 051 2094757; fax: +39 051 209474.

E-mail addresses: elena.bellavista2@unibo.it (E. Bellavista), morena.martucci3@unibo.it (M. Martucci), francesco.vasuri@aosp.bo.it (F. Vasuri), aurelia.santoro@unibo.it

Contents lists available at ScienceDirect

Mechanisms of Ageing and Development

jo ur n al ho mep ag e: www .e lsev ier . c om / lo cate /m ec hag ed ev

(A. Santoro), michele.mishto@charite.de (M. Mishto), alex.kloss@charite.de (A. Kloss), elisa.capizzi@libero.it (E. Capizzi), alessio.degiovanni@gmail.com (A. Degiovanni),

catia.lanzarini2@unibo.it (C. Lanzarini), daniel.remondini@unibo.it (D. Remondini), alled78@yahoo.it (A. Dazzi), sarapellegrini@hotmail.com (S. Pellegrini),

matteo.cescon@aosp.bo.it (M. Cescon), miriam.capri@unibo.it (M. Capri), stefano.salvioli@unibo.it (S. Salvioli), derrico@aosp.bo.it (A. D’Errico-Grigioni),

burkhardt.dahlmann@charite.de (B. Dahlmann), grazi@ifo.it (G.L. Grazi), claudio.franceschi@unibo.it (C. Franceschi).

Please cite this article in press as: Bellavista, E., et al., Lifelong maintenance of composition, function and cellular/subcellulardistribution of proteasomes in human liver. Mech. Ageing Dev. (2014), http://dx.doi.org/10.1016/j.mad.2014.09.003

http://dx.doi.org/10.1016/j.mad.2014.09.003

0047-6374/� 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-

nd/3.0/).

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E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx2

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Introduction

Liver is a vital organ supporting a multitude of differentnctions and plays a central role in human physiology. Studies indents and humans indicate the occurrence of some structuralanges in liver morphology and a reduced regenerative capacityring aging, but liver function tests failed to identify significante-related deficits (Schmucker and Sanchez, 2011). Additional-, orthotopic liver transplants where the age of the donor isnsiderably higher than that of the recipients have notablycreased in the last years, and clinical data indicate that liveraft from aged subjects has, in specific conditions, function andration comparable to those achievable with liver grafts fromung donors (Cescon et al., 2003, 2008; Schmeding et al., 2010).is evidence seems to indicate for the liver a peculiar agingttern where the biological age does not correspond to theronological age of the organ/donor, and indirectly confirms itspacity to rejuvenate when in presence of the appropriateicroenvironment (Conboy et al., 2005). The molecular mecha-sms involved in this peculiar aging pattern are still largelyknown but efficient protein degradation machineries, respon-

ble for the maintenance of cellular homeostasis, might play avotal role (Chondrogianni and Gonos, 2010). One of the mostportant intracellular protease is represented by proteasomes,e central catalytic units of the ubiquitin-proteasome systemPS) (Hershko and Ciechanover, 1998), which, because of itstal functions, has become a therapeutic target for differentseases (Bellavista et al., 2013). 20S standard proteasome (s-oteasome) is a cylinder-shaped multicatalytic complex that ismposed of four stacked rings, each consisting of seven proteinbunits. The two inner rings contain b subunits (b1–b7), three

which (b1, b2, b5) harbor the proteolytic active sitestalyzing, by their N-terminal threonine residues, a caspase-e (C-like), trypsin-like (T-like) and chymotrypsin-like (CT-like)tivity, respectively. The a subunits (a1–a7), which form theo outer rings, have other functions such as gating the centralamber and binding regulator complexes like PA700, PA28 and200 thereby leading to formation of multiple forms ofoteasomes like 30S (19S-20S-19S) proteasomes, single anduble PA28-capped proteasomes as well as hybrid proteasomesA28-20S-19S) (Hershko and Ciechanover, 1998). In presence ofo-inflammatory stimuli, such as gamma-interferon (IFN-g),mor necrosis factor alpha (TNF-a) or lipopolysaccharide (LPS),e three catalytic subunits of s-proteasome b1, b2, b5 areplaced by the homologous immunosubunits b1i (or LMP2), b2ir MECL-1) and b5i (or LMP7) in the neo-synthesized 20Smplex named immunoproteasome (i-proteasome). This iso-rm is constitutively expressed in organs and cells related to themune system functions, as well as in a wide array of normalsues, such as colon, retina and also liver (Ebstein et al., 2012;rrington and Gregerson, 2012 ; Gohlke et al., in press; Vasuri

al., 2010). At present, despite the central role of proteasomes inysiological as well as pathological conditions, no data areailable on their age-related modifications in human liver andntroversial results emerged in rodents. Indeed, in livers of agedice and rats all or some of proteasome activities measured byort fluorogenic peptides have been found to decrease (Breusing

al., 2009; Conconi et al., 1996; Dasuri et al., 2009; Hayashi andto, 1998; Rodriguez et al., 2010, 2012; Shibatani et al., 1996)

temporarily decrease before increasing again (Keller et al.,

whether aging impinges upon proteasome functionality, compo-sition and subcellular distribution.

2. Material and methods

2.1. Reagents

Suc-Leu-Leu-Val-Tyr-MCA (3120-v), Z-Leu-Leu-Glu-MCA (3179-v) and free MCA

(3099-v) fluorogenic substrates were from Peptide Institute INC (Japan), Bz-Val-

Gly-Arg-MCA (BW 9375) was from Biomol (Enzo Life Sciences Inc, Farmingdale,

New York, US). Proteasome inhibitor epoxomicyn (E3652) was purchased from

Sigma (Saint Louis, Missouri, US). SynergyTM fluorimeter (Bio-Tek Instruments,

Winooski, Vermount, US) was used to detect MCA fluorescence. Mouse monoclonal

and rabbit polyclonal antibodies anti-a4 (PW8120, clone MCP34), anti-b1

(PW8140, clone MCP421), anti b2 (PW9300, clone MCP165), anti-b1i (PW8840,

clone LMP2-13), anti-b5i (PW8845, clone LMP7-1), anti-b5 (PW8895), anti-b2i

(PW8200), anti-PA28a (PW8185), anti-Rpt2 (PW8305) proteasome subunits as

well as the antibody against mono and polyubiquitin conjugates (PW8810, clone

FK2) were from Biomol (Enzo Life Sciences Inc, Farmingdale, New York, US).

OxyblotTM protein oxidation detection kit (S7150) for analysis of oxidized proteins

was from Chemicon International from Pierce (#78833; Thermo Fisher Scientific,

Walthman, MA). All the apparatus and reagents for SDS-PAGE and western blotting

(WB), anti-mouse and -rabbit horseradish peroxidase-conjugated secondary

antibodies and the QuantityOne software were from Bio-Rad (Hercules, CA,

USA). ECL reagent (sc-2048, Santa Cruz Biotechnology Inc, Santa Cruz, CA, US),

Hyperfilm ECL sheets (GEH28906837, GE Healthcare, Chalfont St. Giles, UK) and

Development and Fixer processing (Kodak P7042 and P7167, Sigma) were used for

the detection of immunoblot staining. For immunohistochemistry, EnVision Plus

System HRP Labelled polymer (anti-mouse, K4003; anti-rabbit K4001) and 30 , 30

Diaminobenzidine Substrate Chromogen System (K3468) from Dako (Glostrup,

Denmark) were used for staining reaction.

2.2. Human liver samples

The study has been carried out in accordance with Declaration of Helsinki and

approved by the local Ethical Committee at S. Orsola-Malpighi Hospital (2008,

June 26 th; code: 44/2008/O/Tess). Liver biopsies were obtained in the framework

of the protocol of donor-recipient allogeneic liver graft (orthotopic liver

transplantations) performed at General Surgery and Transplant Unit (S. Orsola-

Malpighi Hospital, Bologna, Italy) between 2008 and 2010. Donor death causes

were the following: (i) 66% of spontaneous vascular failure; (ii) 21% of trauma;

(iii) 13% of different causes such as post-anoxic encephalopathy and meningitis.

Biopsies were obtained from heart-beating brain death donors, just after midline

incision and thus before aorta cross clamping and flushing. All liver grafts were

used for transplantation and none of them was discarded as non-suitable. No

donor organs were obtained from executed prisoners or other institutionalized

persons.

Fifty-six samples were used in the present studies; age and gender distribution of

the donors are summarized in Table 1. The biopsies were subdivided according to

the age of the donor in three groups (age group 1: �40 years; age group 2: 41–70

years; age group 3: >70 years), on the basis of previous literature (Vo et al., 2011).

2.3. Histology and immunohistochemistry

Liver biopsies were collected in 54 cases for routine evaluation of graft

suitability for transplantation on frozen sections, following the pre-transplant

protocol for transplant safety (Fiorentino et al., 2009). In 2 cases no liver biopsy

was performed. After evaluation biopsies were formalin fixed, paraffin embedded

and routinely processed. Two-mm-thick slides were cut and stained with

Haematoxylin-Eosin. The following histological parameters were collected:

occurrence of portal and/or lobular inflammation, fibrosis (according to Ishak

et al., 1995), arteriolar myointimal thickening, biliocyte regression, occurrence

and amount of cholestasis, hepatocyte polymorphism, sinusoid dilatation,

occurrence and percentage of steatosis (both micro- and macrovesicular),

lipofuscin accumulation.

Immunohistochemistry (IHC) for antibodies against b1, b1i, b5i, and PA28a was

performed in the 54 cases with routine biopsy available as previously described

Table 1Age and gender distribution of the liver biopsies collected in the context of donor-

recipient orthotopic liver transplantation and analyzed in the study. M = male

donors; F = female donors; SD = standard deviation.

Age group 1:

<40 years

Age group 2:

41–70 years

Age group 3:

>70 years

Mean age � SD (years) 24.1 � 6.3 56.5 � 9.1 77.6 � 4.7

Range (years) 16–37 43–70 72–87

Number of biopsies N = 9 N = 31 N = 16

Donor gender (M = 9, F = 0) (M = 14, F = 17) (M = 10, F = 6)

00; Petersen et al., 2010) or be preserved (Ding et al., 2006;ber et al., 2009). Very recently, a shift toward proteasomes

ntaining immunosubunits in aged rat liver has been describedohlke et al., 2014a,b). In this scenario and in the context of theotocol donor-recipient allograft we extended our previousservations in human liver (Vasuri et al., 2010) by investigating

Please cite this article in press as: Bellavista, E., et al., Lifelong maintenance of composition, function and cellular/subcellulardistribution of proteasomes in human liver. Mech. Ageing Dev. (2014), http://dx.doi.org/10.1016/j.mad.2014.09.003

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E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx 3

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(Mishto et al., 2011; Vasuri et al., 2010). Nuclear and cytoplasmic intensity of

positivity was semi-quantitatively assessed in 0 (absent), 1+ (mild/average) and 2+

(strong), as previously described (Vasuri et al., 2010).

2.4. Protein crude extract preparation

Protein crude extracts were prepared as previously described (Schmidt et al.,

2006; Vasuri et al., 2010). Fifty milligrams of frozen tissue were lysed in TEAD buffer

(Tris–HCl 20 mM pH 7.5, EDTA 1 mM, NaN3 1 mM, DTT 1 mM), homogenized using

a motor-driven homogenizer and centrifuged at 25,000 � g for 1 h at 4 8C. The

supernatant containing the soluble proteins (crude extract) was collected,

quantified by Bradford’s method and proteasomes activities were measured (see

below); the remaining material was stored at �80 8C.

2.5. Proteasome purification

Purification of proteasomes was performed similar as described elsewhere

(Schmidt et al., 2006). Briefly, two pools of liver specimens from middle age (n = 4;

mean age 48 � 4.5 years) and old (n = 3; mean age 85 � 3.5 years) male donors were

homogenized in TEAD buffer, as above described and the undissolved material pelleted

by centrifugation for 20 min at 15,000 � g and 4 8C. Proteins in the supernatant fraction

were loaded onto a DEAE-Toyopearl column equilibrated with TEAD buffer and bound

proteins then eluted with a linear gradient of 0–0.5 M NaCl/TEAD buffer. Proteasomes

containing fractions were pooled and concentrated by adding (NH4)2SO4 to obtain a

final concentration of 75% (w/v) with respect to the salt. Proteins were spun down and

then dissolved in TEAD buffer before loaded onto a Superose 6 gel filtration column for

chromatography in TEAD buffer in a FPLC System (GE Healthcare). Fractions containing

20S proteasome were pooled and further purified by subsequent chromatographies on

Mono Q and Phenyl-Superose. Finally, proteasomes were dialysed against TEAD buffer.

2.6. Proteasome activity assay

Proteasome activities were measured in freshly-prepared crude protein extracts

and purified 20S proteasome fractions with the fluorogenic substrates Suc-LLVY-

MCA, Bz-VGR-MCA and Z-LLE-MCA, specific for chymotrypsin- (CT-), trypsin- (T-),

and caspase-(C-) like activities, as described elsewhere (Dahlmann et al., 2000;

Mishto et al., 2006a). Briefly, 10 mg of proteins in crude extracts were incubated

with 200 mM of Suc-LLVY- and Z-LLE-MCA and 600 mM of Bz-VGR-MCA in TEAD

buffer, at 37 8C for 1 h, in a 96-well plate. The enzymatically released MCA was

fluorometrically measured at 360 nm excitation and 460 nm emission. Proteasomal

activity was expressed as specific activity (nmol of free MCA/min per mg protein).

The specificity of the reaction was assessed measuring the fluorescence released in

presence of the proteasome inhibitor epoxomycin (10 mM).

2.7. Western blotting analysis

Western blotting analysis of protein crude extracts and purified 20S proteasomes

was performed as previously described (Bellavista et al., 2008; Mishto et al., 2006a).

Briefly, 40 mg of crude extracts or 0.5 mg of 20S purified proteasomes were

separated in a 16% polyacrylamide gel and transferred to a nitrocellulose filter.

Proteasome subunits were detected using the primary antibody conditions

reported in Suppl. Table 1, overnight at 4 8C shaking in a 5% non-fat dry milk/

0.01% Tween20 TBS solution. Densitometry analysis (INT*mm2) of the band

corresponding to each subunit was performed using QuantityOne Software. Each

experiment was repeated three times and we considered the mean of results. In

order to compare samples loaded in different gels, a liver sample was used as

internal control and loaded on each gel. Data are reported as relative content

between the samples and the control’s specific band density. Protein isoloading was

assessed by SDS-PAGE and Coomassie dye staining.

2.8. Oxidized proteins and polyubiquitin conjugates detection

Protein carbonyl content was determined in 15 mg of crude extracts of liver

tissue from different age donors, by using the OxyblotTM protein oxidation

detection kit according to manufacturer’s instruction, SDS-PAGE and immunoblot-

ting as above described.

Levels of polyubiquitin conjugated proteins were measured in 20 mg of liver

crude extracts by SDS-PAGE and immunoblotting, as above described, by using the

FK2 monoclonal antibody, specific for K29-, K48-, and K63-linked conjugates.

2.9. 2D-PAGE of 20S proteasomes

Purified 20S proteasomes were precipitated with an equal volume of ethanol and

then subjected to two-dimensional non-equilibrium pH polyacrylamide gel

electrophoresis as previously described (Mishto et al., 2006b).

2.10. Statistical analysis

All values are reported as mean + standard deviation (SD). Data were tested for

normality distribution and homoscedasticity by Shapiro-Francia, Shapiro–Wilk and

Please cite this article in press as: Bellavista, E., et al., Lifelong

distribution of proteasomes in human liver. Mech. Ageing Dev. (201

Skewness-Kurtosis tests. One-way ANOVA, Mann–Whitney or K-Wallis tests were

performed depending on the underlying distribution, to assess differences among

age groups. Spearman’s test was applied to assess the correlations between age and

histological parameters, as well as age and immunohistochemistry data. Chi-square

test was also applied to analyze the distribution of histological parameters among

age groups. Descriptive statistics were carried out with STATA v. 9.0 (Stata Corp.,

Texas, US) and a p-value <0.05 was considered as statistically significant. After

applying Bonferroni correction method for multiple comparisons, the p value

threshold for significance was set at p = 0.017.

3. Results

3.1. Histological profile of human liver

The histological analysis of the 54 liver biopsies (2 out of56 were not available) showed the occurrence of a mild portalchronic infiltrate in 17 (31%) cases, with no piecemeal necrosis orlobular necrosis. Fibrosis was scored as absent in 14 (26%) cases,Ishak grade 1 in 19 (35%) and Ishak grade 2 in 21 (39%), myointimalthickening was present in 33 (61%) cases, biliocyte regression in 15(37%), signs of mild cholestasis in 3 (5%), sinusoid dilatation in 8(15%) cases. Hepatocytic polymorphism was absent in 18 (33%)cases, mild in 26 (48%) and pronounced in 10 (18%) cases. Amoderate-to-severe lipofuscin accumulation was observed in 20(37%) cases. Some amount of steatosis was present in 29 cases,with a mean percentage of 5.42 � 10.7 (range 0–60%).

The histological parameters that directly correlated with agewere fibrosis (p < 0.001), myointimal thickening (p < 0.001),biliocyte regression (p = 0.012), hepatocytic polymorphism(p = 0.01), and lipofuscin accumulation (p < 0.001) (Suppl. Fig. 1,Suppl. Table 2). Remarkably, none of these parameters correlatedwith proteasome composition, distribution and functionalitydescribed below (data not shown).

3.2. Proteasomes activities in human liver during aging

Increased levels of oxidized proteins directly correlated toimpaired proteasomes activities have been described in the liver ofaged mice (Breusing et al., 2009). Accordingly, we investigated thecontent of oxidized proteins in bioptic samples from different agedonors and no changes were observed (Fig. 1A and B). Anotherindirect index of proteasomes functionality, the amount ofpolyubiquitin conjugates, showed no significant differences amongage groups (Fig. 1C and D). Donor’s gender did not contribute tooxidized and poly-ubiquitin protein levels (data not shown).

The analysis of proteasomes activities, measured by shortfluorogenic peptides assay and reported as specific activity pertotal protein (Rodriguez et al., 2010) showed that the CT-like andthe C-like activities were preserved during aging, while a trend toincrease was observed for the T-like activity (Suppl. Fig. 2A).However, by normalizing all three proteolytic activities for a4subunit, a non-replaceable component of the catalytic core andthus representative of total proteasome content, no difference wasobserved (Suppl. Fig. 2B). Of note, by stratifying our data accordingto gender we observed that proteasome activities were differentlyaffected by aging in the liver of men and women. Indeed, no age-related modifications of proteasomes activities were observed inbiopsies from female subjects (Fig. 2A and B). In liver from maledonors the T-like activity/a4 relative content and C-like activity/a4 relative content were increased in the ‘‘old’’ group compared tothe ‘‘middle’’ age group, along with no statistically significantdifferences between ‘‘young’’ and ‘‘old’’ age groups (Fig. 2C and D).

3.3. Age-related modifications of proteasomes composition in human

liver

We then investigated the possible role of proteasome subunitsexpression in the maintenance of proteasomes functionality.

maintenance of composition, function and cellular/subcellular4), http://dx.doi.org/10.1016/j.mad.2014.09.003

275 W276 an277 an278 co279 (F280 fr281 (n

282 co283 w284 pr285 al286 in287 tio288 (F289

290 fo291 b2292 a

293 on294 sa295 ca296 in

297298299300301302303304

305306

307308309310311312313314315316

Fig. 1. Oxidized and polyubiquitinated proteins do not accumulate in aged human liver. Western blotting analyses of oxidized proteins and polyubiquitin conjugates were

performed on crude protein extracts from different age human livers (n = 42; liver biopsies from female donors n = 19, age group 2: 41–70 years n = 16, age group 3: >70 years

n = 3; liver biopsies from male donors n = 23 age group 1: <40 years n = 7, age group 2: 41–70 years n = 10, age group 3 <70 years n = 6). An example of oxyblot (A) and

poliubiquitin staining (C) is given on the following subjects of different age: 1 = 20 years, 2 = 37 years, 3 = 43 years, 4 = 50 years, 5 = 58 years, 6 = 67 years, 7 = 78 years,

8 = 87 years. Data are reported as mean of the relative content + SD of three independent experiments. Relative contents of oxidized proteins (B) and polyubiquitin conjugates

(D) were not different among age groups (one-way ANOVA p = 0.70 and p = 0.19 respectively).

E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx4

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estern blotting analysis showed that the standard (b1, b2, b5)d inducible (b1i, b2i, b5i) catalytic subunits, as well as PA28ad Rpt2, representative of PA28ab and PA700 regulatorymplexes respectively, were expressed in all liver biopsiesig. 3A and C). Additionally, the purification of 20S proteasomeom a pool of liver biopsies from middle age ( n = 4) and elderly

= 3) male donors and subsequent western blotting analysisnfirmed that both standard and inducible catalytic subunitsere incorporated into mature and proteolytically active 20Soteasomes (Fig. 3B and D; Suppl. Table 3). The 2D-PAGE mapsso showed that most of the proteasome subunits were present

multiple isoforms, likely due to post-translational modifica-ns (Schmidt et al., 2006; Sixt et al., 2012), in both age groups

ig. 3E and F).By performing semi-quantitative western blotting analyses we

und no age-related variation in the expression levels of a4, b1i,i, b5i and b5 subunits (Suppl. Fig. 3A). In particular we observed

b1 subunit linear decrease with age in the liver of male donorsly (Fig. 4A) and a non-monotonic change of b2 in the wholemple size (Fig. 4B). However, when the ratio between eachtalytic subunits and a4 subunit was calculated, obtaining andex of the proportion of standard and inducible subunits on total

Please cite this article in press as: Bellavista, E., et al., Lifelongdistribution of proteasomes in human liver. Mech. Ageing Dev. (20

proteasomes (Rodriguez et al., 2010), only an increase in b5i/a4ratio was observed (Fig. 4C; Suppl. Fig. 3B).

The content of PA28ab and PA700 proteasome regulators,measured by PA28a and Rpt2 subunits, showed that Rpt2 subunitlevel t was not affected by aging in human liver (Suppl. Fig. 3C).Conversely, the PA28a subunit decreased with age, and inparticular a trend was observed in biopsies from male donors(Fig. 4D).

3.4. Subcellular distribution of proteasomes in human liver during

aging

Immunohistochemical analysis was performed to assesscellular (cell types) and subcellular distribution of the above-mentioned age-related modifications of proteasome subunits. Aspreviously described ( Gohlke et al., 2014a,b; Vasuri et al., 2010),b1 and PA28a subunits were expressed both at nuclear andcytoplasmic level, in all cell types, including biliocytes. At varianceb1i and b5i showed a cytoplasmic positivity in all cell types,in particular in lymphocytes and Kupffer cells. In particular, inhepatocytes, the main cell population of the liver, a strong nuclearpositivity for b5i was recorded in 8 (15%) cases and b1i was

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3413423433443453463473483493503513523533543553563573583593

360361362363364365366

Fig. 2. Proteasomes activity is differently affected in the liver of female and male during aging. Proteasome activities by short fluorogenic peptides were measured in crude

protein extracts from human liver biopsies (n = 42; liver biopsies from female donors n = 19, age group 2: 41–70 years n = 16, age group 3: >70 years n = 3; liver biopsies from

male donors n = 23 age group 1: <40 years n = 7, age group 2: 41–70 years n = 10, age group 3 <70 years n = 6). In female donors, no age-related variation in CT-like, T-like, and

C–like specific activities (A) (one-way ANOVA p = 0.38, p = 0.06, p = 0.22 respectively) as well as specific activities normalized for a4 subunit relative content (B) (one-way

ANOVA p = 0.35, p = 0.13, p = 0.18 respectively) were observed. Similar results emerged in male donors when CT-like, T-like, and C–like specific activities were considered (C)

(one-way ANOVA p = 0.5, p = 0.45, p = 0.25 respectively), while a non-monotonic age-related modification of the T-like and C-like specific activities per amount of a4 relative

content was observed (D) (T-like specific activity/a4 relative content: one-way ANOVA p = 0.04, age group 2 vs 3 *p < 0.017; C-like specific activity/a4 relative content: one-

way ANOVA p = 0.026, age group 2 vs 3 *p < 0.017). One-way ANOVA was applied and after Bonferroni correction for multiple comparison a p value <0.017 was considered

statistically significant.

E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx 5

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strongly expressed in 1 (2%) case, whereas a mild-to-moderatecytoplasmic positivity was observed in all cases. A strong (2+)nuclear immunoreactivity for b1 was seen in 47 (87%) cases, whilecytoplasm was 2+ positive in 26 (48%) cases. The samedistribution was observed in biopsies from female and maledonors (data not shown). Notably, no changes in the expressionlevel and localization of b1, b1i, b5i subunits were observed inhuman hepatocytes during aging (Fig. 5A –F). Finally, the PA28asubunit of the PA28ab proteasomes regulator, showed a 2+nuclear and cytoplasmic positivity in 38 (70%) and 23 (43%) casesrespectively. Remarkably, its cytoplasmic positivity was signifi-cantly decreased with age ( p = 0.011) (Fig. 5G and H), confirmingthe loss of PA28a subunits recorded in crude protein extracts.

4. Discussion

In the present study we report for the first time the impact ofaging on proteasomes in human liver, thus extending our previousobservation on fetuses and normal adult subjects (Vasuri et al.,2010). We took advantage of a biobank of liver biopsies wecollected during orthotopic liver transplantation from heart-beating donors spanning a wide range of age (16–87 years),including graft from elderly subjects which are usually considered‘‘extended criteria donors’’ (Harring et al., 2011). In optimal setting,clinical data indicate that the function of such transplanted livers iscomparable to that obtained from younger donors (Cescon et al.,

Please cite this article in press as: Bellavista, E., et al., Lifelong

distribution of proteasomes in human liver. Mech. Ageing Dev. (201

2003, 2008; Schmeding et al., 2010). Due to the high percentage ofthese graft we use, we decided to adopt a strategy to minimize thedamage due to the cold ischemic storage of these livers. Thestrategy has been defined as ‘‘low liver-damage’’ and consists inthe performance of two sub-glissonian biopsies on donor liver atthe time of procurement and postpones the aorta clamping only atthe acceptance of the organ based on histological review of theobtained biopsies, thus reducing the total ischemic time (Ravaioliet al., 2009). In this setting, successful outcomes are reachedindependently on the ‘‘classical’’ morphological alterations com-monly found in livers from old subjects (Tajiri and Shimizu, 2013),including those of our sample set. Indeed, there is a consensus thatparenchymal abnormalities (e.g. fibrotic portal expansion, hepa-tocytic polymorphism) do not influence graft survival, and are notexclusion criteria for donation (Fiorentino et al., 2009; Grazi et al.,2001). To explain this apparent paradox we thought worthwhile toperform a comprehensive study (composition, functionality andcellular/subcellular distribution) on one of the most importantcellular machinery for proteostasis such as proteasomes, Qinvolvedin the control of a variety of cellular functions (Hersko andCiechanover, 1998).

The main findings of our research regarding possible age-related changes in human liver proteasomes can be summarized asfollows: (i) no accumulation of oxidized proteins and polyubiquitinconjugates; (ii) maintenance of proteasomes proteolytic activitiesin livers from old donors; (iii) an increase of the b5i/a4 ratio,

maintenance of composition, function and cellular/subcellular4), http://dx.doi.org/10.1016/j.mad.2014.09.003

367 su368 ni369

370 pr371 po372 Ly373 no374 30375 co376 pr377 ac378 ac379 di380 an

381382383384385386387388389390391392393394

Fig. 3. Proteasome composition of different age human livers. Example of western blotting analysis on crude protein extracts from male liver biopsies of different age donors

(1 = 23 years, 2 = 30 years, 3 = 43 years, 4 = 44 years, 5 = 58 years, 6 = 58 years, 7 = 70 years, 8 = 84 years) (A) and on 20S proteasomes (B) purified from a pool of human liver

samples from ‘‘middle age’’ (marked as 1; n = 4, mean age 48 � 4.5 years) and old (marked as 2; n = 3, mean age 85 � 3.5 years) male donors. (C, D) Coomassie gel staining of

samples in picture (A) and (B) was used to assess protein isoloading. 2-D PAGE maps of 20S purified proteasomes from adult (E) and old (F) pooled liver samples as in (B). Different

protein species of 20S proteasome subunits have been identified.

E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx6

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ggesting a shift toward proteasomes containing immunosubu-ts and a decrease of PA28ab regulator in hepatocyte cytosol.

It is noteworthy that two indirect hallmarks linked tooteasome functionality and aging, such as the accumulation oflyubiquitin substrates (Tomaru et al., 2012), including those ons48, and oxidized proteins (Breusing et al., 2009) respectively dot occur in aged human liver. This suggest that globally both 26S/S and 20S proteasomes catalytic properties are preserved, thusntributing to maintenance of an homeostatic balance betweenoduction and degradation of these physiological substrates. Incordance, it is remarkable that the three major proteolytictivities against short fluorogenic peptides do not significantlyffer between livers from young and old donors, considering maled female all together, at variance of some reports in the same

Please cite this article in press as: Bellavista, E., et al., Lifelongdistribution of proteasomes in human liver. Mech. Ageing Dev. (20

organ of animal models. We could have missed other possible,important gender differences, besides those observed in protea-some activity in livers from middle aged male because of the lack ofbiopsies from young women (<40 years). At present, sex-specificdifferences regarding proteasome activities have been reported inDrosophila (Hansen et al., 2012) and in NOD mice concerning theproteasome-dependent NF-kB activation (Hayashi and Faustman,1999), thus this issue deserves more attention. Further investiga-tions are also needed regarding the functional role of the differentproteasomes subtypes during aging, as reported in rats (Gohlkeet al., 2014a). Indeed, owing to shortage of material, we measuredan average of the activity of the different proteasome subpopula-tions and subtypes present in human liver, which can varydepending on specific substrates (Gohlke et al., 2014b).

maintenance of composition, function and cellular/subcellular14), http://dx.doi.org/10.1016/j.mad.2014.09.003

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423424425426427428429430431432433434435436437438439440441442443444445446447448449450

Fig. 4. Modification of proteasomes composition during human liver aging. Semi-quantitative densitometric analysis of the expression levels of proteasomes subunits in

crude protein extracts from human liver biopsies (n = 40; liver biopsies from female donors n = 17, age group 2: 41–70 years n = 14; age group 3: >70 years n = 3; male donors

n = 23, age group 1: �40 years n = 7; age group 2: 41–70 years n = 10; age group 3: >70 years, n = 6). (A) An age-related decrease of b1 subunit was observed considering all

biopsies (one-way ANOVA p = 0.0022; age group 1 vs 2 and 1 vs 3 *p < 0.017) and samples from male donors (one-way ANOVA p = 0.008; age group 1 vs 3 *p < 0.017) but only

a trend to increase was observed in those from female (Student’s t-test p = 0.07). (B) A non-monotonic age-related variation of b2 emerged in the whole sample set (one-way

ANOVA #p = 0.019). (C) b5i/a4 ratio increased during aging in the whole cohort (Kruskal–Wallis test #p = 0.0027) and in both male (Kruskal–Wallis test p = 0.039; age group

1 vs 3 and 2 vs 3 *p < 0.017) and female donors (Student’s t-test p = 0.019). (D) A decreased PA28a content was observed in the whole sample set (one way ANOVA p = 0.05;

age group 1 vs 3 *p < 0.017) and a trend in male donors subset (one way ANOVA p = 0.048), even if Bonferroni correction did not allow to reach the significance; on the

contrary no change in female donors was observed (Student’s t-test p = 0.32). Data are reported as mean of the relative content + SD of three independent experiments. One-

way ANOVA or Kruskal–Wallis tests were applied; after Bonferroni correction for multiple comparison a p value <0.017 was considered statistically significant.

E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx 7

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Regarding proteasome composition and cellular/subcellulardistribution, we found that i-proteasome catalytic subunits,concomitantly to their homologous of s-proteasome, wereexpressed in different cell types of human liver, includinghepatocytes (Vasuri et al., 2010) and incorporated into active20S proteasomes. In our liver samples no signs of necrotic damageor morphologically evident inflammation were present (asexpected in the general donor population), and thus the presenceof i-proteasomes even in liver of young donors free frominflammatory infiltrates indicates that a basal level of pro-inflammatory ‘‘tone’’ can be considered physiological, likelysustained by the continuous exposure to antigenic stimuli comingfrom the gut microbiota via the portal vein. In fact hepatocytesexpress a number of pattern recognition receptors (PRR), such asTLR3, RIG-I and MDA5, that can sustain the production ofinflammatory cytokines (Bottcher et al., 2011; Broering et al.,2001). At the same time, a permanent activation of intracellularsignalling pathways can be envisaged (Ebstein et al., 2012; Grimmet al., 2012). The peculiar position of the liver, which drains bloodfrom the gut, can explain at least in part the presence of i-proteasome subunits even in organs from healthy children (Vasuriet al., 2011) and young subjects (this paper), at variance with otherorgans such as the brain where i-proteasome is never detectableexcept where pathological inflammatory processes occur (Mishtoet al., 2006a, 2010, 2011). This consideration is reinforced by thesemi-quantitative analysis by western blotting of standard andinducible catalytic subunits showing subtle age-related modifica-tions such as the increase in b5i/a4 ratio in all samples. These data

Please cite this article in press as: Bellavista, E., et al., Lifelong

distribution of proteasomes in human liver. Mech. Ageing Dev. (201

represent further evidence that a shift toward mixed-typeproteasomes containing immunosubunits, which exist in humanlivers (Gohlke et al., 2014b), occurs and also increase in aged ratliver (Gohlke et al., 2014a). On the whole, our data on i-proteasomeadd further evidence to the notion that the rate of aging in differentorgans, such as liver and brain, is dictated by specific anatomicaland functional characteristics (Cevenini et al., 2013).

Of note, PA28a subunit representative of PA28ab proteasomeregulator decreased with age in whole sample size and particularlyin elderly male biopsies and in the cytosol of hepatocytes. As novariation in the amount of PA700 regulator emerged from ourstudy, we can hypothesize that a remodelling of liver proteasomecomposition and activity occurs with age, by a variation in theamount of hybrid as well as single and double PA28-cappedproteasomes. This fact might lead to different substrate preferenceand increased antigenic diversity (Shibatani et al., 2006), as well asaltered response to oxidative stress (Pickering et al., 2010) and inany case fits the general remodelling theory of aging (Franceschiet al., 2000a).

In conclusion our data suggest that, despite morphologicalalteration, proteasome activity is well preserved in livers fromaged donors, concomitantly with subtle changes in proteasomescomposition, which might reflect the occurrence of a functionalremodelling to maintain an efficient proteostasis. Genderdifferences are emerging and they deserve further investigationowing to the different aging trajectories between man andwomen (Franceschi et al., 2000b). Finally, our data support thesafe use of livers from old donors for transplantation, especially in

maintenance of composition, function and cellular/subcellular4), http://dx.doi.org/10.1016/j.mad.2014.09.003

451 co452 st

453 Co

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455 Ac

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457 GL458 AGQ7

459 Ec460 M461 fe

462 Ap

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466467468469470471472473474475476477478479480481482483484485486487488

Fig. 5. Immunohistochemical analysis of proteasome distribution showed an age-related decrease of PA28a subunit in the cytosol of hepatocytes. A young (on the left) and an

old (on the right) liver donors are represented. b1 subunit (A, B) showed the same strong nuclear and cytoplasmic positivity, while b1i (C, D) and b5i (E, F) were always

negative or weakly expressed in the nucleus. Note the decreased cytoplasmic positivity of PA28a in older livers (H), compared to younger (G) (Spearman test p = 0.011). 20�magnification, scale bar = 100 mm.

E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx8

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mbination with ad hoc setting such as the low liver-damagerategy adopted by our surgery unit.

nflict of interest

None.

knowledgments

This work was financed in part by MIUR project PRIN2008 toG, by the European Commission Integrated Project PROTEOM-E (FP6-518230) and IDEAL (FP7-259679), by Italian Ministry of

onomic Development project MIAOVER50 to CF. Morenaartucci was supported by Fondazione Italiana Sclerosi Multiplallowship (2011/B/5).

pendix A. Supplementary data

Supplementary data associated with this article can be found, ine online version, at http://dx.doi.org/10.1016/j.mad. 2014.09.003.

Please cite this article in press as: Bellavista, E., et al., Lifelongdistribution of proteasomes in human liver. Mech. Ageing Dev. (20

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