1
2
3
4 Q1
5
6
7
8
910
111213
1415161718192021
22
Mechanisms of Ageing and Development xxx (2014) xxx–xxx
G Model
MAD 10735 1–9
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: [email protected] (E. Bellavista), [email protected] (M. Martucci), [email protected] (F. Vasuri), [email protected]
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), [email protected] (M. Mishto), [email protected] (A. Kloss), [email protected] (E. Capizzi), [email protected] (A. Degiovanni),
[email protected] (C. Lanzarini), [email protected] (D. Remondini), [email protected] (A. Dazzi), [email protected] (S. Pellegrini),
[email protected] (M. Cescon), [email protected] (M. Capri), [email protected] (S. Salvioli), [email protected] (A. D’Errico-Grigioni),
[email protected] (B. Dahlmann), [email protected] (G.L. Grazi), [email protected] (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/).
23 1.
24
25 fu26 ro27 ch28 du29 ag30 ly31 co32 in33 gr34 du35 yo36 Th37 pa38 ch39 ca40 m41 ni42 un43 si44 pi45 im46 th47 (U48 vi49 di50 pr51 co52 su53 of54 ca55 lik56 ac57 tw58 ch59 PA60 pr61 do62 (P63 pr64 tu65 th66 re67 (o68 co69 fo70 imQ2
71 tis72 Fe73 et74 ph75 av76 co77 m78 sh79 et80 Go81 or82 2083 Hu84 co85 ( G86 pr87 ob
8889
90
91
9293949596979899100101102103104105106107108109110111112113114115
116
117118119120121122123124125126127128129130131132133
134
135136137138139140141142143144145146147
E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx2
G Model
MAD 10735 1–9
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
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214215216217218219220221222
223
224
225226227228229230231232233234235236237238239240241242243
244
245246247248249250251252253254255256257258259260261262263264265266267268269270
271272
273274
E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx 3
G Model
MAD 10735 1–9
(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
G Model
MAD 10735 1–9
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
maintenance of composition, function and cellular/subcellular14), http://dx.doi.org/10.1016/j.mad.2014.09.003
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
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
G Model
MAD 10735 1–9
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
G Model
MAD 10735 1–9
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
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409 Q4
410
411
412
413
414 Q5
415
416
417
418
419
420
421
422
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
G Model
MAD 10735 1–9
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
454
455 Ac
456 Q6
457 GL458 AGQ7
459 Ec460 M461 fe
462 Ap
463
464 th
465
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
G Model
MAD 10735 1–9
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
References
Bellavista, E., Mishto, M., Santoro, A., Bertoni-Freddari, C., Sessions, R.B., Franceschi,C., 2008. Immunoproteasome in Macaca fascicularis: no age-dependent modifi-cation of abundance and activity in the brain and insight into an in silicostructural model. Rejuvenation Res. 11 (1), 73–82.
Bellavista, E., Andreoli, F., Parenti, M.D., Martucci, M., Santoro, A., Salvioli, S., Capri,M., Baruzzi, A., Del Rio, A., Franceschi, C., Mishto, M., 2013. Immunoproteasomein cancer and neuropathologies: a new therapeutic target? Curr. Pharm. Des. 19(4), 702–718.
Bottcher, J.P., Knolle, P.A., Stabenow, D., 2011. Mechanisms balancing tolerance andimmunity in the liver. Dig. Dis. 29 (4), 384–390.
Breusing, N., Arndt, J., Voss, P., Bresgen, N., Wiswedel, I., Gardemann, A., Siems, W.,Grune, T., 2009. Inverse correlation of protein oxidation and proteasomeactivity in liver and lung. Mech. Ageing Dev. 130 (11–12), 748–753.
Broering, R., Lu, M., Schlaak, J.F., 2001. Role of Toll-like receptors in liver health anddisease. Clin. Sci. (Lond.) 121 (10), 415–426.
Cescon, M., Grazi, G.L., Ercolani, G., Nardo, B., Ravaioli, M., Gardini, A.,Cavallari, A., 2003. Long-term survival of recipients of liver grafts fromdonors older than 80 years: is it achievable? Liver Transpl. 9 (11), 1174–1180.
Cescon, M., Grazi, G.L., Cucchetti, A., Ravaioli, M., Ercolani, G., Vivarelli, M., D’Errico,A., Del Gaudio, M., Pinna, A.D., 2008. Improving the outcome of liver transplan-tation with very old donors with updated selection and management criteria.Liver Transpl. 14 (5), 672–679.
maintenance of composition, function and cellular/subcellular14), http://dx.doi.org/10.1016/j.mad.2014.09.003
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631632633634635636637
E. Bellavista et al. / Mechanisms of Ageing and Development xxx (2014) xxx–xxx 9
G Model
MAD 10735 1–9
Cevenini, E., Monti, D., Franceschi, C., 2013. Inflamm-ageing. Curr. Opin. Clin. Nutr.Metab. Care 16 (1), 14–20.
Chondrogianni, N., Gonos, E.S., 2010. Proteasome function determines cellularhomeostasis and the rate of aging. Adv. Exp. Med. Biol. 694, 38–46.
Conboy, I.M., Conboy, M.J., Wagers, A.J., Girma, E.R., Weissman, I.L., Rando, T.A.,2005. Rejuvenation of aged progenitor cells by exposure to a young systemicenvironment. Nature 433 (7027), 760–764.
Conconi, M., Szweda, L.I., Levine, R.L., Stadtman, E.R., Friguet, B., 1996. Age-relateddecline of rat liver multicatalytic proteinase activity and protection fromoxidative inactivation by heat-shock protein 90. Arch. Biochem. Biophys. 331(2), 232–240.
Dahlmann, B., Ruppert, T., Kuehn, L., Merforth, S., Kloetzel, P.M., 2000. Differentproteasome subtypes in a single tissue exhibit different enzymatic properties. J.Mol. Biol. 303 (5), 643–653.
Dasuri, K., Zhang, L., Ebenezer, P., Liu, Y., Fernandez-Kim, S.O., Keller, J.N., 2009.Aging and dietary restriction alter proteasome biogenesis and composition inthe brain and liver. Mech. Ageing Dev. 130 (11–12), 777–783.
Ding, Q., Martin, S., Dimayuga, E., Bruce-Keller, A.J., Keller, J.N., 2006. LMP2 knock-out mice have reduced proteasome activities and increased levels of oxidativelydamaged proteins. Antioxid. Redox Signal. 8 (1–2), 130–135.
Ebstein, F., Kloetzel, P.M., Kruger, E., Seifert, U., 2012. Emerging roles of immuno-proteasomes beyond MHC class I antigen processing. Cell. Mol. Life Sci. 69 (15),2543–2558.
Ferrington, D.A., Gregerson, D.S., 2012. Immunoproteasomes: structure function,and antigen presentation. Prog. Mol. Biol. Transl. Sci. 109, 75–112.
Fiorentino, M., Vasuri, F., Ravaioli, M., Ridolfi, L., Grigioni, W.F., Pinna, A.D., D’Errico-Grigioni, A., 2009. Predictive value of frozen-section analysis in the histologicalassessment of steatosis before liver transplantation. Liver Transpl. 15 (12),1821–1825.
Franceschi, C., Valensin, S., Bonafe, M., Paolisso, G., Yashin, A.I., Monti, D., DeBenedictis, G., 2000a. The network and the remodeling theories of aging:historical background and new perspectives. Exp. Gerontol. 35 (6–7), 879–896.
Franceschi, C., Motta, L., Valensin, S., Rapisarda, R., Franzone, A., Berardelli, M.,Motta, M., Monti, D., Bonafe, M., Ferrucci, L., Deiana, L., Pes, G.M., Carru, C.,Desole, M.S., Barbi, C., Sartoni, G., Gemelli, C., Lescai, F., Olivieri, F., Marchegiani,F., Cardelli, M., Cavallone, L., Gueresi, P., Cossarizza, A., Troiano, L., Pini, G.,Sansoni, P., Passeri, G., Lisa, R., Spazzafumo, L., Amadio, L., Giunta, S., Stecconi, R.,Morresi, R., Viticchi, C., Mattace, R., De Benedictis, G., Baggio, G., 2000b. Do menand women follow different trajectories to reach extreme longevity? ItalianMulticenter Study on Centenarians (IMUSCE). Aging (Milano) 12 (2), 77–84.
Gohlke, S., Mishto, M., Textoris-Taube, K., Keller, C., Giannini, C., Vasuri, F., Capizzi,E., D’Errico-Grigioni, A., Kloetzel, P.M., Dahlmann, B., 2014a. Molecular altera-tions in proteasomes of rat liver during aging result in altered proteolyticactivities. Age (Dordr) 36 (1), 57–72.
Gohlke, S., Kloß, A., Tsokos, M., Textoris-Taube, K., Keller, C., Kloetzel, P.M., Dahl-mann, B., 2014b. Adult human liver contains intermediate-type proteasomeswith different enzymatic properties. Ann. Hepathol. 13 (4), 429–438.
Grazi, G.L., Cescon, M., Ravaioli, M., Ercolani, G., Pierangeli, F., D’Errico, A., Ridolfi, L.,Cavallari, A., Mazziotti, A., 2001. A revised consideration on the use of very ageddonors for liver transplantation. Am. J. Transplant. 1 (1), 61–68.
Grimm, S., Ott, C., Horlacher, M., Weber, D., Hohn, A., Grune, T., 2012. Advanced-glycation-end-product-induced formation of immunoproteasomes: involve-ment of RAGE and Jak2/STAT1. Biochem. J. 448 (1), 127–139.
Hansen, T.O., Sarup, P., Loeschcke, V., Rattan, S.I., 2012. Age-related and sex-specificdifferences in proteasome activity in individual Drosophila flies from wild type,longevity-selected and stress resistant strains. Biogerontology 13 (4), 429–438.
Harring, T.R., O’Mahony, C.A., Goss, J.A., 2011. Extended donors in liver transplan-tation. Clin. Liver Dis. 15 (4), 879–900.
Hayashi, T., Goto, S., 1998. Age-related changes in the 20S and 26S proteasomeactivities in the liver of male F344 rats. Mech. Ageing Dev. 102 (1), 55–66.
Hayashi, T., Faustman, D., 1999. NOD mice are defective in proteasome productionand activation of NF-kappaB. Mol. Cell. Biol. 19 (12), 8646–8659.
Hershko, A., Ciechanover, A., 1998. The ubiquitin system. Annu. Rev. Biochem. 67,425–479.
Huber, N., Sakai, N., Eismann, T., Shin, T., Kuboki, S., Blanchard, J., Schuster, R.,Edwards, M.J., Wong, H.R., Lentsch, A.B., 2009. Age-related decrease in protea-some expression contributes to defective nuclear factor-kappaB activationduring hepatic ischemia/reperfusion. Hepatology 49 (5), 1718–1728.
Ishak, K., Baptista, A., Bianchi, L., Callea, F., De Grootes, F., Gudat, F., Denk, H., Desmet,V., Korb, G., MacSween, R.N.M., Phillips, J., Portmann, B.G., Paulsen, H., Scheuer,P.J., Schmid, M., Thaler, H., 1995. Histological grading and staging of chronichepatitis. J. Hepatol. 22, 696–699.
Keller, J.N., Hanni, K.B., Markesbery, W.R., 2000. Possible involvement of protea-some inhibition in aging: implications for oxidative stress. Mech. Ageing Dev.113 (1), 61–70.
Please cite this article in press as: Bellavista, E., et al., Lifelong
distribution of proteasomes in human liver. Mech. Ageing Dev. (201
Mishto, M., Bellavista, E., Santoro, A., Stolzing, A., Ligorio, C., Nacmias, B., Spazza-fumo, L., Chiappelli, M., Licastro, F., Sorbi, S., Pession, A., Ohm, T., Grune, T.,Franceschi, C., 2006a. Immunoproteasome and LMP2 polymorphism in agedand Alzheimer’s disease brains. Neurobiol. Aging 27 (1), 54–66.
Mishto, M., Santoro, A., Bellavista, E., Sessions, R., Textoris-Taube, K., Dal Piaz, F.,Carrard, G., Forti, K., Salvioli, S., Friguet, B., Kloetzel, P.M., Rivett, A.J., Franceschi,C., 2006b. A structural model of 20S immunoproteasomes: effect of LMP2 codon60 polymorphism on expression, activity, intracellular localisation and insightinto the regulatory mechanisms. Biol. Chem. 387 (4), 417–429.
Mishto, M., Bellavista, E., Ligorio, C., Textoris-Taube, K., Santoro, A., Giordano, M.,D’Alfonso, S., Listı, F., Nacmias, B., Cellini, E., Leone, M., Grimaldi, L.M., Fenoglio,C., Esposito, F., Martinelli-Boneschi, F., Galimberti, D., Scarpini, E., Seifert, U.,Amato, M.P., Caruso, C., Foschini, M.P., Kloetze l, P.M., Franceschi, C., 2010.Immunoproteasome LMP2 60HH variant alters MBP epitope generation andreduces the risk to develop multiple sclerosis in Italian female population. PLoSONE 5 (2), e9287.
Mishto, M., Ligorio, C., Bellavista, E., Martucci, M., Santoro, A., Giulioni, M., Martucci,G., Franceschi, C., 2011. Immunoproteasome expression is induced in mesialtemporal lobe epilepsy. Biochem. Biophys. Res. Commun. 408 (1), 65–70.
Petersen, A., Honarvar, A., Zetterberg, M., 2010. Changes in activity and kineticproperties of the proteasome in different rat organs during development andmaturation. Curr. Gerontol. Geriatr. Res. 230697.
Pickering, A.M., Koop, A.L., Teoh, C.Y., Ermak, G., Grune, T., Davies, K.J., 2010. Theimmunoproteasome, the 20S proteasome and the PA28ab proteasome regula-tor are oxidative-stress-adaptive proteolytic complexes. Biochem. J. 432 (3),585–594.
Ravaioli, M., Grazi, G.L., Cescon, M., Cucchetti, A., Ercolani, G., Fiorentino, M., Panzini,I., Vivarelli, M., Ramacciato, G., Del Gaudio, M., Vetrone, G., Zanello, M., Dazzi, A.,Zanfi, C., Di Gioia, P., Bertuzzo, V., Lauro, A., Morelli, C., Pinna, A.D., 2009. Livertransplantations with donors aged 60 years and above: the low liver damagestrategy. Transpl. Int. 22 (4), 423–433.
Rodriguez, K.A., Gaczynska, M., Osmulski, P.A., 2010. Molecular mechanisms ofproteasomes plasticity in aging. Mech. Ageing Dev. 131 (2), 144–155.
Rodriguez, K.A., Edrey, Y.H., Osmulski, P., Gaczynska, M., Buffenstein, R., 2012.Altered composition of liver proteasome assemblies contributes to enhancedproteasomes activity in the exceptionally long-lived naked mole-rat. PLoS ONE7 (5), e35890.
Schmeding, M., Sauer, I., Hartwig, K., Theruvath, T., Pratschke, J., Neuhaus, R.,Neuhaus, P., Neumann, U.P., 2010. Aging of the liver graft and functional qualityin the absence of recurrent disease: a 10 year histological follow-up. Ann.Transplant. 15 (1), 5–13.
Schmidt, F., Dahlmann, B., Janek, K., Kloß, A., Wacker, M., Ackermann, R., Thiede, B.,Jungblut, P.R., 2006. Comprehensive quantitative proteome analysis of 20Sproteasome subtypes from rat liver by isotope coded affinity tag and 2-Dgel-based approaches. Proteomics 6, 4622–4632.
Schmucker, D.L., Sanchez, H., 2011. Liver regeneration and aging: a current per-spective. Curr. Gerontol. Geriatr. Res. 526379.
Shibatani, T., Nazir, M., Ward, W.F., 1996. Alteration of rat liver 20S proteasomeactivities by age and food restriction. J. Gerontol. A Biol. Sci. Med. Sci. 51 (5),B316–B322.
Shibatani, T., Carlson, E.J., Larabee, F., McCormack, A.L., Fruh, K., Skach, W.R., 2006.Global organization and function of mammalian cytosolic proteasome pools:implications for PA28 and 19S regulatory complexes. Mol. Biol. Cell. 17 (12),4962–4971.
Sixt, S.U., Alami, R., Hakenbeck, J., Adamzik, M., Kloss, A., Costabel, U., Jungblut, P.R.,Dahlmann, B., Peters, J., 2012. Distinct proteasome subpopulations in thealveolar space of patients with the acute respiratory distress syndrome.Mediat. Inflamm. 204250.
Tajiri, K., Shimizu, Y., 2013. Liver physiology and liver diseases in the elderly. WorldJ. Gastroenterol. 19 (46), 8459–8467.
Tomaru, U., Takahashi, S., Ishizu, A., Miyatake, Y., Gohda, A., Suzuki, S., Ono, A.,Ohara, J., Baba, T., Murata, S., Tanaka, K., Kasahara, M., 2012. Decreased pro-teasomal activity causes age-related phenotypes and promotes the develop-ment of metabolic abnormalities. Am. J. Pathol. 180 (3), 963–972.
Vasuri, F., Capizzi, E., Bellavista, E., Mishto, M., Santoro, A., Fiorentino, M., Capri,M., Cescon, M., Grazi, G.L., Grigioni, W.F., D’Errico-Grigioni, A., Franceschi, C.,2010. Studies on immunoproteasome in human liver. Part I: absence infetuses, presence in normal subjects, and increased levels in chronicactive hepatitis and cirrhosis. Biochem. Biophys. Res. Commun. 397 (2),301–306.
Vo, T.K., Godard, P., de Saint-Hubert, M., Morrhaye, G., Debacq-Chainiaux, F., Swine,C., Geenen, V., Martens, H.J., Toussaint, O., 2011. Differentially abundant tran-scripts in PBMC of hospitalized geriatric patients with hip fracture compared tohealthy aged controls. Exp. Gerontol. 46 (4), 257–264.
maintenance of composition, function and cellular/subcellular4), http://dx.doi.org/10.1016/j.mad.2014.09.003
Top Related