Molecular Biology Reports 26: 89–93, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Changes in 20S proteasome activity during ageing of the LOU rat Fawzia Bardag-Gorce, Luc Farout, Christelle Veyrat-Durebex, Yves Briand∗ & Mariele Briand Universit´e Blaise Pascal Clermont 2, Laboratoire de Biochimie Appliqu´ee - EA 995, 63177 Aubiere Cedex, France; ∗ Author for correspondence (Phone: 33 4 73 40 74 19; Fax: 33 4 73 40 70 42; E-mail:
[email protected])
Key words: ageing, LOU rat, muscle atrophy, proteasome
Abstract Muscular functions decline and muscle mass decreases during ageing. In the rat, there is a 27% decrease in muscle protein between 18 and 34 months of age. We examined age-related changes in the proteasome-dependent proteolytic pathway in rats at 4, 18, 24, 29 and 34 months of age. The three best characterised activities of the proteasome (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) increased to 29 months and then decreased in the senescent animal. These variations in activity were accompanied by an identical change in the quantity of 20S proteasome measured by Western blot, whereas the S4 subunit of the 19S regulator and the quantity of ubiquitin-linked proteins remained constant. mRNA of subunits C3, C5, C9, and S4 increased in the senescent animal, but ubiquitin mRNA levels were unchanged. These findings suggest that the 20S proteasome may be partly responsible for the muscular atrophy observed during ageing in the rat.
Introduction Ageing is accompanied by a progressive loss of muscle function and a substantial decrease in muscle mass [1, 2]. This deterioration is caused by an imbalance between the synthesis and degradation of proteins. Muscle proteolysis is known to increase in certain diseases and during fasting. Activation of the ubiquitinproteasome pathway has been observed in models such as cancer [3, 4], sepsis [5], acidosis [6, 7], glucocorticoid treatment [7, 8], denervation [9] and weightlessness [10], whereas there is probably little stimulation of two other main proteolytic pathways: the lysosomal pathway and the Ca++ -dependent pathway [11]. However, Ilian & Forsberg [12] have shown that fasting increases mRNAs coding for calpains I and II, for cathepsins D, and for proteasome subunit C2, but noted no change in the activity of calpain. Huang & Forsberg [13] also recently provided evidence for a role of calpains in destabilisation of sarcomeric proteins of cultured L8 cells. There is direct in vitro evidence that the proteasome degrades rabbit [14, 15] and bovine myofibrillar proteins [16, 17]. The proteasome also seems to be involved in the hydrolysis
of myofibrillar structures in the moulting lobster [18], and in Manduca sexta on transformation of the larva during programmed cell death [19]. Few studies have examined proteolysis and its role in muscle degradation during ageing. Conconi et al. [20] have shown that the age-linked accumulation of oxidised proteins in the liver may be partly due to a drop in 20S proteasome activity. Hayashi & Goto [21] and Shibatani & Ward [22] have reported a decrease in PGPH activity of liver proteasomes. But liver and muscle tissues differ in protein catabolism and no agerelated weight loss is seen in the liver. Further, the 20S proteasome in the liver probably degrades preferentially oxidised proteins, whose concentration rises during ageing [23]. We studied the effects of ageing on (1) the three best-characterised peptidase activities of the proteasome: chymotrypsin-like (ChT-L), trypsin-like (TL) and peptidylglutamyl peptide hydrolase (PGPH), (2) cellular levels of proteasomes (using an antibody against the iota subunit) and of polyubiquitinated proteins, and (3) the mRNAs of different subunits of the 20S proteasome α (C3, C9), and β (C5), the 19S regulator (S4), and ubiquitin.
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Figure 1. Changes in the body weight of LOU rats during ageing (means of 6 to 8 values).
Figure 2. Ratio of gastrocnemius muscle mass to body mass as a marker of muscle deterioration at different ages.
Our animal model was the LOU rat, which has a longer lifespan than other common rat species and which does not become obese. We studied the gastrocnemius medialis, a biochemically well characterised mixed muscle subject to substantial locomotor demands.
Material and methods Animals Male and female rats of the inbred LOU strain had free access to standard food (pellets) and water. They were killed by decapitation, and the gastrocnemius muscles were excised, kept at −80 ◦ C for RNA purification, or used immediately for protein and enzyme assays, and Western blot analysis.
Figure 3. Peptidase activities of the proteasome in the gastrocnemius medialis muscle. µmoles/30 min/50µg protein (×10−1 for AAF substrate).
Bovine serum albumin was used as the protein standard. Peptidase activities
Preparation of muscle extracts Extracts were prepared according to Solomon & Goldberg [15] with slight modifications. Excised male rat gastrocnemius muscle was immediately placed in 50 mm Tris (pH 8.0), 10% glycerol, 1 mm EDTA, 1 mm EGTA, 50 nM E64, and 2.5 µm pepstatin (1:10 W:V). It was homogenised in a Polytron homogenise and centrifuged for 1 h at 100 000 g and 4 ◦ C. The supernatant was recovered and this crude extract was set aside for protein and enzyme assays and for Western blot analysis. Protein assay Protein concentration was measured according to the method of Bradford [24] using BioRad assay reagent.
Peptidase activities were measured in gastrocnemius muscle of rats aged 4, 24, 29 and 34 months. Reaction mixtures (200 µl of crude extract containing 50 µg of protein) contained 50 mm Tris HCl buffer pH 8, 1mM DTT and AAF-MCA (40 µm) or LSTR-MCA (40 µm) or LLE-NA (100 µm). The reaction was allowed to run for 30 min at 37 ◦ C. Fluorescence was monitored on a Hitachi F-2000 fluorimeter (excitation 370 nm, emission 420 nm for MCA-substrates and excitation 333 nm, emission 410 nm for NA-substrate). We verified that these activities were inhibited by the inhibitors MG 132 and lactacystine and were sedimented after centrifugation for 4 h at 100 000 g.
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Figure 4. Western blot assay of the iota subunit of the proteasome in the gastrocnemius medialis muscle. An age of 4 months is taken as the 100% reference.
Western blot analysis Gastrocnemius muscle extracts were electrophoresed, and polyubiquitinated proteins were revealed by Western blot, according to Towbin et al. [25], using antiubiquitin antibodies as probe. Replicas on PVDF sheets were saturated with 10% skimmed milk, 0.1% Tween in PBS buffer for 2 h at room temperature before overnight incubation with the first antibody in PBS. After washing in PBS-0.1% Tween, the second antibody coupled to peroxidase was added for 1 h at room temperature. Membranes were revealed by chemoluminescence using the ECLT M kit from Amersham. RNA purification and Northern blot analysis Total RNA was isolated from female rat gastrocnemius muscle using the method of Chomczynski and Sacchi [26]. Northern blotting was done according to Taillandier et al. [10]. Variations in RNA loading were corrected by the use of 18S RNA. Results Muscle and body mass Between 2 and 34 months of age, male rat body mass initially rose substantially due to growth, was then stable up to 18 months, and by 34 months had decreased by 27% compared with 18 months (Figure 1). The ratio of gastrocnemius muscle mass to body mass was stable during the early months of life and then decreased greatly up to 29 months, thereafter remaining relatively stable (Figure 2). This decrease corresponds to substantial muscular atrophy, which is also seen in different catabolic states.
Figure 5. Western blot assay of ubiquitin-linked proteins.
Figure 6. Expression of proteasome subunits 20S C3, C5, C9, of the S4 subunit of the 19S regulator, and of ubiquitin, in the gastrocnemius muscle during ageing. An age of 4 months is taken as the 100% reference.
Proteasome expression and activities The chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase activities of the proteasome followed a similar pattern, with an increase at 24 and 29 months followed by a decrease during the last months of life (Figure 3). Western blot analysis using an antibody directed against the iota subunit revealed the proteasome α subunit, which should be representative of the quantity of 20S proteasome in the cell, since the iota subunit has never been found in the isolated state. Levels of the α subunit gradually rose from 4 to 29 months and then decreased in the senescent animal (Figure 4). This means that the observed variations in activity are essentially due to increased proteasome levels.
92 Identical measurements were made with the antibody specific for the S4 subunit of the 19S regulator, and no significant age-related change was noted (data not shown), suggesting that levels of this regulator do not vary. Proteins degraded by the ATP-ubiquitin-dependent pathway should be bound to several ubiquitin molecules and will therefore be rapidly degraded by the 26S proteasome. Western blotting revealed two main protein bands at approximately 97 kDa and 31 kDa (Figure 5), as reported by Baracos et al. [3] in the muscles of rats bearing cancerous tumours. Other much weaker bands were also noted. Band densitometry revealed no significant age-related differences and no new band was detected. This contrasts with the finding of Baracos et al. [3] that ubiquitin-protein conjugates increased after 3 and 5 days of tumour implantation, demonstrating that 26S proteasome was involved in this tumour-induced muscle deterioration. Lastly, we investigated whether the above variations were associated with changes in mRNA, using Northern blots and hybridisation with the cDNAs of proteins alpha C3 and C5, beta C9 of the 20S proteasome, and subunit S4 of the 19S regulator. The RNA blots were also hybridised with the cDNA of ubiquitin (Figure 6). A slight increase was noted in subunits C9 and C5, and a larger increase in subunit C3. No change was seen in subunit S4. Levels of the four subunits studied increased between 24 and 34 months. Ubiquitin mRNA levels were unchanged over the three study periods.
suggested by Grune et al. [23], the 20S proteasome represents a second line of defence and selectively eliminates damaged proteins, whose concentration increases with age, resulting in marked loss of protein and hence muscle atrophy.
Acknowledgements This work was supported in part by the Conseil Régional Auvergne, the Ministère de la Recherche et de la Technologie, and the Institut National de la Recherche Agronomique.
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Conclusions Proteasome activity increased greatly during ageing up to 29 months and then decreased in the senescent rat at 34 months. These activity variations seem to result from a parallel change in the quantity of 20S proteasome. Levels of subunit S4 of the 19S regulator and ubiquitin-linked proteins were unchanged. The decrease in 20S proteasome between 29 and 34 months corresponds to a clear slowing of atrophy, as indicated by the muscle mass to body mass ratio, which is virtually constant over this period. The constant level of subunit S4 and of ubiquitinlinked proteins may indicate that the ubiquitindependent pathway is not responsible for this muscle atrophy seen in some diseases and the 20S proteasome could explain the atrophy observed during ageing. As
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