Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle
Introduction
The ubiquitin–proteasome system (UPS) is involved in most catabolic states and is the main actor of muscle wasting. The 26S proteasome is formed by the 20S proteolytic core capped by two 19S regulatory complexes (RCs). The 19S RC confers the recognition specificity of ubiquitin conjugates and the ATP dependence of the 26S proteasome. We and others previously reported that most of the components of the UPS pathway were up-regulated at the mRNA levels in skeletal muscles during highly catabolic situations (ubiquitin, 14 kDa-E2, muscle-specific E3s and 20S proteasome subunits) (Bodine et al., 2001, Taillandier et al., 1996). However, while the 20S proteasome subunits are coordinately elevated during muscle atrophy, studies reported either a concomitant elevation of mRNA levels of 19S subunits (Combaret et al., 2004) or an independent regulation (Dawson et al., 1995, Combaret et al., 2002, Tilignac et al., 2002, Lecker et al., 2004, Arlt et al., 2007). These discrepancies could be related to mild vs. strong situations of muscle wasting and/or not optimal measurement schedule. Another possibility could be that the different 19S subunits are independently controlled at different levels (transcriptional vs. translational), thus implicating differential translational efficiency for mRNAs. The 19S RC can be separated into two entities, the base and the lid, and possesses ATPase and non-ATPase subunits that are believed to accomplish specific roles. It is thus possible that different functions could implicate different levels of recruitment during atrophy processes. We wanted in this study to verify in a well-established model of severe muscle atrophy whether the 19S RC subunits were coordinately regulated at the mRNA levels. To further determine whether the 19S RC subunits are differently regulated, the translational efficiency of different components of the UPS pathway was also studied in both basal and atrophy states, and compared to another proteolytic system also involved in the decrease in muscle mass. Finally, using a 19S subunit, we purified and analyzed substrates of the 26S proteasome and found that at least a protein involved in muscular dystrophy was polyubiquitinated.
Section snippets
Animals and experimental design
The experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. In the present study, muscle atrophy was induced by hindlimb suspension, a well-known model of muscle deconditioning that mainly affects slow-twitch skeletal muscles. Forty male Wistar rats (Charles River) of an average body weight of 120 g were randomly assigned to either a control (CT) or a hindlimb suspended (HS) group. After 4 days of standard housing, rats of the
mRNA levels of the 19S regulatory complex are coordinately up-regulated during a catabolic state induced by unloading atrophy
The 19S RC is formed by at least 18 different subunits that confer the ATP and ubiquitin dependence of the UPS. It comprises 6 ATPases (S4, S6, S6′, S7, S8 and S10b) and 12 non-ATPases subunits, and can be divided into two subcomplexes, the base and the lid. Our experiments were performed at day 9 of hindlimb suspension where marked proteolysis (Thomason et al., 1989, Taillandier et al., 1996) and alterations of the metabolic and contractile properties of the soleus muscle occur (Yu et al., 2007
Discussion
Muscle atrophy induced by unloading is mainly driven by proteolysis through the activation of the Ca2+-dependent (Tischler et al., 1990), lysosomal (Judge et al., 2007) and UPS proteolytic pathways (Table 1, Taillandier et al., 1996). Total proteolysis peaks around day 9 of suspension (Thomason et al., 1989) and several components of the UPS are coordinately up-regulated (20S proteasome subunits, ubiquitin, E2s, E3s, …) (Attaix and Taillandier, 1998, Taillandier et al., 1996, Tilignac et al.,
Acknowledgements
We would like to thank Pr. M. Rechsteiner (University of Utah, Salt Lake City, USA) for the gift of the plasmids encoding for the human 19S RC subunits, Pr. Simon S. Wing (McGill University, Montréal, Canada) for providing us with the cDNA of the testis USP2-core, Pr. K. Tanaka for the C9 proteasome subunit cDNA and the anti-S8 antibody (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), and Pr. P. Cottin for the m-calpain cDNA (ISTAB, University of Bordeaux, France). This work was
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These authors contributed equally to this work.