Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle

doi:10.1016/j.biocel.2008.04.013

The International Journal of Biochemistry & Cell Biology

Volume 40, Issue 11, 2008, Pages 2544-2552

https://doi.org/10.1016/j.biocel.2008.04.013 Get rights and content

Abstract

Catabolic stimuli induce a coordinate expression of the 20S proteasome subunits in skeletal muscles. However, contradictory data have been obtained for the 19S regulatory complex (RC) subunits, which could reflect differential regulation at the transcriptional and/or translational level. To address this point we used a well-established model of muscle atrophy (hindlimb suspension) and determined the mRNA levels for 19S subunits belonging to both the base (non-ATPase S1, ATPases S7 and S8) and the lid (S14) of the 19S RC. Concomitant increased mRNA levels were observed for all studied subunits in rat soleus muscles after 9 days of unloading. In addition, analysis of polysome profiles showed a similar proportion of actively translated mRNA (50%) in unloaded and control soleus muscle. Furthermore, the repressed pool of messenger ribonucleoparticles (mRNPs) was low in both control (14%) and unloaded (15%) animals. Our data show that representative 19S subunits (S7 and S8) were efficiently translated, suggesting a coordinate production of 19S RC subunits. The 19S RC is responsible for the binding of polyubiquitin conjugates that are subsequently degraded inside the 20S proteasome core particle. We observed that soleus muscle atrophy was accompanied by an accumulation of ubiquitin conjugates. Purification of ubiquitin conjugates using the S5a 19S subunit followed by deubiquitination identified telethonin as a 26S proteasome substrate. In conclusion, muscle atrophy induces a concomitant expression of 26S proteasome subunits. Substrates to be degraded include a protein required for maintaining the structural integrity of sarcomeres.

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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Coordinate expression of the 19S regulatory complex and evidence for ubiquitin-dependent telethonin degradation in the unloaded soleus muscle

Abstract

Introduction

Section snippets

Animals and experimental design

mRNA levels of the 19S regulatory complex are coordinately up-regulated during a catabolic state induced by unloading atrophy

Discussion

Acknowledgements

Int J Biochem Cell Biol

Anal Biochem

Biochem Biophys Res Commun

Int J Biochem Cell Biol

Metabolism

FEBS Lett

J Biol Chem

J Biol Chem

In vitro proteolytic processing of a diubiquitin and a truncated diubiquitin formed from in vitro-generated mRNAs

Proc Natl Acad Sci USA

Immediate early gene-X1 interferes with 26S proteasome activity by attenuating expression of the 19S proteasomal components S5a/Rpn10 and S1/Rpn2

Biochem J

Identification of ubiquitin ligases required for skeletal muscle atrophy

Science

The base of the proteasome regulatory particle exhibits chaperone-like activity

Nat Cell Biol

Glucocorticoids regulate mRNA levels for subunits of the 19S regulatory complex of the 26S proteasome in fast-twitch skeletal muscles

Biochem J

Torbafylline (HWA 448) inhibits enhanced muscle ubiquitin–proteasome-dependent proteolysis in cancer and septic rats

Biochem J

Developmental changes of the 26S proteasome in abdominal intersegmental muscles of Manduca sexta during programmed cell death

J Biol Chem

Ubiquitin–proteasome pathway on intracellular protein degradation: implications for muscle atrophy during unloading

Exerc Sport Sci Rev

Myosin accumulation and striated muscle myopathy result from the loss of muscle RING finger 1 and 3

J Clin Invest

The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction

Physiol Rev

Mapping subunit contacts in the regulatory complex of the 26S proteasome S2 and S5b form a tetramer with ATPase subunits S4 and S5

J Biol Chem

The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity

J Cell Biol

Translational control in mammalian cells

Annu Rev Biochem