In the ubiquitin-proteasome system (UPS), proteins are targeted for degradation by the 26S proteasome through covalent attachment of a chain of ubiquitin molecules. The ubiquitin ligase enzyme, or E3, binds the protein substrate and catalyzes the movement of the ubiquitin from the E2 enzyme to the substrate. This is the rate-limiting step of the ubiquitination process, which affects the subsequent proteasome-dependent degradation. Once the protein is ubiquitinated, it is docked to the proteasome for degradation, unless the polyubiquitin chain is removed by the deubiquitinating enzymes. Among the different E3s, only a few have been found to regulate the atrophic process and to be transcriptionally induced in atrophying muscle.

The regulation of skeletal muscle mass largely depends on protein synthesis and degradation processes. Two muscle-specific E3 ubiquitin ligases, muscle RING finger 1 (MuRF1) and muscle atrophy F-box (atrogin-1/MAFbx), are believed to be key regulators of proteasomal proteolysis in skeletal muscle, particularly under atrophy-inducing conditions [9–11]. These two E3s are specifically expressed in muscle atrophy [12, 13]. Atrogin-1/MAFbx and MuRF1 knockout mice are resistant to muscle atrophy induced by denervation [12]. Also, knockdown of atrogin-1 spares muscle mass in fasted animals [14]. Thus far, MuRF1 ubiquitinates several muscle structural proteins, including troponin I [15], myosin heavy chains [16], actin [17], myosin-binding protein C, and myosin light chains 1 and 2 [18], whereas the substrates of atrogin-1 that have been identified appear to be involved in growth-related processes or survival pathways. Atrogin-1 promotes degradation of MyoD, a key muscle transcription factor, and of eIF3-f, an important activator of protein synthesis [19, 20].

Another E3 ubiquitin ligase found to play a critical role in atrophy is TNF receptor-associated factor 6 (TRAF6) [21], which mediates the conjugation of Lys63-linked polyubiquitin chains to target proteins. Lys48-linked polyubiquitin chains are a signal for proteasome-dependent degradation, but Lys63-linked polyubiquitin chains play other roles, such as regulating autophagy-dependent cargo recognition by interacting with the scaffold protein p62 (also known as SQSTM1) [22–24]. Notably, muscle-specific TRAF6 knockout mice have a decreased amount of polyubiquitinated proteins, with almost no Lys63-polyubiquitinated proteins in starved muscles [25], and are resistant to muscle loss induced by denervation, cancer, or starvation [21, 25, 26]. The mechanism of this protection involves both direct and indirect effects of TRAF6 on protein breakdown. Inhibition of TRAF6 reduces the induction of atrogin-1 and MuRF1, thereby preserving muscle mass under catabolic conditions. Moreover, TRAF6-mediated ubiquitination may have an additional function on modulating intracellular signaling. In fact, TRAF6 is required for the optimal activation of c-Jun N-terminal kinase (JNK), AMP-activated protein kinase (AMPK), and forkhead box O3 transcription factor (FoxO3) pathways [25]. The effects on FoxO3 and nuclear factor κB (NF-κB) may explain why atrogin-1 and MuRF1 are less induced in TRFAF6 knockout mice.

In skeletal muscle, E3 ligases have important regulatory functions in signaling pathways. For example, it was recently found that anabolic signals were regulated by the ubiquitin ligase F-box protein 40 (Fbxo40) [27]. Fbxo40 ubiquitinates and affects the degradation of insulin receptor substrate 1 (IRS1), a downstream effector of insulin receptor-mediated signaling. Inhibition of Fbxo40 by RNA interference induces hypertrophy in myotubes, and Fbxo40 knockout mice display bigger muscle fibers [27].

Proteasomal degradation is mediated by an ATP-dependent protease complex, the 26S proteasome, which is present in both the cytoplasm and nucleus. The 26S proteasome consists of the 20S proteasome and the 19S regulatory particles. The 19S regulatory particles unfold ubiquitin-conjugated proteins to enable their entry into the 20S proteasome. The 19S regulatory particles contain several putative ATPases, such as PSMC1–PSMC6 (proteasome 26S subunit, ATPase, 1–6). These subunits form a large family with a highly conserved ATPase domain [28]. PSMC4, also known as Rpt3 (proteasomal regulatory particle AAA-ATPase-3; Fig. 2.1), is an essential subunit of the 26S proteasome and is required for the degradation of most proteasomal substrates. In particular, Rpt3-deficient mice die before implantation because of a defect in blastocyst development [28]. Interestingly, an insertion/deletion variant in intron 5 of the Rpt3 gene was frequently found in a cohort of patients with Parkinson’s disease [29]. Recently, Tashiro et al. reported that the conditional knockout of the proteasome subunit Rpt3 in motor neurons caused locomotor dysfunction that was accompanied by progressive motor neuron loss and gliosis in mice [30].

We also generated conditional Rpt3-knockout mice to specifically block proteasomal activity in skeletal muscle to clarify the role of the proteasomal system in skeletal muscle tissue (Fig. 2.2). Accordingly, we reported that muscle-specific deletion of a crucial proteasomal gene, Rpt3, resulted in profound muscle growth defects and a decrease in force production in mice with the accumulation of abnormal proteins [31]. In contrast to previous studies using proteasome inhibitors on dystrophic or myostatin-defective mice, in which muscle hypertrophy was demonstrated [32], the specific deletion of the proteasome component Rpt3 resulted in a significant loss of muscle mass with premature death and significantly reduced physical activity. Furthermore, our study suggests that both the UPS and autophagy systems are required to maintain myocellular homeostasis and integrity. The protein and organelle degradation by both autophagy and UPS may provide resources, such as oligopeptides and amino acids, for maintaining cellular integrity in skeletal muscle tissue [33]. Therefore, Rpt3 deficiency may result in a far more serious condition that deprives the cells of two major pathways responsible for pooling resources for cellular maintenance. We hypothesize that the skeletal muscle Rpt3 deficiency may have resulted in blocking the cellular “recycling system” that is essential for maintaining skeletal muscle fibers; this question needs to be further examined.

As reviewed above, some diseases present UPS dysfunction, while others represent excessive UPS activity. The suppression of excessive UPS activity is a promising method for some muscle diseases.

The 427 kDa protein dystrophin is expressed in striated muscle where it physically links the interior of the muscle fibers to the extracellular matrix. A range of mutations in the gene encoding dystrophin can lead to a severe muscular dystrophy known as Duchenne muscular dystrophy (DMD) or a typically milder form known as Becker muscular dystrophy. Dystrophin missense mutations, however, cause a wide range of phenotypic severities in patients. When stably expressed in mammalian muscle cells, the mutations caused steady-state decreases in dystrophin protein levels that are inversely proportional to the tertiary stability and are directly caused by proteasomal degradation. Proteasome inhibitors are able to increase the mutant dystrophin to wild-type levels, establishing the new cell lines as a platform to screen for potential therapeutics personalized for patients with destabilized dystrophin [57]. Using systemic treatment with an osmotic pump, the administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin in skeletal muscle fibers from dystrophin-deficient mdx mice. Proteasomal inhibitors reduced muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle that was isolated from treated mdx mice; it also ameliorated the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice [58]. Bortezomib was systemically administered in mdx mice over a 2-week period. In this system, bortezomib restored the membrane expression of dystrophin and dystrophin-glycoprotein complex members and improved the dystrophic phenotype [59]. Golden retriever muscular dystrophy (GRMD) is a genetic myopathy that corresponds to DMD in humans. Five GRMD dogs were examined; two were treated with the proteasome inhibitor bortezomib, and three were control dogs. The skeletal tissue from the treated dogs had lower levels of connective tissue deposition and inflammatory cell infiltration that in the control animals as determined by histology, collagen morphometry, and ultrastructural analysis. Significant inhibition of 20S proteasome activity was observed 1 h after bortezomib administration. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition, and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane [60].

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding the laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. Proteasome activity is increased in laminin α2 chain-deficient muscle and treatment with the proteasome inhibitor MG-132 reduces the muscle pathology in laminin α2 chain-deficient dy(3 K)/dy(3 K) mice. The proteasome inhibitor bortezomib (currently used for the treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3 K)/dy(3 K) mice and in congenital muscular dystrophy type 1A muscle cells can also be effective in the improvement of muscle pathology. Bortezomib improved several histological hallmarks of the disease, partially normalized microRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of the dy(3 K)/dy(3 K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes [61]. The same group explored the use of bortezomib in dy2J/dy2J animals. However, bortezomib neither improved the histological hallmarks of disease nor increased muscle strength or locomotive activity in dy2J/dy2J mice. It is possible that proteasome inhibition could be useful as a supportive therapy in patients with a complete absence of the laminin α2 chain[62].

Dysferlin is a transmembrane protein implicated in the surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, LGMD2B, and distal anterior compartment myopathy. A marked reduction in dysferlin in patients harboring missense mutations implies that dysferlin is degraded by the cell’s quality control machinery. One study revealed that dysferlin-deficient human myoblast cultures harbored the common R555W missense allele and a dysferlin-null allele and that control human myoblast cultures harbored either two wild-type or two null alleles. Dysferlin protein and messenger RNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability were measured after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). Inhibition of the proteasome increased the levels of R555W missense mutated dysferlin. This salvaged protein was functional as it restored the plasma membrane, resealing in the patient-derived myoblasts, and reversed their deficits in myotube formation. These results raised the possibility that inhibition of the degradation pathway of missense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin missense mutations [63]. In the other study, the authors described dysferlinopathy due to a homozygous Arg555Trp mutation in dysferlin in patients treated with the proteasome inhibitor bortezomib and monitored dysferlin expression in monocytes and in skeletal muscle by repeated percutaneous muscle biopsy. Expression of the missense mutated dysferlin in the skeletal muscle and monocytes of the three patients increased markedly, and dysferlin was correctly localized to the sarcolemma of the muscle fibers on histological sections. The salvaged missense mutated dysferlin was determined to be functional in a membrane resealing assay in patient-derived muscle cells treated with three different proteasome inhibitors [64].