UBR5: A New Player in Protein Quality Control for Skeletal Muscle Growth and Remodeling

Abstract

A balance between protein synthesis and degradation regulates skeletal muscle size. Proteolytic mechanisms, like the ubiquitin–proteasome system, are critical processes in protein quality control. Counterintuitively, the E3 ubiquitin ligase, UBR5, appears to be involved in skeletal muscle hypertrophy and regrowth and interacts with protein synthesis. We present the novel hypothesis for protein quality control being critical for skeletal muscle growth and remodeling.

UBR5 is pivotal in maintaining proteome integrity for skeletal muscle adaptation and function.

Key Points.

• Protein quality control involves a surveillance system to maintain proteome integrity under cellular stress, perturbations in protein synthesis, and defects in mRNA translation.

• The ubiquitin–proteasome system plays a pivotal role in the clearance of damaged and misfolded proteins via the coordinated function of E1, E2, and E3 ubiquitin ligases.

• The E3 ubiquitin ligase, UBR5, is important in skeletal muscle homeostasis and its adaptation to mechanical loading and growth.

• Other UBR-box E3 ubiquitin ligases have been reported to regulate skeletal muscle size.

• This brief review seeks to broaden our knowledge and identify gaps related to E3 ubiquitin ligases and protein quality control mechanisms under growth conditions.

INTRODUCTION

The maintenance of protein homeostasis centers around the synchronous interworking of multiple cellular processes that oversee the building or breakdown of new or damaged proteins (1). The four processes of folding, trafficking, synthesis, and degradation rely on pathway systems (i.e., proteostasis network) to maintain the proteome within the cell (1). Protein quality control encompasses the ability for the cell system to adapt to new environmental stimuli and conditions, with the goal of preventing prolonged damage to the cell due to altered protein dynamics and turnover (2,3).

Skeletal muscle is a highly adaptable tissue that can respond to various stimuli (e.g., inactivity, disease, and exercise) and has the ability to increase (i.e., hypertrophy) or decrease (i.e., atrophy) in size when required (4,5). The regulation of skeletal muscle mass is orchestrated by the activity of key signaling pathways that control protein breakdown and synthesis within myofibers (5,6). Skeletal muscle growth and remodeling under various stimuli, such as exercise and mechanical load, place cellular stress on the system, which adapts via an influx of newly synthesized proteins and the necessary removal of damaged proteins (7). Recent evidence has alluded to protein synthesis and degradation being elevated at the same time in different rodent models of skeletal muscle remodeling, and approaches via isotope tracer labeling have shed light on this dynamic (8,9). Indeed, there is evidence of the importance of increased autophagic and unfolded processing and unfolded protein responses upon aerobic and resistance exercise stimuli, emphasizing the need for protein degradation, without a subsequent loss of muscle mass (10–13). There is a need to expand our knowledge of proteolytic systems on skeletal muscle health and function, to develop and refine therapeutic strategies that promote proteostasis towards improving the quality of life during aging and disease.

This brief review will discuss the novel hypothesis for protein quality control as being a critical process during periods of skeletal muscle growth and remodeling when the synthesis of new proteins is prominent (Fig. 1). We will highlight unique aspects of the ubiquitin–proteasome system (UPS) (e.g., N-degron and C-degron pathways) and how proteolysis pertains to maintaining proteome integrity under cellular stress, plus recent advances in our understanding of novel E3 ligases (e.g., UBR5) in skeletal muscle growth and exercise adaptation. Our recent studies have formed the foundation for this hypothesis and emphasized the need to expand our understanding of proteolytic systems in different conditions and contexts outside those they have been historically investigated.

Figure 1.

Protein quality control of newly synthesized proteins: E3 surveillance perspective. Under growth conditions, demand for protein synthesis is placed within cells and tissues. Newly synthesized proteins from ribosomes will typically form into protein complexes where degron signals will be inaccessible for recognition [comprehensive review, refer to Pla-Prats and Thomä (2)]. However, not all proteins may be formed into complexes, leaving the likelihood of increased protein misfolding or excess orphan proteins to be present. In this scenario, the orphan protein presents a degron signal (depicted as red spots) that can be recognized by E3 ubiquitin ligases and the proteasome system for targeted ubiquitination and degradation. We hypothesize that E3s, like UBR5, function to provide protein surveillance for the degradation of excess or misfolded proteins during growth stimuli to maintain proteome integrity. (The figure was generated using BioRender.)

PROTEIN QUALITY CONTROL: A GROUP OF IMPORTANT SURVEILLANCE SYSTEMS

The UPS and the lysosome–autophagy system are key in maintaining proteostasis and aid in deciding the fate of unfolded or misfolded and damaged proteins through either nonproteolytic or proteolytic signals (1,14). Chaperones also play a key part in maintaining proteome health by assisting with conformational changes of proteins throughout the lifespan, from promoting proper folding, complex assembly, and transport across membranes, to targeting proteins for degradation (1). Together, these systems finely tune and coordinate a surveillance system designed to recognize and respond to destabilized proteins, peptides, and orphan proteins that require clearance when there are perturbations in protein homeostasis (15,16). Indeed, the finely tuned nature of the UPS and LAS is exemplified by the ability of these systems to target short-lived versus long-lived proteins/organelles, respectively, for protein turnover (17). In human and rodent models, our understanding of these systems is still limited, and studying these systems in different contexts or diseases may extend our knowledge of protein quality control.

The UPS contributes to the proteostasis network by utilizing ubiquitination, a process that involves covalently linking a small protein called ubiquitin to substrate proteins. The transfer of ubiquitin to a target protein is mediated by a series of reactions involving ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2; ∼40 in the human genome), and ubiquitin-protein ligases (E3; ∼600 in the human genome) (18), the lattermost of which are considered the dominant determinants of substrate-specific ubiquitin application. E3 ubiquitin ligases are classified into families based on characteristic domains and the mechanism of ubiquitin transfer to the substrate protein (19,20). The RING (Really Interesting New Gene) and HECT (Homologous to E6AP C-terminus) E3s are two families of E3 ubiquitin ligases that differ in their mechanism of ubiquitin transfer (19,20). RING E3s catalyze a direct transfer of ubiquitin from the E2 to the target protein, whereas transfer of ubiquitin by HECT E3s requires an intermediate step where the ubiquitin is first transferred from the E2 to an active-site cysteine residue on the HECT E3 ligase before substrate ubiquitination (21). However, the SCF (Skip-Cullin-F-box) complexes, also known as the Cullin-RING family complexes, use a family of F-box proteins as substrate adaptors to target substrates for ubiquitination (22). For example, the skeletal muscle-specific E3, MAFbx (muscle atrophy F-box; Fbxo32), can bind both Skp1 and Cullin1, and this interaction is dependent on the F-box domain (23). F-box proteins can target protein substrates in multiple ways, such as domain-based recognition and priming phosphorylation, which allows for diverse processes to be regulated (22). The nature and function of these E3 families may allow for diversity and adaptability for the degradation system to regulate the proteome under different conditions.

Pertinent to this review is that E3 ubiquitin ligases can be divided into subclasses as well, due to distinct properties and potential functions that they exhibit within the cell. For instance, the C-degron and N-degron pathways (Fig. 2) encompass a specific set of E3 ubiquitin ligases that will target protein substrates for ubiquitination based on a destabilizing signal (termed Degron) detected at the C-terminal or N-terminal end of the protein substrate (24). In terms of the N-degron pathway, the destabilizing signal at the N-terminus occurs as a result of an amino acid residue presenting as a degron, which previous studies have highlighted can be any of the 20 amino acids (e.g., arginine, lysine, and leucine) (25). E3 ubiquitin ligases in the N-degron pathway, specifically Arg/N-degron, contain a UBR-box domain that is critical for substrate recognition, and they function by targeting nascent proteins or protein fragments, as these are more likely to develop a degron signal early after being newly synthesized (3,25). In terms of protein assembly, other E3 ubiquitin ligases (e.g., NOT4, Fbxl17) can aid in quality control through targeting orphan proteins for degradation by cotranslational modifications (2,15). Finally, E3 ubiquitin ligases (e.g., ZNF598, Hel2) provide a key step in regulating ribosome quality control by contributing to the removal of stalled ribosomes or surveillance and degradation of defective mRNA or nascent peptides (15). Defects within these pathways can lead to increased cytotoxicity, protein aggregation, and subsequently an impairment in tissue function and overall health (1,3).

Figure 2.

N-degron and C-degron pathways in protein quality control. Protein substrates can develop destabilizing amino acid residues at the N-terminus and C-terminus that act as degradation signals [comprehensive review, refer to Sherpa et al. (25) and Varshavsky (25)]. The destabilization can occur at the primary or secondary structure level of the protein and be induced by an array of factors such as hypoxia, oxidative stress, and calpain activity. Modifications on the N-terminal residue, such as acetylation, can allow for subclasses of E3 ubiquitin ligases to recognize the protein substrates for ubiquitination and proteasomal degradation. The protein substrate upon ubiquitination is processed through the proteasome complex into short peptides for amino acid recycling. The N-degron pathway has received extensive investigation, whereas our understanding of C-degron pathways is in its infancy. In the C-degron pathway, complexes like the Kelch repeat complex (KLHDC) can form with E3 ubiquitin ligases (e.g., Cul2) to recognize the C-degron signal, leading to ubiquitination of the protein substrate. Our knowledge of these pathways has stemmed from cell model systems, with in vivo evidence illustrating how important components (e.g., E3 ubiquitin ligases) are in maintaining proteome integrity. However, very little is known about the role and function of E3s, like UBR7, Gid4, or Not4, in skeletal muscle health and proteostasis. (The figure was generated using BioRender.)

THE UBIQUITIN–PROTEASOME SYSTEM AND SKELETAL MUSCLE REMODELING

Our knowledge of the proteasome system and ubiquitination in skeletal muscle biology is limited. Although progress has been made in studying the UPS in models of skeletal muscle atrophy, and the refinement of tools such as proteomics has contributed to our advancement, the vastness of the UPS is still under-characterized in relation to exercise adaptation, skeletal muscle remodeling, and growth (26). In terms of exercise response, a recent study by Parker and colleagues (27) assessed diGly tag enrichment in quadricep [vastus lateralis (VL)] muscle biopsies from healthy untrained adults at pre-exercise, immediately after exercise, and 2 and 5 h post exercise. The authors reported a rapid clearance of ubiquitinated proteins, consistent with proteasome activation, which was returned to pre-exercise levels after 2 h. In addition, recent studies from the Molecular Transducers of Physical Activity Consortium study group have reported in tissues exposed to an exercise stimulus, an increased abundance of proteins that are ubiquitinated, and other important posttranslational modifications such as acetylation and phosphorylation (28). It is worth noting that protein ubiquitination does not necessarily equal a degradation event, and advances in omics technology have allowed for the proteome and posttranslational modifications to be explored (29,30). Overall, these data highlight the bioenergetic stress that exercise places on the whole-body system and the response of posttranslational modifications, such as ubiquitination, in activating cellular processes to maintain proteostasis.

There is evidence for E3 ubiquitin ligases and the UPS to be important for skeletal muscle remodeling in response to acute exercise, and during recovery of mass following a period of disuse atrophy (27,31–37). MuRF1 and MAFbx are the most commonly studied E3 ubiquitin ligases in skeletal muscle biology and were originally identified in a variety of atrophy models in 2001 (18,23). Since then, our understanding of E3 ligases in skeletal muscle biology has been attributed to their role in the process of atrophy. However, there is data within the literature that alludes to a role of MuRF1 and MAFbx for skeletal muscle remodeling under growth conditions and in response to exercise (31–33). A study by You et al. (38) observed MuRF1 protein quantity to be increased in the early stages of mechanical overload, with no change in MAFbx. Other studies have reported increases in MuRF1 expression using a mechanical overload model in rodent hindlimb muscles, and in humans following an acute bout of resistance exercise, which is suggestive of the importance of MuRF1 in the early remodeling process of myofibers required for growth (31–33). The change in MuRF1 expression in differing models of growth may indicate a possible action of MuRF1 in regulating mechanical tension through its modulation of proteins, such as Titin (39). During periods of growth, a temporal response for increased proteasome activity has been observed using in vitro activity assays for proteasome subunits (20s and 26s) (31,35). For instance, in skeletal muscle from functional overload studies induced by synergist ablation, proteasome activity was elevated from 1 to 14 d post overload. This increase in proteasome activity appeared to be independent of MuRF1 and MAFbx expression, as these E3s were only observed to be transcriptionally increased after 1 d of functional overload (31). Examination of functional overload in MuRF1 and MAFbx knockout (KO) mice revealed that MAFbx, but not MuRF1 was potentially important for the remodeling process since the MAFbx KO mice had an attenuated growth response, whereas the MuRF1 KO mice had a normal growth response (31). There is a gap in knowledge and a need to explore the roles of understudied E3 ubiquitin ligases in skeletal muscle remodeling and growth.

Proteasomal activity throughout the time course of a supraphysiological growth model, such as functional overload or during a regrowth period following atrophy, points to the importance of protein clearance under growth conditions, alongside activation of the protein synthetic machinery. Given that newly synthesized proteins might display a shortened half-life or orphan state, proteasome activity during the growth period could be suggestive of protein clearance for those proteins that are not required for incorporation or unable to form protein complexes (2,3,7). However, the extent to which changes in proteasome activity are observed may be influenced by the model used to induce skeletal muscle growth. For example, in a model of maximal intensity contractions, induced by electrical stimulation of the sciatic nerve, proteasomal activity has not been observed to be elevated in rat skeletal muscles following an acute bout (40,41). Future studies are warranted to investigate the contribution of the UPS in skeletal muscle remodeling or intracellular amino acid recycling in models of growth.

UBR5 AS AN IMPORTANT REGULATOR OF SKELETAL MUSCLE MASS

As nearly 600 E3 ligases have been identified to date, numerous of which exhibit differential expression in transcriptional data sets of skeletal muscle adaptation, it is plausible, and in likelihood probable, that there are uncharacterized E3 ubiquitin ligases that contribute to the regulation of skeletal muscle mass (Fig. 3). Recent work on the N-degron pathway has led to the identification of the UBR-box domain subclass of E3 ubiquitin ligases as being important for protein quality control in the context of skeletal muscle hypertrophy and regrowth following inactivity (36,37,42–44).

Figure 3.

Overview of experimental approaches used where UBR-box E3 ubiquitin ligases have been identified in skeletal muscle. UBR5 was first reported in resistance-trained human skeletal muscle. Subsequent studies utilizing human and rodent models have found UBR4, UBR5, and UBR7 mRNA expression and protein levels to be elevated under skeletal muscle growth conditions. Genetic manipulation of mouse and Drosophila models has uncovered roles for UBR4 and UBR5 in protein quality control and interactions with protein synthesis signaling, which are key in proteostasis and skeletal muscle function. Future studies are warranted to identify UBR-box E3 ligase substrates and explore how these E3s contribute to skeletal muscle growth. (The figure was generated using BioRender.)

The UBR-box family of E3 ubiquitin ligases encompasses part of the UPS, but their action on protein substrate recognition is also critical for the N-degron pathway (25). The N-degron pathway (also known as the N-end rule pathway) identifies degradation signals on destabilized proteins through the presence of ubiquitin and acetyl motifs (24,25). The UBR-box family of E3 ubiquitin ligases is part of the N-degron pathway and has seven family members, named UBR1 through UBR7 (Fig. 2). Despite differences in ubiquitin transfer (HECT vs RING vs F-box), all UBRs have a UBR-box domain that allows for the identification of unacetylated protein substrates for ubiquitination (24,25,45,46). In the ubiquitin biology field, published studies have highlighted a diverse role of cellular processes for the function of UBR-box E3s, with protein quality control and protein clearance typically being referenced (44,47). However, very little is known about this family of E3 ubiquitin ligases in skeletal muscle. In an early study from the Goldberg lab (48), various inhibitors targeting UBR1 (also known as E3α) showed an ~60% contribution of this proteolytic pathway to the turnover of endogenous proteins in mammalian skeletal muscle. It has been observed that this subset of E3 ubiquitin ligases may be important in skeletal muscle health and the regulation of skeletal muscle mass (37,42–44,49,50). Emerging evidence on this subfamily of E3 ubiquitin ligases has highlighted the responsiveness of these UBR-box E3s to mechanical loading and exercise (36,37,49,50). It should be noted that UBR2, like MuRF1 and MAFbx, has to date been reported to be upregulated under different atrophy conditions such as cancer cachexia (51–54).

The E3 ubiquitin ligase, UBR5, a HECT E3 and member of the N-degron pathway, was identified in skeletal muscle of human subjects performing resistance exercise training. UBR5 was found to be responsive to periods of loading and displayed a temporal transcriptional profile that mimicked the repeat loading model that was implemented (36). This observation by Seaborne and colleagues (36) opened the potential idea that E3 ubiquitin ligases are not only involved in the process of skeletal muscle atrophy but also could contribute towards the development of hypertrophy. In collaboration with the Sharples Laboratory, we further developed the idea that UBR5 might be involved in skeletal muscle growth by assessing UBR5 in different models of mechanical loading (37). In models of skeletal muscle atrophy and regrowth, such as hindlimb unloading and nerve crush injury, we observed an increase in UBR5 protein abundance during the regrowth phase. In addition, in tissue and cell culture models exposed to mechanical stimulation and hypertrophy, we reported increases in UBR5 mRNA expression and protein levels (37). Together, these findings supported the idea that upregulation of UBR5 is an adaptive response to loading and is potentially involved in the skeletal muscle remodeling process when the proteome is challenged.

In a recent study, we mechanistically assessed the importance of UBR5 in skeletal muscle mass regulation via in vivo electroporation and targeting UBR5 for knockdown in adult mouse TA muscle (42). The knockdown approach resulted in a loss of skeletal muscle mass and fiber cross-sectional area and, surprisingly, coincided with perturbations in Akt-mTORC1-p70S6K1 and ERK/p90RSK signaling pathways, which suggested an interaction between UBR5 and protein synthesis. Interestingly, with acute UBR5 suppression, we saw an increase in newly synthesized peptides as assessed by puromycin labeling (55) and chronic mTORC1 hyperactivation after 7- and 30-d post electroporation. The incidence of mTORC1 hyperactivation has been observed in various disease conditions and age-related loss of muscle mass and function (56,57), and, thus, is often referred to as a loss of proteostasis hallmark (1). How UBR5 interacts with mTORC1 and protein synthesis remains to be determined, but recent evidence suggests that UBR5 may regulate Akt phosphorylation in other cell types (58). Future studies are required to understand the relation between UBR5 and protein turnover and how it might function to maintain proteome integrity in skeletal muscle.

Although the substrates for UBR5 have yet to be identified, numerous studies from the ubiquitin biology field have been published detailing the possible role and function of UBR5 in protein quality control. In vitro cell systems suggest possible UBR5 targets to be nuclear receptors and transcription factors (16,47). Additional studies have shown that UBR5 can promote degradation of its substrates through the formation of a scaffold (dimer or tetramer) that allows for the formation of K48-linked polyubiquitin chains (59,60). Given this suggested function and structural organization of UBR5, it is interesting that changes in its mRNA expression and protein levels have commonly been observed under periods of skeletal muscle growth and response to mechanical loading, where K48-mediated proteasomal degradation might not be prominent. However, one possibility is that the influx of newly synthesized proteins during a period of cellular growth increases the potential for protein fragmentation to occur and activates important mechanisms of protein quality control (2,7,61). Thus, the development of a degron signal on newly synthesized proteins acts as an attractant for UBR5 to target these unidentified protein fragments for clearance via a K48 ubiquitination event (59,60). It might be postulated that the N-degron pathway and the UBR-box E3 ubiquitin ligases act as a protein surveillance system for protein substrates that are susceptible to protein fragmentation, and during periods of increased protein synthesis, the likelihood of this occurring might be increased. Other observations made in cell-based systems have alluded to the C-degron pathway having a similar protein surveillance role where cotranslational modifications occur on newly synthesized proteins and those proteins are targeted for proteasomal degradation by other E3 ubiquitin ligases specialized in C-terminal degradation to maintain protein homeostasis (2,24,25). However, these hypotheses require further testing in vivo, but they could explain the sophistication of the UPS for why E3 ligases might be required to maintain the proteome under mechanical loading and hypertrophy. Future studies are required to expand our UPS knowledge and interrogate how protein synthesis and degradation may interplay for skeletal muscle remodeling and exercise adaptation, as recent evidence alludes to a direct interaction between these systems (42,62,63).

ARE UBR-BOX E3 UBIQUITIN LIGASES INVOLVED IN SKELETAL MUSCLE ADAPTATION UNDER AN EXERCISE STIMULUS?

Early Identification of UBR5 in Skeletal Muscle Adaptation and Exercise Response

Early studies showcased UBR5 as a potentially important factor in skeletal muscle adaptation to exercise (36). As previously highlighted, initial observations by Seaborne and colleagues (36) reported the response of UBR5 in VL skeletal muscle to periods of resistance exercise. In another study by Blazev et al. (34), UBR5 displayed increased phosphorylation in VL skeletal muscle from participants who performed endurance and resistance exercise, with the temporal dynamics of UBR5 phosphorylation being different between the exercise modes. Moreover, the finding that UBR5 phosphorylation might be important for its activity in response to mechanical overload and muscle growth highlights that E3 ligases are subject to their own posttranslational modifications, which have the potential to regulate activity (64,65). This phosphorylation observation has been made in another E3 ligase, TRIM28, which is associated with the regulation of skeletal muscle size (66). An acute exercise study in female participants by Horwath et al. (67) observed no change in UBR5 protein under this performance testing stimulus. Indeed, the authors biopsied the VL at 1 h post exercise, and, thus, the action of UBR5 might be through its own phosphorylation status versus increasing UBR5 protein abundance. In rodent models, data on UBR5 phosphorylation status have been collected in response to maximal intensity contractions with observations alluding to an elevated change that occurs independently of mTORC1 activity (66). Thus, the change in protein abundance for UBR5 may occur over time and with prolonged exercise training, but future studies are required to understand the turnover rate for UBR5 and how it might influence training status and adaptation.

Other UBR-Box E3 Ligases in Skeletal Muscle Mass and Exercise Adaptation?

Since the initial observations for UBR5 and exercise, other E3 ubiquitin ligases of the N-degron pathway (Fig. 3) have been observed to increase at the transcriptome and proteome level in response to an acute or chronic exercise stimulus (49,50). A recent study by Roberts and colleagues (50) utilized a deep proteomic approach to assess the effect of training and age on protein abundance in human VL skeletal muscle. The authors noted that the E3 ubiquitin ligase, UBR7, was increased in response to resistance training, further supporting a potential role for the N-degron pathway in protein quality control under growth conditions. An earlier study by Raue and colleagues (68) identified UBR7 in human VL skeletal muscle as a transcriptional signature that displayed an increased expression in response to resistance exercise plus appeared to show a modest correlation with gains in muscle mass size and strength. However, very little is known about the role of UBR7 in skeletal muscle or other tissues; except for the UBR-domain in the protein, it is not classified as an HECT or F-box E3 ubiquitin ligase, but does contain a RING-related plant homeodomain (45,46). Thus, the extent to which UBR7 regulates proteome integrity and targets protein substrates for ubiquitination remains unknown. Studies have suggested that UBR7 acts as a histone chaperone, regulates NOTCH and TGF-β signaling, and potentially is important for extracellular matrix remodeling (69,70). It is worth noting that evidence does exist that UBR5 and UBR7 may function together (70) and, thus, could contribute to skeletal muscle adaptation with resistance exercise via a similar function or role. However, future studies are required to study UBR7 and UBR5 in exercise adaptation and mechanistically determine their importance in the process of skeletal muscle remodeling.

Another UBR-box E3 ubiquitin ligase, UBR4, has been observed to be critical in the regulation of skeletal muscle size and potentially exercise adaptation (43,44,49). UBR4 is the largest protein of the UBR-box E3 ubiquitin ligases at ~600 kda size and it does not contain a HECT or F-box domain. However, recent evidence suggests that the E3 module and activity for UBR4 is dependent on a “hemiRING” (RING-related structure) and zinc finger domain, which allows for ubiquitination of N-degron signals on protein substrates (71). Like UBR5, UBR4 has been implicated to be critical for protein quality control in the cytoplasm and targets nascent proteins for degradation (3). A recent study by Chambers et al. (49) used a progressive weighted wheel running intervention in aged mice and observed UBR4 protein abundance to be exercise-responsive and may be controlled epigenetically. Interestingly, UBR4 KO mice display increased skeletal muscle myofiber size, and this may occur through regulating the components of the UPS system, such as UBE2B, STUB1, ASB8, KLHL30, and targeting a histone complex consisting of HAT1/RBBP4/RBBP7 proteins (43). Notably, increases in skeletal muscle fiber size with UBR4 removal did not proportionally enhance muscle strength. The muscle size and strength disconnect becomes evident as UBR4 KO mice are aged, where loss of muscle-specific force and reduced protein quality are evident, even though age-related loss of muscle mass is spared (44). A similar observation was made by Hwee and colleagues (72), where aged MuRF1 KO mice displayed sparing of age-related muscle mass compared with wild type, yet muscle force was not protected. This study from our laboratory also reported increased proteasome activity (20S and 26S β5) in aged MuRF1 KO mice versus age-matched wild-type mice, suggesting global proteasome function to be critical for maintaining protein clearance with age (73). The study from the Judge laboratory (73) noted different ubiquitination patterns between MuRF1 KO versus wild-type mice under cancer conditions, and, thus, provided insight into how E3 ligases can regulate the proteome. It is worth noting that many of the ubiquitinated targets identified by the Judge Laboratory, we also identified in our previous study using MuRF1 overexpression to induce skeletal muscle atrophy (29). Overall, these data highlight the importance of proteome integrity being maintained and how E3 ubiquitin ligases contribute to the regulation of skeletal muscle size and exercise adaptation (Table).

TABLE. .

Summary of UBR-box E3 ubiquitin ligases changes in skeletal muscle adaptation

E3 Ubiquitin Ligase	Organismal Model	Stimulus Model	Skeletal Muscle Analyzed	Expression	Reference
UBR4	Mouse	Progressive weighted wheel running	Gastrocnemius	Increased protein abundance	Chambers et al. (49).
UBR5	Human	Resistance exercise (long term)	Vastus lateralis	Increased mRNA expression. Altered DNA methylation status	Seaborne et al. (36).
	Human	Exercise (performance tests)	Vastus lateralis	No change in protein abundance after 1 h	Horwath et al. (67).
	Rat	Regrowth following atrophy period (hindlimb unloading)	Triceps surae	Increased protein abundance	Seaborne et al. (37).
	Mouse	Regrowth following atrophy period (nerve crush injury)	Triceps surae	Increased protein abundance	Seaborne et al. (37).
UBR7	Human	Resistance exercise (long term)	Vastus lateralis	Increased protein abundance	Roberts et al. (50).
	Human	Resistance exercise	Vastus lateralis	Increased mRNA expression	Raue et al. (68).

CONCLUSIONS

Protein quality control provides a surveillance system to maintain proteome integrity under cellular stress, perturbations in protein synthesis, and defects in mRNA translation. The specialization of the UPS and E3 ubiquitin ligases extends beyond our current understanding of this protein quality control system, only being confined to models of skeletal muscle atrophy. UBR5 is an example of a newly identified E3 ubiquitin ligase in skeletal muscle biology that appears pertinent for the response of skeletal muscle to exercise and growth stimuli. Future studies are warranted to broaden our knowledge of the UPS and other protein quality control systems (e.g., autophagy, unfolded protein response) to develop strategies and identify proteostasis-promoting interventions for maintaining or improving skeletal muscle health and function.

Acknowledgments

We would like to acknowledge our colleague, Dr. Leslie M. Baehr, for her insightful feedback and discussions when preparing this manuscript.

Funding: D.C.H. was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under award numbers K01AR077684 and R03AR083980. The contents are solely the responsibility of the authors and do not necessarily represent the official view of the NIH.

Conflicts of interest: S.C.B. is on the scientific advisory board and has equity in Emmyon Inc. D.C.H. declares no conflict of interest exist.