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Skeletal muscle wasting is a major reason for morbidity and mortality in many chronic disease states, disuse conditions and aging. The ubiquitin-proteasome and autophagy-lysosomal systems are the two major proteolytic pathways involved in regulation of both physiological and pathological muscle wasting. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an important adaptor protein involved in receptor-mediated activation of various signaling pathways in response to cytokines and bacterial products. TRAF6 also possesses E3 ubiquitin ligase activity causing lysine-63-linked polyubiquitination of target proteins. We have uncovered a novel role of TRAF6 in regulation of skeletal muscle mass. Muscle-wasting stimuli upregulate the expression, as well as the auto-ubiquitination, of TRAF6 leading to downstream activation of major catabolic pathways in skeletal muscle. Muscle-specific depletion of TRAF6 preserves skeletal muscle mass in a mouse model of cancer cachexia or denervation. Inhibition of TRAF6 also blocks the expression of the components of the ubiquitin-proteasome system (UPS) and autophagosome formation in atrophying skeletal muscle. While more investigations are required to understand its mechanisms of action in skeletal muscle, our results indicate that blocking TRAF6 activity can be used as a therapeutic approach to preserve skeletal muscle mass and function in different disease states and conditions.
Proteolytic degradation of skeletal muscle proteins occurs through enhanced activity of the UPS and the autophagy-lysosome system, which is preceded by activation of certain “atrogenes” (specifically MAFbx/Atrogin-1 and MuRF1), and catabolic pathways including but not limited to those regulated by nuclear factor-kappaB, adenosine monophosphate-activated protein kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. In spite of the identification of these molecular events, a common point of convergence upstream or proximal to all these cascades in atrophying muscle was yet to be discovered.
Looking for proteins that play regulatory roles in the activation of various signaling pathways in atrophying muscle, we identified TRAF6. TRAF6 is an E3 ubiquitin (Ub) ligase that mediates a lysine 63 (Lys63)-linked polyubiquitination of its target proteins through its association with the dimeric ubiquitin-conjugating enzyme Ubc13/Uev1A. Though Lys63-linked polyubiquitination is involved in cell signaling, it is not yet fully understood whether this also primes molecules and subcellular compartments for interaction with the components of major proteolytic pathways. The idea gains more importance since recent investigations have now suggested that the proteasome also accepts other ubiquitin-chain types, in addition to the fact that ubiquitination plays an important role in selective autophagy.
Muscle-specific TRAF6-knockout (TRAF6mko) mice generated for our studies show significantly inhibited muscle atrophy which could be primarily attributed to the suppressed levels of “atrogenes” and components of proteasomal and lysosomal degradation pathways. Furthermore, deletion of TRAF6 also rescues the normal distribution of intermyofibrillar and subsarcolemmal mitochondria and prevents the formation of autophagic vacuoles and autophagosomes upon denervation.
It is increasingly evident that the relative contribution of autophagy and the UPS may vary significantly between target sites and catabolic conditions, and a general belief is that in normal conditions, proteasomal degradation predominates. But in atrophying skeletal muscle, both pathways are coordinately upregulated and autophagy can account for ~40% of degradation of long-lived proteins and defunct subcellular compartments. Our results reach a similar conclusion. Prevention of skeletal muscle atrophy in TRAF6mko mice seems to be attributable to both reduced ubiquitination and autophagy. Accumulating evidence further suggests that TRAF6 may have a direct role in selective autophagy. Interaction of TRAF6 with p62/SQSTM1 has already been highlighted as essential for RANKL-NFκB signaling that regulates osteoclastogenesis. The C-terminal ubiquitin binding domain of p62 can bind both Lys48-linked and Lys63-linked Ub chains but has a higher affinity for Lys63 chains. The prototypical autophagic adaptor p62 also contains a LIR motif (LC3/GABARAP interacting region). With these features, p62 is justifiably purported to have a role in protein aggregate (having a high Ub concentration) removal and selective autophagy of depolarized mitochondria, the latter of which undergo ubiquitination through Lys63 or Lys27 linkages. While it is yet to be determined whether E3 Ub ligases in or at mitochondrial membrane regulate the process or it is done by recruiting cytosolic E3 ligases, in the absence of any published evidence for a clearly identified signal or mechanism of autophagic degradation of damaged or superfluous mitochondria, it can be envisaged that TRAF6 and p62 provide a mechanistic link between ubiquitination and mitophagy.
Another interaction of TRAF6 that may connect it to autophagic degradation is with Beclin 1. Beclin 1 is a Bcl-2 interacting protein that has a role in autophagic degradation of protein aggregates. Beclin 1 has two TRAF6 binding sites and Ub-binding domains. The Ub-binding domains of Beclin 1 interact preferentially with Lys63-linked ubiquitination, and its TRAF6 binding sites are instrumental in recruiting TRAF6 to Beclin 1, suggesting that TRAF6 is critical for Beclin 1-mediated autophagy. Our results support all of the above and indicate that TRAF6 could be an important link between the UPS and autophagy, and that it plays a critical role as a regulatory molecule in autophagic degradation of proteins and beyond. We have determined that in different catabolic conditions TRAF6 gets activated potentially through auto-ubiquitination, and further regulates several mediators of critical importance to the UPS. In response to catabolic stimuli, ubiquitination of total protein increases significantly along with elevated expression of markers of both proteolytic pathways such as MAFbx, MuRF1, Beclin 1, LC3 and GABARAPL1. In muscle of TRAF6mko mice, expression of these markers is significantly inhibited in response to atrophic stimuli. This difference was translated proportionally at the tissue level as our histological and morphometric analyses confirmed significant inhibition in muscle atrophy.
Complicating the matter further, recent studies indicate that in addition to conventional proteolytic pathways, ubiquitination is involved in several other types of selective autophagies (with targets of mitochondria, ribosomes, peroxisomes and intracellular bacteria). Ubiquitination also marks internalized membrane proteins for lysosomal degradation by engagement with endosomal sorting complexes required for transport (ESCRTs), and Lys63 is the primary ubiquitin-chain type involved in endosomal sorting.
Recent insights have added to the fast-growing idea that all the major cellular degradation pathways rely heavily upon ubiquitination and that it might be the common signal for proteasomal, autophagic and endolysosomal targeting of proteins, protein aggregates, and subcellular compartments. This also creates a shift in long-held belief that autophagy is nonselective. With mounting evidence that Lys63-linked mono/polyubiquitination is critical in target marking in all three major degradation pathways, a preliminary conclusion can be drawn that TRAF6 as an E3 Ub ligase is involved in several physiological processes including degradative, homeostatic, cytoprotective and remodeling. This places TRAF6 at the crossroads of the proteasomal and autophagy pathways. Our work adds to the expansion of this field by revealing a regulatory role of TRAF6 in models of skeletal muscle atrophy. With increasing consensus on the interdependence of proteasomal, autophagic and endolysosomal degradation and the importance of ubiquitination in other selective autophagies, the diversified influence of TRAF6 further enhances this network. Future studies will determine how a particular target is marked for a particular pathway or how the same linkage type can create a bias for activation or degradation or even a specific type of degradation. We believe that new techniques and further investigation into specific localization of E3 ligases and other Ub-binding mediators will bring about an understanding of protein turnover as a function of stress stimuli and disease states, and will open more vistas to a finer and comprehensive appreciation of protein degradation.
This work was supported by funding from National Institutes of Health (RO1 AG029623) to A.K.
Punctum to: Paul PK, Gupta SK, Bhatnagar S, Panguluri SK, Darnay BG, Choi Y, Kumar A. Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J Cell Biol. 2010;191:1395–1411. doi: 10.1083/jcb.201006098.