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The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins

2012; Springer Nature; Volume: 31; Issue: 15 Linguagem: Inglês

10.1038/emboj.2012.178

1460-2075

Rosanna Piccirillo, Alfred L. Goldberg,

Ubiquitin and proteasome pathways

Article6 July 2012free access Source Data The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins Rosanna Piccirillo Rosanna Piccirillo Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rosanna Piccirillo Rosanna Piccirillo Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Alfred L Goldberg Corresponding Author Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Rosanna Piccirillo1 and Alfred L Goldberg 1 1Department of Cell Biology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 432 1855; Fax: +1 617 232 0173; E-mail: [email protected] The EMBO Journal (2012)31:3334-3350https://doi.org/10.1038/emboj.2012.178 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The p97/VCP ATPase complex facilitates the extraction and degradation of ubiquitinated proteins from larger structures. We therefore studied if p97 participates to the rapid degradation of myofibrillar proteins during muscle atrophy. Electroporation of a dominant negative p97 (DNp97), but not the WT, into mouse muscle reduced fibre atrophy caused by denervation and food deprivation. DNp97 (acting as a substrate-trap) became associated with specific myofibrillar proteins and its cofactors, Ufd1 and p47, and caused accumulation of ubiquitinated components of thin and thick filaments, which suggests a role for p97 in extracting ubiquitinated proteins from myofibrils. DNp97 expression in myotubes reduced overall proteolysis by proteasomes and lysosomes and blocked the accelerated proteolysis induced by FoxO3, which is essential for atrophy. Expression of p97, Ufd1 and p47 increases following denervation, at times when myofibrils are rapidly degraded. Surprisingly, p97 inhibition, though toxic to most cells, caused rapid growth of myotubes (without enhancing protein synthesis) and hypertrophy of adult muscles. Thus, p97 restrains post-natal muscle growth, and during atrophy, is essential for the accelerated degradation of most muscle proteins. Introduction The size and functional capacity of a muscle are determined by the balance between overall rates of protein synthesis and degradation. In response to fasting, denervation or inactivity and various systemic diseases (e.g., diabetes, cancer, sepsis, cardiac failure), skeletal muscles undergo a rapid debilitating loss of weight and contractile capacity (Mitch and Goldberg, 1996; Glass and Roubenoff, 2010). During these various types of atrophy, a common transcriptional programme is activated that leads to an increased overall rate of protein degradation that is primarily responsible for the loss of muscle mass (Mitch and Goldberg, 1996; Lecker et al, 2004). Among the atrophy-related genes (atrogenes) that are coordinately induced are a number of components of the ubiquitin-proteasome system, including two key muscle-specific ubiquitin ligases (E3), MuRF1 and atrogin-1, and also components of the autophagic/lysosomal pathway, such as Gabarapl1 and LC3 (Lecker et al, 2004; Mammucari et al, 2007; Sacheck et al, 2007; Zhao et al, 2007). This transcriptional programme is induced by activation of FoxO-transcription factors (Sandri et al, 2004). Molecules that block this activation of proteolysis could represent novel pharmacological agents to combat muscle wasting. The major constituents of muscle are myofibrillar proteins. These highly organized components of the sarcomere are normally degraded by the ubiquitin-proteasome pathway (Solomon and Goldberg, 1996; Cohen et al, 2009), but more slowly than most cellular proteins (Zak et al, 1977). This process is markedly accelerated in atrophy, where they are disassembled and degraded in an ordered manner (Cohen et al, 2009). Moreover, since muscles continue to function even during rapid atrophy (e.g., in a fasting animal), the increased proteolysis must occur in a way that does not dramatically alter the relative amounts and functional capacities of different myofibrillar proteins. While the ubiquitin-proteasome pathway is responsible for the accelerated loss of myofibrillar proteins during atrophy (Solomon and Goldberg, 1996), autophagy is also activated and causes the degradation of some soluble proteins and organelles, especially mitochondria (Mammucari et al, 2008; Zhao et al, 2008). During disuse atrophy, thick filament components (e.g., myosin) are selectively ubiquitinated by MuRF1 while in the myofibrils and degraded (Cohen et al, 2009), but other ubiquitin ligases appear to catalyse the degradation of thin filament components (e.g., actin) (Kudryashova et al, 2005). However, the mechanisms for extraction of ubiquitinated proteins from the myofibrils and their delivery to the proteasome are still unclear, nor is it known what cellular factors catalyse this process, which must be highly selective and allows disassembly of some sarcomere components without blocking contractile function. One attractive candidate for such a role in myofibrillar disassembly is the p97/VCP/Cdc48 ATPase complex, since it binds multiple ubiquitin ligases and ubiquitinated proteins and catalyses the ATP-driven disassembly of other protein complexes, including the extraction and degradation of endoplasmic reticulum-associated proteins (ERAD) (Rape et al, 2001; Ye et al, 2001) as well as of mitochondrial proteins (Xu et al, 2011), membrane fusion events (Ye, 2006), myofibril biogenesis (Kim et al, 2008), chromatin dynamics (Ramadan et al, 2007) and autophagy (Ju et al, 2009; Krick et al, 2010). We therefore examined whether p97 could be involved in muscle atrophy when myofibrillar proteins are rapidly degraded. p97 is a ubiquitous ATPase complex containing six identical subunits (Peters et al, 1990). A common function of p97 in these various cellular processes is to extract ubiquitinated proteins from complexes or membranes, often leading to their proteasomal degradation (Ye, 2006). The p97 complex has been proposed to disassemble also aggregates of misfolded proteins, because p97 overexpression reduces the content of polyglutamine inclusions (Nishikori et al, 2008), and p97 is found in ubiquitinated inclusions in several models of neurodegenerative diseases (Ishigaki et al, 2004). A number of proteins, including soluble cytosolic and nuclear proteins, require p97 for their degradation by the proteasomes, such as UNC45B (Janiesch et al, 2007), Aurora B kinase (Ramadan et al, 2007), the transcription factors Spt23 in yeast (Rape et al, 2001) and HIF1α in mammals (Alexandru et al, 2008), cyclin E (Dai and Li, 2001), IκB (Dai et al, 1998), misfolded secretory proteins (Ye et al, 2003), oxidatively damaged cytosolic proteins (Medicherla and Goldberg, 2008) and substrates of the N-end rule and ubiquitin-fusion degradation (UFD) pathways (Wojcik et al, 2006; Beskow et al, 2009). The variety of p97's actions is probably due to its ability to interact with diverse substrate-recruiting and -processing cofactors (Jentsch and Rumpf, 2007). The recruiting cofactors include p47 and Ufd1/Npl4 dimer, which associate with the N-terminus of p97 in a mutually exclusive way (Meyer et al, 2000). Substrates bound to p97 can undergo further processing by ubiquitination, deubiquitination or deglycosylation. Among these substrate-processing cofactors are many ubiquitin ligases (Zhong et al, 2004; Alexandru et al, 2008). One E3 that is of special importance in muscle is Ufd2/E4B, which binds to p97's C-terminus and appears to add further ubiquitin molecules (termed 'E4 activity') to the substrate (Richly et al, 2005). In C. elegans, Ufd2 has been implicated with another E3, CHIP and p97 in the degradation of UNC45B, a myosin assembly factor (Janiesch et al, 2007). Another strong reason for studying p97 function in skeletal muscle is that mutations in human p97 cause the multisystem disorder inclusion-body myopathy with Paget's disease of the Bone and Frontotemporal Dementia (IBMPFD) (Watts et al, 2004), which is characterized by ubiquitin-positive inclusions in the muscle and brain (Guinto et al, 2007), abnormal vacuolation, sarcomeric disorganization, muscle weakness and functional impairment (Kimonis et al, 2008). The molecular bases for these various changes in muscle in IBMPFD are uncertain (Weihl et al, 2006; Hubbers et al, 2007). In this study, we investigated whether p97 and its cofactors play a critical role in muscle atrophy, specifically in the rapid breakdown of the highly organized myofibrillar apparatus, during the rapid muscle wasting induced by denervation or fasting. We have studied (1) whether overproduction of WTp97 or an ATPase-deficient p97 mutant influences the atrophy process through effects on overall rates of protein synthesis and degradation; (2) whether the accelerated disassembly and degradation of myofibrillar proteins during atrophy is dependent on p97 and its cofactors; (3) how expression of p97, its cofactors and substrate, UNC45B, may change in atrophying muscles and (4) whether p97, a dominant negative p97 mutant or UNC45B also influences the sizes of normal myotubes or adult muscle fibres. These studies show that p97 plays a critical role during atrophy in the accelerated degradation of muscle proteins via both the proteasomal and autophagic pathways, and most likely functions in the disassembly and proteasomal degradation of ubiquitinated components of the myofibrillar apparatus. Moreover, in normal adult muscles and myotubes, p97 by promoting protein degradation helps limit muscle growth. Results Inhibition of p97 decreases muscle atrophy induced by denervation or fasting To establish whether p97 function is essential for atrophy, we tested whether inhibition of p97 can reduce muscle wasting induced by denervation or food deprivation, by comparing the effects of expression of WTp97 and the p97K524A mutant, a dominant negative inhibitor (DNp97) (Ye et al, 2003). Mutation of lysine 524 to alanine in the D2 ATPase domain of p97 allows the binding of p97 to substrates while substantially decreasing p97's ATPase activity, and thus preventing substrate release and degradation by proteasomes (Ye et al, 2003). In these constructs, p97 is fused to GFP, which enabled us to identify the transfected fibres. Fusion of GFP to p97's C-terminus does not perturb its subcellular localization, propensity to form hexamers, ATPase activity or association with its cofactors p47 and Ufd1/Npl4 (Kobayashi et al, 2002). The WT and DNp97 plasmids as well as a control (GFP) plasmid were then electroporated bilaterally into Tibialis Anterior (TA) of adult mice, and at the same time, muscles of one hindlimb were denervated by sectioning the sciatic nerve. Nine days later, the muscles were collected and cryosectioned, and the areas of the transfected fibres were determined by fluorescence microscopy. At this time, denervated fibres decreased in mean area by about 40%, and muscle weight by about 35% (Figures 1A and 10C). Figure 1.Electroporation of DNp97 in TA blocks both denervation and starvation-induced atrophy. (A) Frequency histograms showing the distribution of cross-sectional areas of muscle fibres of TA either innervated or 9 days denervated and transfected or not with DNp97GFP. Muscles were electroporated with DNp97GFP plasmids at the same time as section of the sciatic nerve. According to the Kruskal–Wallis Test, differences (P<0.001) were found between innervated versus denervated and innervated versus denervated+DNp97. (B) Frequency histograms showing the distribution of cross-sectional areas of muscle fibres of TA either from fed or 2 days fasted mice and transfected or not with DNp97GFP. Muscles were electroporated with DNp97GFP plasmids and after 4 days mice were deprived of food for 2 days. According to the Kruskal–Wallis Test, differences (P<0.0001) were found between fed versus fasted, fed versus fasted+DNp97 and fasted versus fasted+DNp97. (C) A representative field of a transverse section of fibres expressing DNp97GFP from food-deprived mice. Scale bar represents 50 μm. Download figure Download PowerPoint Although electroporation of GFP or WTp97 had little or no effect on fibre cross-sectional areas in the denervated muscles (i.e., <8% change in median area, 1946 μm2 for denervated and 2100 for denervated+WTp97, P<0.05), electroporation of the DN to inhibit p97 resulted in a marked reduction of atrophy (Figure 1A). The electroporated fibres (which comprised 60–80% of the total fibres in the different animals) had a median cross-sectional area 60% greater than that of untransfected fibres in the same denervated muscle (Figure 1A). In fact, the median cross-sectional area of denervated fibres overexpressing DNp97 did not differ from that of innervated fibres of the contralateral limb. Thus, p97 inhibition seemed to prevent completely denervation atrophy. We therefore investigated whether p97 function is also essential for the more rapid loss of muscle mass induced by fasting. Mice were electroporated with the DNp97 plasmid, and 4 days later when no change in muscle weight was evident (data not shown), the mice were deprived of food for 48 h, and their muscles then collected. The muscles of fed mice, which had the same initial mean body weights as the fasted animals were used as controls (Figure 1B). Muscle fibres of food-deprived mice had a median cross-sectional area 33% smaller than that of fibres of fed animals (Figure 1B). Strikingly, in the starved animals, inhibition of p97 by electroporation of the DN resulted in fibres with a median cross-sectional area 70% greater than that of untransfected fibres in the same muscles (Figure 1B and C). Surprisingly, expression of DNp97 not only caused a clear inhibition of fasting-induced atrophy, but also resulted in even larger fibres than those in the TA of fed mice (Figure 1B). Inhibition or depletion of p97 in normal muscles causes rapid fibre growth To determine if p97 function is essential to maintain normal muscle size or if blocking it may even induce growth (as suggested in Figure 1B), we electroporated into the contralateral TA muscles of fed adult mice plasmids for either the WTp97 or DNp97 fused to GFP. Because the WTp97 was expressed more efficiently in initial control experiments, we determined by immunoblotting the amount of plasmid electroporated that gives equal expression of WT and DNp97 proteins (Figure 2E). Seven days later, the median cross-sectional area and size distribution of the TA fibres expressing WTp97 resembled that of the surrounding non-electroporated fibres (Figure 2A). By contrast, fibres expressing DNp97 had a 60% greater median area than untransfected fibres (Figure 2B). Furthermore, even though 20–40% of the fibres were not electroporated, the average weight of the TA electroporated with DNp97 in 7 days exceeded by 15% that of the muscles expressing the WTp97 (P<0.05) (Figure 2G). Thus, normal p97 function inhibits muscle growth, but increasing WTp97 content per se does not induce muscle wasting. Figure 2.Electroporation of DNp97 or depletion of p97 using shRNA in TA increases fibre size in 7 days. (A–D) Frequency histograms showing the distribution of cross-sectional areas of TA muscle fibres either untransfected (control) or transfected for 7 days with WTp97 (A), DNp97 (B), a non-silencing shRNA (C) or a shRNA against p97 (D). According to the Mann–Whitney Test, the differences between DNp97 versus control and p97shRNA versus control were highly significant (P<0.0001). (E) Sixty micrograms of protein lysates from TA muscles electroporated with WTp97GFP (right leg) or DNp97GFP (left leg) were loaded. The anti-GFP antibody detection shows that equal amounts of WTp97GFP or DNp97GFP were expressed per muscle. The anti-p97 antibody detection reveals two bands: the endogenous (lower band) and the exogenous (upper band) p97. Rpl26 was used as loading control. (F) Seven days after electroporation with a plasmid for a non-silencing shRNA (right leg) or a shRNA against p97 (left leg), the contralateral TA muscles were frozen in isopentane cooled-liquid nitrogen from the same animal. Scale bar represents 1 cm. (G) The average weights of TA muscles 7 days after electroporation are plotted. Error bars indicate s.e.m. Paired t-test: *P<0.05 and ***P<0.003, n=5. Download figure Download PowerPoint Although it was quite surprising to find that reducing p97 function with the DNp97 induces fibre growth, we confirmed this conclusion by electroporating into TA muscles plasmids encoding either a shRNA for murine p97 or a non-silencing shRNA as the control (Supplementary Figure S2A, B and E). Both vectors co-expressed a GFP marker to allow the identification of the transfected fibres. Seven days later, the median cross-sectional area of fibres expressing the shRNA for p97 was ∼30% larger than untransfected fibres and those expressing the control shRNA (Figure 2C and D). Also, the average weight of the muscles electroporated with the shRNA for p97 increased by 25% in 7 days above that of controls expressing non-silencing shRNA (P<0.003) (Figure 2F and G). Because only 60–80% of fibres were transfected, these changes in muscle weight must underestimate the actual increase in the mass of the fibres when p97 is inhibited or depleted. In contrast to the DNp97 or shRNA for p97, electroporation of a disease-associated p97 mutant gene, p97R155H (Watts et al, 2004), that is not known to reduce ATPase activity, to trap substrates or function as a dominant negative inhibitor (Halawani et al, 2009; Fernandez-Saiz and Buchberger, 2010; Manno et al, 2010), did not induce appreciable fibre growth in 7 days (i.e., <5% change in median area, 2931 μm2 for transfected fibres versus 2805 for untranfected ones, P=0.05) nor significant change in muscle weight (44.7 mg±4.1 with p97R155H versus 41.9±7.1 in controls, n=5). p97 inhibition stimulated myotube growth by decreasing protein degradation without enhancing overall protein synthesis In the transfected muscle fibres in mice, it is not possible to measure precisely protein synthesis and degradation as is possible with cultured cells. To test whether DNp97 also increases fibre size in myotubes as it does in adult muscles, C2C12 myotubes were infected with adenoviruses encoding GFP, WTp97 or DNp97. Increased levels of p97 protein were evident at 24 h, and subsequently p97 content continued to increase (Figure 4C). By 48 h, overexpression of DNp97 caused a 30% increase in total protein content (P<0.02) (Table I) and in mean cell diameter (P<0.0005) (Figure 3A and B) in accord with the growth-promoting effects of DNp97 in adult muscle (Figure 2B and G). By contrast, no change in cell growth occurred upon overexpression of WTp97. Figure 3.DNp97 increases cell diameter of myotubes. (A) Brightfield images of myotubes expressing GFP or DNp97 for 72 h are shown. Scale bar represents 50 μm. (B) The average diameter of myotubes expressing GFP or DNp97 for 72 h is plotted. ***P<0.0005, n=100. Error bars indicate s.e.m. Download figure Download PowerPoint Table 1. In myotubes, DNp97, but not WTp97, increases total protein content, without perturbing protein synthesis Infection with Protein content (μg protein/well) Protein synthesis (CPM/μg protein) GFP 323.7±18.3 161.0±12.3 WTp97 292.4±16.7 168.7±10.1 DNp97 409.2±19.4 164.3±14.4 Total protein content of myotubes expressing GFP, WTp97 or DNp97 for 48 h in a six-well plate was measured per well. DNp97-expressing myotubes display increased overall protein content by about 30% in 2 days, *P<0.02 versus GFP, n=5. Protein synthesis was determined by measuring the incorporation of 3H-tyrosine for 2 h after 48 h from adenoviral infection, n=10. No differences were found. s.e.m. is reported. We then examined whether the increase in myotube size caused by the DNp97 was due to an overall enhancement of protein synthesis as is characteristic of growing cells, and/or a reduction in overall protein degradation. Although we anticipated finding an increase in protein synthetic rate, when we assayed the rates of incorporation of 3H-tyrosine into total cell proteins, no clear difference was observed upon overexpression of GFP, WTp97 or DNp97 (Table I). Furthermore, no increase in the content or phosphorylation of AKT or mTOR's downstream effectors was found in the myotubes overexpressing the DNp97 (Supplementary Figure S5A and B). This lack of a clear increase in protein synthesis raised the possibility that the DNp97 induced growth primarily by inhibiting overall proteolysis. In fact, by 36 h after adenoviral infection of DNp97, but not of WTp97, polyubiquitinated proteins began to accumulate in the myotubes (as shown using the FK1 antibody, which does not recognize monoubiquitinated or multiple monoubiquitinated proteins) (Figure 4C). Such an accumulation of ubiquitin chains would be expected if p97 plays a general role in the degradation of ubiquitinated proteins by proteasomes, and because the DNp97 complex binds ubiquitin conjugates but, unlike the WT, fails to release them (Ye et al, 2003; Wojcik et al, 2004). We therefore measured the rates of degradation of long-lived proteins (the bulk of cell proteins) as well as short-lived ones by pulse-chase method using 3H-tyrosine (Zhao et al, 2007). Myotubes were infected for 36 h with adenoviruses expressing GFP, WTp97 or DNp97. To follow the degradation of short-lived cell components, myotube proteins were labelled with a 20-min-pulse of 3H-tyrosine, and to selectively label the long-lived ones, cells were exposed to the labelled precursor for 20 h. The cells were then washed and resuspended in a large excess of not labelled tyrosine. Overexpression of WTp97 did not increase the degradation of either short- or long-lived proteins (Figure 4A and B). Therefore, the level of p97 is not rate-limiting for overall protein degradation. By contrast, expression of the DNp97 for 36 h, but not 24 h (not shown), decreased the degradation of short- (Figure 4A) and also long-lived proteins (Figure 4B). These 20–35% reductions in rates of proteolysis were highly reproducible, and if sustained for several days, should have major effects on muscle size. Thus, inhibition of p97 function seems to increase the protein content of myotubes primarily by decreasing overall proteolysis without significantly altering rates of synthesis. Figure 4.In myotubes, DNp97, but not WTp97, decreases degradation of both short- and long-lived proteins. (A, B) Myotubes were infected with adenoviruses encoding GFP (dotted line), WTp97 (dashed line) or DNp97 (full line) for 36 h and then incubated with 3H-tyrosine for the last 20 min (A) or 20 h (B) to differentially label short- and long-lived proteins, respectively. Error bars indicate s.d. n=6. (C) Equal amounts of protein lysates from myotubes expressing WTp97 or DNp97 were loaded and blotted with antibodies against: RGSH4, polyubiquitinated proteins (FK1) and GAPDH as loading control. Thirty-six hours after infection with DNp97-expressing virus, myotubes start to accumulate ubiquitinated proteins. Download figure Download PowerPoint p97 inhibition decreases FoxO3-stimulated proteasomal and lysosomal proteolysis A critical factor in the induction of muscle atrophy is the activation of the transcription factor FoxO3 (Sandri et al, 2004). Overexpression of a constitutively active FoxO3 mutant (caFoxO3) by itself causes profound atrophy by enhancing protein degradation and the expression of genes involved in the ubiquitin-proteasome pathway and autophagy (Mammucari et al, 2007; Zhao et al, 2007). To test whether inhibition of p97 can block the increased proteolysis induced by FoxO3, we compared myotubes expressing GFP, WTp97 or DNp97 for 24 h and then labelled these same cells for 24 h with 3H-tyrosine and concomitantly infected the cells with adenoviruses encoding GFP or caFoxO3. In myotubes, DNp97 overexpression blocked the increase in degradation of long-lived proteins by caFoxO3 (Figure 5A). Thus, p97 function appears essential for the stimulation of overall proteolysis by FoxO3 that is critical in multiple types of atrophy. Figure 5.In myotubes, DNp97, unlike WTp97, completely blocks the stimulation of proteolysis by caFoxO3, without altering the content of some atrogenes. (A) Myotubes expressing GFP, WTp97 or DNp97 for 24 h were given fresh media containing a second adenovirus expressing GFP or caFoxO3 supplemented with 3H-tyrosine to label long-lived proteins. Error bars indicate s.d. ANOVA was performed followed by Tukey's HSD procedure. *P<0.05 DN–GFP versus GFP–GFP, **P<0.05 GFP–caFoxO3 versus GFP–GFP, ***P<0.05 DN–caFoxO3 versus GFP–caFoxO3, n=4. The DNp97-induced decrease in proteolysis was calculated by subtracting the protein degradation rate in the DNp97 overproducing cells from that in the controls (GFP–GFP or GFP–caFoxO3 expressing cells). Error bars indicate s.d. *P<0.001. (B) Overexpression of caFoxO3 in myotubes does not change the endogenous level of p97 protein. Equal amounts of protein from myotubes expressing for 48 h GFP (first three lanes) or caFoxO3 (last three lanes) were loaded. Endogenous p97, FoxO3 and GAPDH were immunoblotted. (C) The content of some atrogenes (atrogin-1, MuRF1, LC3 and Gabarapl1) increases upon overexpression of caFoxO3 but does not change in myotubes expressing WTp97 or DNp97 for 48 h. A lysate of cells treated with Bortezomib for 2 h has been also loaded as additional control for accumulation of polyubiquitin conjugates. Bortz: Bortezomib. Download figure Download PowerPoint Because caFoxO3 overexpression nearly doubled p97-dependent proteolysis (Figure 5A), but did not change the amount of p97 in the cell (Figure 5B), the capacity of the p97 complex to support proteolysis in the myotubes is not saturated. It is also noteworthy that this inhibitory effect of DNp97 on proteolysis did not involve any alteration in the content of FoxO3-induced atrogenes, including the key muscle-specific ubiquitin ligases, atrogin-1 and MuRF1, and the proteins involved in autophagy, Gabarapl1 and LC3 (Figure 5C). Thus, inhibiting p97 did not reduce FoxO-mediated gene expression but blocks the ability of these atrogenes to catalyse proteolysis. Although in atrophying adult muscles, the ubiquitin-proteasome pathway is important in myofibrillar degradation (Solomon and Goldberg, 1996; Cohen et al, 2009), in myotubes, where such proteins are not abundant, the autophagic pathway makes a greater contribution to overall proteolysis (Zhao et al, 2007). To learn how p97 influences these two processes, we measured the FoxO3-induced degradation of long-lived proteins in myotubes and dissected the relative importance of lysosomal and proteasomal pathways using inhibitors of the proteasome (1 μM Bortezomib/Velcade) or lysosomal acidification (10 mM NH4Cl) (Zhao et al, 2007). DNp97 expression decreased both proteasomal and lysosomal pathways in control myotubes as well as ones atrophying due to the overexpression of caFoxO3 (Figure 6A–C). Figure 6.In myotubes, DNp97 blocks the FoxO3-induced proteolysis by inhibiting both proteasomal and lysosomal degradation. (A) Myotubes treated as described in Figure 5A were given fresh DMEM (control) containing 2% HS with or without the proteasomal (Bortezomib=Bortz) or lysosomal (ammonium chloride=NH4Cl) inhibitors. Rates of proteolysis were determined starting 2 h later. Error bars indicate s.d. n=4. (B) The amount of proteolysis sensitive to each inhibitor represented the amount of proteasome or lysosome-mediated degradation and was calculated from data in Figure 6A by subtracting the rates of proteolysis in cells treated with the inhibitors from those of untreated cells. Error bars indicate s.d. (C) The DNp97-induced decrease in proteolysis was calculated by subtracting the amount of lysosomal or proteasomal protein degradation rate (i.e., the inhibitor-sensitive component) in the DNp97 overproducing cells from that in the controls (GFP-expressing cells). ***P<0.005 and *P<0.05 versus GFP–GFP controls. Error bars indicate s.d. Download figure Download PowerPoint p97 inhibition causes muscle growth independently of the myosin-chaperone UNC45B One possible mechanism by which inhibiting proteolysis might promote muscle growth is by causing a buildup of UNC45B, the chaperone necessary for myosin assembly. In muscles of C. elegans, UNC45B is a substrate of p97, and mutations in p97 impair UNC45B degradation (Janiesch et al, 2007). However, during atrophy induced by either denervation or food deprivation (Figure 7A), we did not observe any change in the content of UNC45B as shown by quantitation of bands in SDS–PAGE. Nevertheless, we tested whether the increased fibre size upon inhibition of p97 was in part due to stabilization of UNC45B and thus, to a possible increase in myosin incorporation into myofibrils. When GFP or UNC45B were overexpressed for 7 days, the fibres overexpressing UNC45B were reproducibly only about 10% larger in median area th