1 Polypeptide Laboratory, Department of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada; and 2 MGC-Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University, Rotterdam, 3000 DR Rotterdam, The Netherlands
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ABSTRACT |
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Activated skeletal muscle proteolysis in catabolic states has been linked to an upregulation of the ATP-ubiquitin-dependent proteolytic system. Previous studies suggested that the N-end rule pathway is primarily responsible for the bulk of skeletal muscle proteolysis. The activity of this pathway is dependent on the 14-kDa ubiquitin-conjugating enzyme E214k (HR6B) and the ubiquitin protein ligase Ubr1. To address the requirement of E214k in muscle proteolysis, we examined muscle protein metabolism in wild-type (WT) mice and mice lacking the E214k gene (KO) in fed and fasted (48 h) states. Baseline body weight, muscle mass, and protein content were similar, and these parameters decreased similarly upon fasting in the two genotypes. There were also no effects of genotype on the rate of proteolysis in soleus muscle. The fasting-induced increase in the amount of ubiquitinated proteins was the same in WT and KO mice. The absence of any significant effect of loss of E214k function was not due to a compensatory induction of the closely related isoform HR6A. Total intracellular concentration of E214k and HR6A in the WT mice was 290 ± 40 nM, but the level in the KO mice (reflecting the level of HR6A) was 110 ± 9 nM. This value is about threefold the apparent Michaelis-Menten constant (Km) of E214k (~40 nM) for stimulating conjugation in muscle extracts. Because the HR6A isoform has a Km of 16 nM for stimulating conjugation, the HR6A levels in the muscles of KO mice appear sufficient for supporting conjugation mediated by this pathway during fasting.
ubiquitin conjugation; starvation; muscle wasting; proteasome; muscle incubation
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INTRODUCTION |
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SKELETAL MUSCLE IS THE MAIN REPOSITORY of body proteins. Increased skeletal muscle protein catabolism occurs in many diseased and malnutritional states. For example, in cancer, sepsis, and diabetes, increases in muscle proteolytic rates of up to 50% are described (24, 35, 48, 51). Because some of these conditions are also associated with suppression of muscle protein synthesis (15, 43, 46, and reviewed in Ref. 7), loss of muscle mass of up to 40% can occur (38, 51).
The ubiquitin system is the main cytosolic proteolytic system in
eukaryotes (reviewed in Refs. 12, 55).
Proteins to be degraded by this pathway are first covalently conjugated
through the -amino group of a lysine residue of the substrate to the carboxyl group of the terminal glycine residue of ubiquitin (reviewed in Refs. 22, 36). Studies with inhibitors of
distinct intracellular proteolytic pathways have shown that, in
experimental tumor implantation (8, 53), starvation
(59), sepsis (24, 48, 51), diabetes (11, 35, 38), and muscle denervation (48),
increased skeletal muscle protein catabolism is attributable largely to
activation of the ubiquitin-dependent proteolytic system.
The conjugation of ubiquitin to proteins requires the serial actions of
at least three classes of enzymes. Ubiquitin is first activated by
ubiquitin-activating enzyme (E1), leading to the formation of a thiol
ester bond between the carboxy-terminal glycine of ubiquitin and the
active-site cysteine of the enzyme. The thiol ester-linked ubiquitin is
then transferred to one member of a family of ubiquitin-conjugating
enzymes (E2), which also transiently carries the ubiquitin as a thiol
ester intermediate. The E2 then interacts with a member of the
ubiquitin protein ligase (E3) family. E3s play important roles by
binding substrates. They fall into one of two broad classes, the HECT
(homology to E6-AP carboxy terminus) domain or RING finger-containing
E3s. Members of the HECT family include E6-AP and Rsp5/Nedd4. The RING
finger-containing E3s may be monomeric enzymes (e.g., Ubr1/E3, Rad
5, Mdm2, c-Cbl) or members of stable protein complexes such as the
anaphase-promoting complex or the Skp1-Cullin-F-box protein
(36). Proteins to be degraded usually require
polyubiquitination, in which a chain of at least four ubiquitin
residues is attached to the protein. Polyubiquitinated proteins are
subsequently recognized and degraded by the 26S proteasome, a 2,500-kDa
barrel-shaped multisubunit protein complex.
There appears to be only one major E1, as its inactivation leads to significant defects in both yeast and mammalian cells (17, 34). However, E2s exist as a large family, with 11 members in Saccharomyces cerevisiae (12, 36, 55). The E2s all contain a common core domain of ~150 amino acids and may, in addition, carry NH2- and/or COOH-terminal extensions (12, 36). These differences in primary structure confer specificities on the activities of these enzymes.
Each of these E2s appears to interact with one or more E3s, resulting
in different pathways of ubiquitin conjugation, each recognizing one or
more protein substrates (22, 36). The earliest described
pathway was the N-end rule pathway (4, 50). This rule
relates the metabolic stability of a protein to the identity of its
NH2-terminal residue. This pathway requires UBC2 or the 14-kDa E2 (E214k) as the E2 and Ubr1/E3 as the
recognition component of the pathway. Ubr1/E3
possesses different
binding sites, one of which recognizes basic amino acids at the
NH2 terminus whereas another recognizes hydrophobic amino
acids. A third site recognizes substrates through features other than
their NH2-terminal residue. E3
/Ubr1 specifically binds
E214k/UBC2 at still another site, suggesting a model in
which this E3 promotes conjugation by spatially approximating the
substrate and the E2 charged with ubiquitin.
The N-end rule pathway is thought to be the main pathway of skeletal
muscle protein ubiquitination (44, 45). In support of
this, we and others have shown that increased proteolysis in skeletal
muscle during catabolic states such as fasting (54, 56),
tumor implantation (8, 14, 49, 53), sepsis (25, 51), diabetes (5, 6, 11, 29, 35), administration of
proteolysis-inducing factor (30, 31), head trauma and burn injuries (16, 33), glucocorticoid administration
(2), and disuse atrophy (47) is associated
with two- to sevenfold increases in the gene expression of the
ubiquitin-conjugating enzyme of the N-end rule, E214k.
Others have reported about twofold increases in the gene expression of
E3 in muscle from diabetic and septic animals (18, 29, and reviewed
in Ref. 26). In vitro ubiquitination assays with soluble
extracts from normal and atrophying muscles also revealed that specific
inhibitors of E3
or dominant negative forms of
E214k attenuate protein ubiquitination in those extracts (44). Although these findings are highly suggestive, the
significance of this pathway in muscle proteolysis in vivo remains to
be firmly established.
A direct approach would be to examine in transgenic animals the effects of deletion of components of the N-end rule pathway on skeletal muscle proteolysis. E214k is the rat/rabbit homolog of the S. cerevisiae DNA repair protein UBC2 (58). Two closely related human isoforms of UBC2, HR6A and HR6B, have been previously described (27). HR6B is identical to E214k. Genetic inactivation of HR6B/E214k has been reported. Mice lacking this enzyme are infertile, exhibiting several defects in spermatogenesis (41). Because we have previously shown that E214k/HR6B expression increased in skeletal muscle upon fasting and is suppressed by insulin and insulin-like growth factor I (IGF-I) (56, 57), we tested, in E214k/HR6B knockout animals, whether the increased skeletal muscle protein catabolism seen in fasting would be attenuated. Our data indicate that this conjugating enzyme is nonessential for this catabolism.
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MATERIALS AND METHODS |
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Animals. The inactivation of the HR6B/E214k gene in the mouse has been described previously (41). Sibling wild-type and knockout mice were obtained by breeding heterozygotes. At 7-10 wk, mice were starved for 48 h, as previous studies in rats indicate significant loss of muscle mass and upregulation of E214k mRNA after such a deprivation (56). After the mice were killed, tissues were isolated and analyzed as described in the following sections. To measure dry carcass weights, the heads, paws, and tails were amputated, and the skin and visceral tissues in the abdominal and thoracic cavities were removed. The remaining carcass was then dried in an oven at 60°C for 3-4 days, at which time tissue weight remained constant. All experiments were performed in accordance with the Canadian Council on Animal Care Guidelines and were authorized by the institutional Animal Policy and Welfare Committee.
Muscle incubations. For in vitro measurements of proteolysis, soleus muscles were dissected and mounted on inert plastic supports derived from plastic tubing (Tygon). Incubation was carried out in Krebs-Ringer bicarbonate buffer (in mM: 120 NaCl, 5 KCl, 1 MgSO4, 1 KH2PO4, 25 NaHCO3) supplemented with 5 mM glucose, 170 µM leucine, 100 µM isoleucine, 200 µM valine, 250 µg/ml cycloheximide, and 4 µg/ml insulin, as previously described (59). Muscles were preincubated in 2 ml of buffer for 30 min. Incubation was then resumed in fresh medium and continued for 2 h. Because skeletal muscle neither synthesizes nor degrades tyrosine, the release of this amino acid, as measured fluorometrically (52), into the incubation medium serves as an index of protein degradation.
Northern hybridization. RNA was prepared from muscle by the guanidinium thiocyanate-CsCl method (3) and hybridization carried out as previously described (40, 56). Briefly, 10 µg of RNA were resolved on 1% formaldehyde-containing agarose gels followed by transfer to nylon membranes and cross-linking with ultraviolet light. Membranes were hybridized with a 32P-labeled 354-bp probe corresponding to bases 538-892 of the mHR6B cDNA, a region in the 3'-untranslated region of mHR6B/E214k (27). The probe from the HR6A cDNA (for HR6A Northern) corresponds to bases 275-584 of the mHR6A cDNA (27). After overnight hybridization, membranes were washed and then subjected to autoradiography. To correct for RNA loading, membranes were stripped and reprobed with a 32P-labeled 18S ribosomal RNA probe.
Western blotting.
Endogenous levels of HR6A and HR6B/E214k proteins were
determined by Western blotting as described previously
(40). Proteins (50 µg) from soluble gastrocnemius muscle
extracts were separated by 15% SDS-PAGE and transferred onto 0.1-µm
polyvinylidene difluoride membrane. Membranes were blocked for 1 h
in 5% skim milk protein in TTNS (25 mM Tris, pH 7.5, 0.15 M NaCl,
0.1% Tween 20), rinsed thoroughly in TTNS, and then incubated
sequentially for 1 h each in primary antibody (9 µg/ml
affinity-purified anti-HR6B/E214k raised against the entire
enzyme) and secondary antibody (~80 µg/ml 125I-labeled
goat anti-rabbit IgG) with washing in between. Antibodies were diluted
in TTNS containing 2.5% BSA. Signals were quantified with a
phosphoimager or by autoradiography followed by quantitative densitometry of appropriate bands on X-ray film. To permit absolute quantification of the sum of the HR6A and HR6B/E214k
proteins, purified HR6B/E214k standards that had been
quantitated by absorbance spectrophotometry were included on the gels.
Because the antibody was raised against the entire E214k
protein and because the two proteins are 95% identical in amino acid
sequence, the antibody recognizes both HR6A and HR6B/E214k
isoforms and does so equally well (data not shown). From the
measurements of E2 levels per unit protein and the total protein per
muscle, total E2 per muscle was calculated. Intracellular
concentrations were then calculated by estimating intracellular
cytoplasmic volume. Cytoplasmic volume was estimated at 0.5 ml/g tissue
wet wt. This is based on previous data that indicate that intracellular
aqueous volume is 0.65 ml/g tissue wet wt (13). Because
these E2s are excluded from organelles (Golgi, mitochondria,
endoplasmic reticulum, etc.), we assumed that cytoplasmic volume
represented ~80% of the published total aqueous volume. In some
studies, an antibody that was raised against a peptide corresponding to
the COOH-terminal end of HR6B/E214k and previously found to
be specific for this particular isoform (41) was used. To
measure levels of ubiquitinated proteins in muscle, the tissue was
homogenized in 5 ml/g of 2% SDS, 50 mM Tris-Cl, pH 7.5, and 1 mM
dithiothreitol (DTT), and the lysate was spun briefly to collect all of
the solution at the bottom of the tube. After addition of
-mercaptoethanol to 5% (vol/vol), the lysates were boiled for 10 min and then stored frozen until use. Aliquots (50 µg of proteins)
were run on a 10% SDS-PAGE gel, transferred onto nitrocellulose
membrane, and blotted as above but with a monoclonal anti-ubiquitin
antibody specific for ubiquitinated proteins (FK2, International
Bioscience) followed by 125I-labeled goat anti-mouse
secondary antibody.
Ubiquitination assays. The chloramine-T method was used to label bovine ubiquitin with Na125I to a specific activity of 6,000 cpm/pmol. Unincorporated 125I was removed by passing the reaction products over a Sephadex G25 column.
HR6B/E214k was expressed in Escherichia coli and purified as previously described (39). HR6A was similarly subcloned into the pET11d expression vector (Novagen), expressed, and purified using the same protocol as for HR6B/E214k. Quantitation of enzymatic activity was by thiol ester assays (37). Soluble gastrocnemius muscle homogenates were prepared from wild-type and knockout mice. Muscles were homogenized in 5 ml/g of 50 mM Tris · HCl, pH 7.5, 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 10 µg/ml pepstatin A with the use of a Polytron tissue homogenizer. Homogenates were spun in a microfuge at 16,000 g for 10 min at 4°C. The supernatants were stored atStatistical analyses. One-way analysis of variance or Student's t-test was used to compare means. In experiments examining the effects of genotype and nutritional states (starved or fed), a 2 × 2 analysis of variance was employed and means were separated by the Student-Newman-Keuls test.
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RESULTS |
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Effects of fasting on body mass, visceral tissues, and skeletal
muscle.
Gross phenotypes of HR6B/E214k knockout mice have been
reported previously (41). These mice appear normal except
for defects in spermatogenesis in the testes. Here, we examined the
responses of these mice to food deprivation. In the fed state (Table
1), there were no effects of loss of
E214k on body weight or weights of most of the visceral
tissues. However, the weights of the liver and small intestine were
found to be ~10% higher in the E214k knockout mice. In
response to fasting, the losses of body and tissue masses were similar
in wild-type and knockout mice. This was true also in the liver and the
small intestine, such that the weights of these two tissues remained
~10% higher in the knockout mice.
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Effects on rates of muscle proteolysis.
Despite the aforementioned observations, subtle differences at the
level of muscle protein degradation might not be easily noticed in the
measurements described. Because the N-end rule pathway appears to be
involved in mediating the ubiquitination of the bulk of muscle proteins
before their degradation by the proteasome (44), we
determined whether the loss of E214k, the conjugating
enzyme of this pathway, would lower rates of muscle proteolysis. We
carried out these measurements in the soleus muscle, which has adequate
tendon sizes in the mouse to permit reproducible stretching to resting
length in in vitro incubations (the importance of which is reported in
Ref. 9) and thereby permit reliable measurements of rates
of proteolysis. Although fast-twitch muscles appear more sensitive to
fasting in the rat than slow-twitch muscles such as the soleus
(59), the use of the soleus in the mouse is probably less
susceptible to this effect because it has a mixture of both fast- and
slow-twitch fibers (23). Rates of proteolysis were similar
in muscles from wild-type and knockout mice in the fed state (Table
3). This was also true in the fasted
state. However, the significance of the measurements in the fasted
state is less clear, because older animals had to be used for these studies to yield muscles that remained large enough after fasting to
technically permit reliable stretching during the incubation. Unfortunately, older animals show less of an increase in proteolysis upon fasting (21, 32); accordingly, we were unable to
detect a significant increase in the rate of proteolysis in the muscles from wild-type animals.
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Effects on ubiquitination of skeletal muscle proteins and
expression of the ubiquitin-conjugating enzymes E214k/HR6B
and HR6A.
Although we could not detect differences between the two genotypes in
overall rates of proteolysis, it remained possible that conjugation of
ubiquitin to proteins might be affected by the loss of this E2.
Therefore, we measured steady-state levels of ubiquitinated proteins in
the muscles in the fed and fasted states (Fig.
1). As previously shown in rats
(60), there is an increase in levels of ubiquitinated
proteins upon fasting. However, despite the absence of
HR6B/E214k in the knockout mice, levels of ubiquitinated proteins were similar in the fed state and rose similarly upon fasting.
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Levels of the HR6A enzyme isoform appear sufficient to maintain overall E214k/HR6B- and HR6A-dependent ubiquitination in skeletal muscle. Because we had previously shown that E214k/HR6B and HR6A isoforms of UBC2 together are responsible for ~50% of the rate of conjugation in muscle extracts (39), we were surprised that the loss of HR6B/E214k did not affect steady-state levels of ubiquitinated proteins. We therefore measured the dependence of conjugation in these muscle extracts on concentrations of E214k/HR6B and HR6A. As seen in Table 4, the apparent Km values of HR6B/E214k for stimulating conjugation are ~40 nM and are similar in muscle extracts of wild-type and knockout mice. Such values are low compared with the estimated muscle concentrations of enzyme both in wild-type (288 nM) and in knockout (114 nM) mice. Thus, if HR6A has a Km similar to E214k/HR6B for stimulating conjugation, the rates of conjugation would likely not decrease to a significant extent. In fact, the apparent Km values of HR6A for stimulating conjugation are ~20 nM and are similar in muscle extracts of wild-type and knockout mice (Table 4).
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DISCUSSION |
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These studies are the first to evaluate the role of E214k in vivo in a condition of skeletal muscle atrophy and show that this enzyme is not essential for mediating the increase in muscle protein catabolism upon fasting. Interestingly, however, there were small increases in the weights of the liver and small intestine in the knockout mice. These observations are consistent with some findings that indicate that the ATP-ubiquitin proteolytic system may be important in the regulation of intestinal mass and protein content (1, 42). We are currently exploring further the basis for these differences.
The lack of effect of inactivation of the E214k/HR6B gene on protein catabolism in skeletal muscle was surprising, because numerous studies suggest strongly that the N-end rule pathway of ubiquitin conjugation plays an important role in mediating the activation of proteolysis in skeletal muscle atrophying in various catabolic conditions. In particular, increased HR6B/E214k expression has been reported in a wide array of catabolic conditions (see introductory remarks for details). Most of these studies demonstrated increased expression of the mRNA. Levels of E214k/HR6B protein have been less widely reported on, as antibodies generally are unable to resolve E214k/HR6B from the closely related but unregulated form HR6A. We have not seen increased levels of total HR6B and HR6A in muscle upon fasting (data not shown). Our studies using antibodies specific to E214k/HR6B suggest that E214k/HR6B levels in muscle are similar in the fed and fasted states (Fig. 2C), so the increased mRNA levels are likely required to maintain levels of this enzyme in the face of overall net protein catabolism in this tissue. All of these previous studies together argued that, whether it is to raise or maintain levels, the induction of E214k/HR6B plays some role in mediating the increased rates of proteolysis seen in these tissues. In the absence of any effect of the gene inactivation, it remains mysterious why the induction of this mRNA is so frequently seen in such a variety of catabolic conditions.
The ability of these knockout mice to activate ubiquitin conjugation and protein catabolism in response to fasting may be explained in several ways. First, our data suggest that, despite the much lower total E214k/HR6B and HR6A levels in the muscle of knockout mice, the residual levels are not limiting and are sufficient to maintain near-maximal rates of ubiquitin conjugation (Table 4). Indeed, total HR6B and HR6A levels appear to be similarly high in most other tissues, exceeding widely the apparent Kms for supporting overall conjugation in extracts of those tissues (39). Thus examination of mice lacking both HR6A and HR6B would appear necessary to further examine the role of these E2s. Interestingly, although total HR6A and HR6B levels are similarly high in the testis, loss of HR6B alone does result in defective spermatogenesis (41). Thus, although levels of HR6A may be sufficient to support overall conjugation, there may be specific E3s in male germ cells that have lower affinities for HR6B or both isoforms and require higher levels to mediate ubiquitination. Alternatively, it remains possible that concentrations of these E2s are different in various subcellular compartments or locations and so may be limiting at some of these intracellular sites.
The lack of effect of loss of E214k/HR6B is consistent with
the recent report (28) that inactivation of the Ubr1/E3
gene in mice also has no major effect on muscle mass in the fed state. Muscle mass was slightly smaller but proportionately so to the decrease
in overall body size in these animals. However, the existence of the
closely related isoforms Ubr2 and Ubr3, as well as the embryonic
lethality of Ubr1/Ubr2 knockout mice, also makes it difficult
to decide conclusively whether the N-end rule pathway is involved in
the activation of muscle proteolysis.
Alternatively, these data may argue that the activation of ubiquitin conjugation seen in such conditions occurs by a non-N-end rule but ubiquitin-dependent mechanism or that other pathways of proteolysis are activated. Although we cannot rule out these possibilities, we did not observe any differences in rates of proteolysis in muscles of wild-type and knockout mice measured in the presence of inhibitors of lysosomal and calcium-dependent proteases (data not shown). Of interest, the induction of two other E3s in atrophying skeletal muscles has been recently reported (10, 20). The precise E2s with which these E3s interact remain unknown. One of these E3s possesses an F-box, suggesting that they likely interact with a member of the UBC4 family of E2s or with CDC34. The other contains a RING finger (10) and so may interact with one of several E2 families, including E214k/HR6A/HR6B. Importantly, inactivation of either of these E3s in mice results in decreased atrophy in response to denervation (10). However, neither gene inactivation completely suppressed wasting, which in denervation has been shown to be due solely to activated proteolysis (19). Thus both and possibly additional pathways of ubiquitin conjugation and potentially non-ubiquitin-dependent proteolytic pathways may contribute synergistically to mediate this process. Thus it appears that there is not one common pathway of proteolysis in atrophying skeletal muscles and that the mechanisms of muscle protein wasting and their regulation are more complex than previously envisioned.
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ACKNOWLEDGEMENTS |
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We thank Jocelyn Reid and Zhiyu Pang for help with some of the experiments, and Susan Samuels for helpful suggestions.
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FOOTNOTES |
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This work was supported by the Canadian Institutes of Health Research Grant MT12121 to S. S. Wing and by the Dutch Organization for Scientific Research (GB-MW) and the Dutch Cancer Society (EUR 99-2003) research grants to H. P. Roest. O. A. J. Adegoke is supported by fellowships from the Canadian Diabetes Association and the McGill University Health Centre Research Institute. S. S. Wing is a recipient of a Chercheur Boursier award from the Fonds de la Recherche en Santé du Québec.
Address for reprint requests and other correspondence: S. S. Wing, Polypeptide Laboratory, Dept. of Medicine, McGill Univ., 3640 Univ. St., Montreal, Quebec H3A 2B2, Canada (E-mail: simon.wing{at}mcgill.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 30, 2002;10.1152/ajpendo.00097.2002
Received 4 March 2002; accepted in final form 25 April 2002.
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