Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin (original) (raw)
Related papers
Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system
Biochemical Journal, 1987
Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This prote...
Proteolytic Activity of the ATP-dependent Protease HslVU Can Be Uncoupled from ATP Hydrolysis
Journal of Biological Chemistry, 1997
HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase. Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins. We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g. casein). Also, the nonhydrolyzable analogs of ATP, 5-adenylyl ,␥-imidodiphosphate (AMP-PNP) and adenosine 5-(␣,-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP. This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together. Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog. The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP. NH 4 ؉ and Cs ؉ had similar effects as K ؉ , but Na ؉ and Li ؉ were ineffective. AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K ؉ , Cs ؉ , or NH 4 ؉ , AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed. In addition, MgCl 2 , MnCl 2 , and CaCl 2 allowed some peptidase and caseinase activity in the absence of any nucleotide. However, Mn 2؉ and Ca 2؉ , unlike Mg 2؉ , abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP. These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process. Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites. However, AMP-PNP binding together with K ؉ can induce a form of HslVU that degrades proteins without energy consumption.
Studies of the ATP-dependent proteolytic enzyme, protease La, from Escherichia coli
Journal of Biological Chemistry, 1982
The ATP-dependent proteolytic activity, protease La, *om Escherichia coli has been partially purified, and the role of ATP investigated. ATP (3 m) stimulated degradation of [methyL3HJcasein and [methyl-'*C]apohemoglobin 8-40-fold but only when M&' was present. The nucleotide had to be continuously present for this process, since rapid depletion of ATP prevented further proteolysis. A concentration of 260 BM MgATP gave half-maximal activity, but the effects of different concentrations of ATP depended also on the Mg' concentration, and high concentrations of nucleotides inhibited. dATP, CTP, and U T P also stimulated proteolysis but not as well as ATP, while GTP, ADP, AMP, pyrophosphate, and the nonmetabolizable analogs a,/3-methylene-ATP, B,y-methylene-ATP, adenosine 5'-0-(3-thiotriphosphate), and 2',3'-dialdehyde-ATP, had little or no effect. These analogs, as well as ADP and AMP, inhibited the effect of ATP, probably by competition for the same binding sites. Both ATP and ADP appeared to bind to and stabilize the protease (even in the absence of M&+), since they prevented the rapid loss of activity that occurred at 42 "C. No protein kinase or protein adenylylase activity was demonstrable in these preparations. ATP-dependent proteolysis also was not affected by the addition of termination factor riw or ubiquitin, and these preparations did not conjugate ubiquitin to protein substrates. Although certain inhibitors of ATPases (NJV" dicyclohexylcarbodiimide, oligomycin, azide) did not affect casein hydrolysis, others (vanadate, Dio-9, quercetin) markedly inhibited this process. Proteolysis ceased rapidly after addition of vanadate; thus, continued splitting of ATP seems necessary, but a simple relationship between ATPase activity and proteolysis was not clear in these impure preparations. At concentrations above 200 m, phosphate markedly inhibited the ATP-dependent activity and stimulated proteolysis slightly in the absence of ATP. These various properties suggest that ATP cleavage is required for proteolysis and distinguish protease La from the recA gene product and the cytoplasmic ATPstimulated proteases of mammalian cells. One of the fundamental and unexplained features about intracellular protein degradation is its apparent requirement * These studies have been supported by research grants from the Juvenile Diabetes Fund, the Kroc Foundation, and the National Institute of Communicative and Neurological Diseases and Stroke. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Journal of Biological Chemistry, 1996
Recent studies have suggested that activation of the ubiquitin-proteasome pathway is primarily responsible for the rapid loss of muscle proteins in various types of atrophy. The present studies were undertaken to test if different classes of muscle proteins are degraded by this pathway. In extracts of rabbit psoas muscle, the complete degradation of soluble proteins to amino acids was stimulated up to 6-fold by ATP. Peptide aldehyde inhibitors of the proteasome or the removal of proteasomes markedly inhibited only the ATP-dependent process. Addition of purified myosin, actin, troponin, or tropomyosin to these extracts showed that these proteins served as substrates for the ubiquitin-proteasome pathway. By contrast, degradation of myoglobin did not require ATP, proteasomes, or any known proteases in muscles. When myosin, actin, and troponin were added as actomyosin complexes or as intact myofibrils to these extracts, they were not hydrolyzed at a significant rate, probably because in these multicomponent complexes, these proteins are protected from degradation. Accordingly, actin (but not albumin or troponin) inhibited the degradation of 125 I-myosin, and actin was found to selectively inhibit ubiquitin conjugation to 125 I-myosin. Also, the presence of tropomyosin inhibited the degradation of 125 I-troponin. However, neither actin nor tropomyosin inhibited the degradation of 125 I-lysozyme or soluble muscle proteins. Thus, specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation, and the ratelimiting step in their degradation is probably their dissociation from the myofibril.
Molecular and cellular biochemistry, 2001
The activity of ATP, ubiquitin (Ub)-dependent proteases partially purified from skeletal muscle (psoas) from alloxan diabetic rabbits was determined at different periods of insulin deficiency. Two days after alloxan injection, no change was observed in the activity of ATP, Ub-dependent proteases, but this activity increased 3 and 5 days after diabetes induction, attaining 181% of control values on the 5th day. However, after this early rise, the activity of muscle ATP, Ub-dependent proteases decreased, returning to values that did not differ significantly from controls 7 and 10 days after alloxan injection. After 15 days, the activity of these proteases was 57% lower than in muscle from control rabbits. Both the initial increase and the subsequent fall in the activity of the enzymes were prevented by insulin treatment of alloxan diabetic rabbits. The data suggest that Ub-proteasome-dependent proteolysis have an important role in the control of muscle protein degradation and may be r...
The mechanism and functions of ATP-dependent proteases in bacterial and animal cells
EJB Reviews, 1993
In eukaryotes and prokaryotes, the degradation of most cell proteins requires metabolic energy (Goldberg and St. John, 1976; Hershko and Ciechanover, 1982). This feature of intracellular proteolysis applies not only for cytosolic proteins in bacterial and animal cells. Mitochondria (Desautels and Goldberg, 1982a) and chloroplasts (Malek et al., 1984) also contain systems for complete degradation of abnormal proteins, and this process also requires ATP. An ATP requirement for proteolysis is surprising on thermodynamic grounds, since hydrolysis of peptide bonds should be a spontaneous, exergonic process, and protein breakdown by traditional proteases does not require energy-rich cofactors (Goldberg and St. John, 1976). Initial speculations concerning the energy requirement for proteolysis in eukaryotic cells led to the suggestion that ATP might be necessary for the function of lysosomes in which protein degradation was assumed to occur. However, bacteria lack such organelles, but do show a similar ATP dependence for proteolysis as animal cells (Olden and Goldberg, 1978). Therefore, this requirement must represent a more fundamental property of the degradative process. For a number of years, our laboratory has been attempting to understand the biochemical basis of this ATP requirement, because it seemed to represent an important clue for identifying the responsible degradative system, and also because this requirement suggested the existence of novel biochemical mechanisms. Both these assumptions have proven valid. Attempts to understand the basis of this requirement led first to the discovery of the soluble ATP-dependent system for protein breakdown in extracts of animal (Etlinger and Goldberg, 1977) and bacterial cells (Murakami et al., 1979). Subsequent work with these cell-free extracts led to the discovery in reticulocyte extracts of the involvement of ubiquitin (Hershko and Ciechanover, 1982) and in Escherichia coli of a new type of proteolytic enzyme whose activity is coupled to ATP hy
Br J Cancer, 2002
The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C 2 C 12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped doseresponse curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 mM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the a-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E2 14k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysisinducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia.
Ubiquitin-proteasome-dependent proteolysis in skeletal muscle
Reproduction Nutrition Development, 1998
The ubiquitin-proteasome proteolytic pathway has recently been reported to be of major importance in the breakdown of skeletal muscle proteins. The first step in this pathway is the covalent attachment of polyubiquitin chains to the targeted protein. Polyubiquitylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyse recent findings in the regulation of this pathway, both in animal models of muscle wasting and in some human diseases. The identification of regulatory steps of ubiquitin conjugation to protein substrates and/or of the proteolytic activities of the proteasome should lead to new concepts that can be used to manipulate muscle protein mass. Such concepts are essential for the development of anti-cachectic therapies for many clinical situations. @ Inra/Elsevier, Paris skeletal muscle / protein breakdown / ubiquitin / proteasome Résumé -Protéolyse musculaire ubiquitine-protéasome dépendante. Il a été récemmment démontré que la protéolyse ubiquitine-protéasome dépendante joue un rôle majeur dans la dégradation des protéines musculaires. La première étape de cette voie protéolytique correspond à la fixation covalente de chaînes polyubiquitylées sur les substrats protéiques. Ces protéines polyubiquitylées sont ultérieurement spécifiquement reconnues et dégradées par le protéasome 26S. Les derniers progrès accomplis dans la compréhension de la régulation de ce système dans les modèles animaux de fontes protéiques musculaires, et dans certaines situations cliniques, sont analysés de façon critique dans cette revue bibliographique. L'identification d'étapes régulatrices de 1'ubiquitylation des substrats protéiques, et/ou des activités protéolytiques du protéasome, devrait conduire à de nouveaux concepts utilisables pour manipuler le dépôt des protéines musculaires. En particulier, ces concepts sont indispensables au développement de traitements anti-cachectiques dans de nombreuses situations cliniques. @ Inra/Elsevier,
Role of ATP hydrolysis in the degradation of proteins by protease la fromEscherichia coli
Journal of Cellular Biochemistry, 1986
Protein degradation in animal and bacterial cells is dependent upon ATP [ 1,2]. In bacteria [3-61 and mammalian mitochondria [7], this energy dependence arises in large part from the involvement of a novel type of proteinase whose function is directly coupled to ATP hydrolysis [2,3]. The best-characterized of these ATPdependent enzymes is protease La from Escherichiu coli [3-71. This unusual enzyme is both a serine proteinase and an ATPase [4,5]. In fact the rate of protein degradation is directly proportional to the rate of ATP consumption [3-51. Protein substrates stimulate ATP cleavage by protease La, but nonhydrolyzable proteins or small peptide substrates have no effect on this ATPase activity [4,5]. Previous studies [5] have shown that ATP hydrolysis is required for the degradation of proteins to acid-soluble products, but the hydrolysis of small peptides requires only the binding of a nucleotide to the enzyme. For example, nonhydrolyzable ATP analogues can support hydrolysis of oligopeptides but not protein degradation [5]. On this basis, a multistep model of protein degradation has been proposed in which ATP binding activates the enzyme for each round of proteolysis (Fig. 1) [5]. Since peptide bond cleavage in small peptides does not require ATP hydrolysis [5], the latter process must serve another important function in the degradation of large polypeptides. For example, ATP consumption may promote the release of the polypeptide products from the enzyme; alternatively, ATP hydrolysis may permit the translocation of the enzyme along the substrate to the next cleavage site [5]. One prediction of such mechanisms is that in the presence of a nonhydrolyzable ATP analogue, a single cleavage or only a limited number of cleavages should occur. Previous studies of the function of protease La have measured the generation of acidsoluble peptides and therefore would not have detected limited proteolysis that resulted in large acid-precipitable fragments.