USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

Deubiquitinating enzymes in skeletal muscle atrophy—An essential role for USP19

The ubiquitin proteasome system is well recognized to be involved in mediating muscle atrophy in response to diverse catabolic conditions. To date, almost all of the genes that have been implicated are ubiquitin ligases. Although ubiquitination is modulated also by deubiquitinating enzymes, the roles of these enzymes in muscle wasting remains largely unexplored. In this article, the potential roles of deubiquitinating enzymes in regulating muscle size are discussed. This is followed by a review of the roles described for USP19, the deubiquitinating enzyme that has been most studied in muscle wasting. This enzyme is upregulated in muscle in many catabolic conditions and its inactivation leads to protection from muscle loss induced by stimuli that are common in many illnesses causing cachexia. It can regulate both protein synthesis and protein degradation as well as myogenesis, thereby modulating the key processes that control muscle mass. Roles for other deubiquitinating enzymes remain possible and to be explored.     

Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome

The post-translational modification of proteins by covalent attachment of ubiquitin targets these proteins for degradation by the proteasome. An astounding number of proteins are involved in ubiquitination and deubiquitination of proteins. The pathways are combinatorial, and selectivity of proteolysis will depend strongly on the exact combination of ubiquitinating and deubiquitinating enzymes present at any time. In addition to temporal control, it is likely that these modifications are also regulated spatially. In this review, we discuss the regulation of ubiquitination by enzymes of this pathway and highlight some of the outstanding problems in understanding this regulation.     

Adaptation of the ubiquitin-proteasome proteolytic pathway in cancer cachexia

The ubiquitin-proteasome proteolytic pathway is 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. Polyubiquitinylated proteins are then recognized and degraded by the 26S proteasome complex. In this review, we critically analyze recent findings in the regulation of ubiquitinylation of protein substrates and of their subsequent proteasome-dependent degradation in animal models of cancer cachexia. In particular, we discuss the influence of various mediators (anorexia, hormones, prostaglandins, cytokines, and proteolysis-inducing factor) in signaling the activation of ubiquitin-proteasome proteolysis in skeletal muscle. These findings have lead to new concepts that are starting to be used for preventing cachexia in cancer and other wasting diseases.     

Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin-proteasome pathway

Ubiquitination of proteins is now recognized to target proteins for degradation by the proteasome and for internalization into the lysosomal system, as well as to modify functions of some target proteins. Although much progress has been made in characterizing enzymes that link ubiquitin to proteins, our understanding of deubiquitinating enzymes is less developed. These enzymes are involved in processing the products of ubiquitin genes which all encode fusion proteins, in negatively regulating the functions of ubiquitination (editing), in regenerating free ubiquitin after proteins have been targeted to the proteasome or lysosome (recycling) and in salvaging ubiquitin from possible adducts formed with small molecule nucleophiles in the cell. A large number of genes encode deubiquitinating enzymes suggesting that many have highly specific and regulated functions. Indeed, recent findings provide strong support for the concept that ubiquitination is regulated by both specific pathways of ubiquitination and deubiquitination. Interestingly, many of these enzymes are localized to subcellular structures or to molecular complexes. These localizations play important roles in determining specificity of function and can have major influences on their catalytic activities. Future studies, particularly aimed at characterizing the interacting partners and potential substrates in these complexes as well as at determining the effects of loss of function of specific deubiquitinating enzymes will rapidly advance our understanding of the important roles of these enzymes as biological regulators.     

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines

Summary. Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.     

自噬系统和蛋白酶体系统之间的相互联系

在真核细胞中已发现两条主要降解途径,即自噬系统和蛋白酶体系统．长期以来,这两条降解途径一直被认作是完全独立的路径,然而最近的证据强烈提示,这两条主要降解途径之间相互联系．其中,发现干扰这两条途径的任一条可影响另一条途径的活性,抑制蛋白酶体可刺激自噬活性．同时发现泛素的作用比先前想象更广泛,不仅具有标记蛋白酶体降解蛋白质这一 “经典作用”,还涉及自噬-溶酶体途径降解底物泛素化,是这些主要降解途径的共同标签．这些降解系统分享某些底物和调节分子,显示协同作用,在某些背景下,显示补偿功能．降解系统之间的协同和补偿作用在许多细胞过程中显得至关重要．因此这些降解系统异常或联系失常不仅导致细胞功能的异常,而且也与多种重要疾病的发生和发展密切相关．对这些降解途径功能及其联系的深入了解可拓展人们对这些降解途径的认识,有助于对多种细胞分解代谢过程的深入理解,也有助于相关新药的研发．     

TRAF6 coordinates the activation of autophagy and ubiquitin-proteasome systems in atrophying skeletal muscle

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.     

Degradation of skeletal muscle protein during growth and development of salmonid fish

Published data and the results of the authors’ own studies on the role of intracellular proteolytic enzymes and the metabolic and signaling processes regulated by these enzymes at certain stages of growth and development of salmonid fishes are analyzed in the present review. The major pathways of intracellular proteolysis relying on autophagy, proteasome activity, and calpain activity are considered, as well as the relative contribution of these pathways to proteolysis in skeletal muscle of the fish. Skeletal muscle accounts for more than half of the weight of the fish and undergoes the most significant changes due to the action of anabolic and catabolic signals. Special attention is paid to the intensity of protein degradation during the active growth period characterized by a high rate of protein synthesis and metabolism in fish, as well as to protein degradation during the reproductive period characterized by predomination of catabolic processes in contrast to the growth period. Skeletal muscle plays a unique role as a source of plastic and energy substrates in fish, and, therefore, the process of muscle protein degradation is regarded as a key mechanism for the regulation of growth intensity in juvenile salmon and for maintenance of viability and reproductive capacity of salmonid fish during the maturation of gametes, starvation, and migration related to spawning. The possibility of using a set of parameters of intracellular proteolysis to characterize the early development of salmonids is demonstrated in the review.     

Insulin-like growth factor-1 and muscle wasting in chronic heart failure

Chronic heart failure is a clinical syndrome of cardiac origin, which affects various organ systems. It is associated with metabolic abnormalities leading to a catabolic syndrome in advanced stages of the disease. As in several other chronic diseases, skeletal muscle dysfunction and structural muscle abnormalities result in progressive muscle wasting and cachexia. These changes are accompanied by increased expression of proinflammatory cytokines, increased rate of apoptosis and activation of the proteolytic ubiquitin-proteasome pathway. Further, reduced expression of the local anabolic insulin-like growth factor-1 has been demonstrated in skeletal muscle of animals and patients with chronic heart failure. This suppression occurs in the presence of normal serum levels of insulin-like growth factor-1. In addition to catabolic effects of proinflammatory cytokines, these recent findings are consistent with reduced anabolism involving altered local insulin-like growth factor-1 levels in progressive muscle atrophy in chronic heart failure. This article describes local effects of insulin-like growth factor-1 on skeletal muscle function and morphology, its role in stem cell recruitment and muscle regeneration as well as its regulation in circumstances of muscle inflammation and wasting.     

Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of "cachectic-like" muscle wasting in mice. The mechanism of action of myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with myostatin promoted dramatic myotubular atrophy. Mechanistically, myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated Forkhead Box-O (FOXO1) and FOXO3. We further show that addition of myostatin results in enhanced activation of atrogin-1 and muscle-specific RING finger protein 1 (MURF1) and reduced expression of both myosin light chain (MYL) and myosin heavy chain (MYH). In addition, we found that myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and atrogin-1. Therefore, these data suggest that the mechanism through which myostatin promotes muscle wasting is very well conserved between species, and that myostatin-induced human myotube atrophy is mediated through inhibition of insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-K)/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.     

Ubiquitin-proteasome-dependent proteolytic activity remains elevated after zymosan-induced sepsis in rats while muscle mass recovers

We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery process. Protein levels of 19S complex and 20S proteasome subunits did not increase throughout the study, pointing to alternative mechanisms regulating proteasome activities. Overall these data support a role for ubiquitin-proteasome dependent proteolysis in the zymosan septic model, in both the catabolic and muscle recovery phases.     

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin‐1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1 but it is not known if atrogin‐1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin‐1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain‐, and caspase‐3‐dependent protein breakdown in addition to proteasome‐dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin‐1 and MuRF1 mRNA levels. The same treatment increased proteasome‐, cathepsin L‐, and calpain‐dependent proteolytic rates by approximately 40% but did not influence caspase‐3‐dependent proteolysis. The expression of atrogin‐1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley‐Liss, Inc.     

Cellular Localization and Associations of the Major Lipolytic Proteins in Human Skeletal Muscle at Rest and during Exercise

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis.     

Analysis of ubiquitination in vivo using a transgenic mouse model

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.     

Insulin action on skeletal muscle protein metabolism during catabolic states

Insulin plays a major role in the regulation of skeletal muscle protein turnover but its mechanism of action is not fully understood, especially in vivo during catabolic states. These aspects are presently reviewed. Insulin inhibits the ATP-ubiquitin proteasome proteolytic pathway which is presumably the predominant pathway involved in the breakdown of muscle protein. Evidence of the ability of insulin to stimulate muscle protein synthesis in vivo was also presented. Many catabolic states in rats, e.g. streptozotocin diabetes, glucocorticoid excess or sepsis-induced cytokines, resulted in a decrease in insulin action on protein synthesis or degradation. The effect of catabolic factors would therefore be facilitated. In contrast, the antiproteolytic action of insulin was improved during hyperthyroidism in man and early lactation in goats. Excessive muscle protein breakdown should therefore be prevented. In other words, the anabolic hormone insulin partly controlled the 'catabolic drive'. Advances in the understanding of insulin signalling pathways and targets should provide information on the interactions between insulin action, muscle protein turnover and catabolic factors.     

Muscle wasting in cardiac cachexia

Cardiac cachexia is a serious complication of chronic heart failure which is characterized by complex changes that overall lead to a catabolic/anabolic imbalance resulting in body wasting and a poor prognosis. The wasting process affects all body components, but particularly the skeletal musculature, causing extreme fatigue and weakness, especially in cachectic heart failure patients. Available evidence suggests that several pathophysiologic pathways play a role in the muscle wasting process. Metabolic, neurohormonal, and immune abnormalities lead to an altered regulation of proliferation, differentiation, apoptosis, and metabolism in skeletal muscle, finally resulting in deterioration of the underlying cause with symptomatic exercise intolerance. Possible treatment strategies against muscle wasting and cachexia in chronic heart failure are also described here. As there is no validated therapy for cardiac cachexia yet, further research is necessary to find more therapeutic options for the wasting process.     

Skeletal muscle metabolism in physiology and in cancer disease

Skeletal muscle is a tissue of high demand and it accounts for most of daily energy consumption. The classical concept of energy metabolism in skeletal muscle has been profoundly modified on the basis of studies showing the influence of additional factors (i.e., uncoupling proteins (UCPs) and peroxisome proliferator activated receptors (PPARs)) controlling parameters, such as substrate availability, cellular enzymes, carrier proteins, and proton leak, able to affect glycolysis, nutrient oxidation, and protein degradation. This extremely balanced system is greatly altered by cancer disease that can induce muscle cachexia with significant deleterious consequences and results in muscle wasting and weakness, delaying or preventing ambulation, and rehabilitation in catabolic patients.     

Ubiquitin-protein ligases in muscle wasting

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.