Didier Attaix

Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.

Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes.

The ubiquitin-proteasome system and skeletal muscle wasting.

The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

We tested the hypothesis that skeletal muscle ubiquitin–proteasome‐dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome‐dependent proteolysis in incubated muscles from 8‐ and 22‐month‐old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22‐month‐old rats compared with 8‐month‐old animals, while the rate of ubiquitination was not altered. Furthermore, the down‐regulation of in vitro proteasome‐dependent proteolysis that prevailed in the postprandial state in 8‐month‐old rats was defective in 22‐month‐old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine‐supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome‐dependent proteolysis in 22‐month‐old animals, by down‐regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long‐lasting effects by comparing 8‐ and 22‐month‐old rats that were fed either a leucine‐supplemented diet or an alanine‐supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22‐month‐old rats fed the leucine‐supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14‐kDa ubiquitin‐conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22‐month‐old animals chronically fed the leucine‐supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down‐regulation of in vitro proteasome‐dependent proteolysis in 22‐month‐old rats was restored in animals chronically fed a leucine‐supplemented diet.

Skeletal muscle proteolysis in aging

Purpose of reviewTo understand age-related changes in proteolysis and apoptosis in skeletal muscle in relation to oxidative stress and mitochondrial alterations. Recent findingsDuring aging, a progressive loss of muscle mass (sarcopenia) has been described in both human and rodents. Sarcopenia is attributable to an imbalance between protein synthesis and degradation or between apoptosis and regeneration processes or both. Major age-dependent alterations in muscle proteolysis are a lack of responsiveness of the ubiquitin–proteasome-dependent proteolytic pathway to anabolic and catabolic stimuli and alterations in the regulation of autophagy. In addition, increased oxidative stress leads to the accumulation of damaged proteins, which are not properly eliminated, aggregate, and in turn impair proteolytic activities. Finally, the mitochondria-associated apoptotic pathway may be activated. These age-induced changes may contribute to sarcopenia and decreased ability of old individuals to recover from stress. SummaryAlterations in proteasome-dependent or lysosomal proteolysis, increased oxidative stress, mitochondrial dysfunction, and apoptosis presumably contribute to the development of sarcopenia.

Ubiquitin-proteasome-dependent proteolysis in skeletal muscle

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.

Pressure support ventilation attenuates ventilator-induced protein modifications in the diaphragm

IntroductionControlled mechanical ventilation (CMV) induces profound modifications of diaphragm protein metabolism, including muscle atrophy and severe ventilator-induced diaphragmatic dysfunction. Diaphragmatic modifications could be decreased by spontaneous breathing. We hypothesized that mechanical ventilation in pressure support ventilation (PSV), which preserves diaphragm muscle activity, would limit diaphragmatic protein catabolism.MethodsForty-two adult Sprague-Dawley rats were included in this prospective randomized animal study. After intraperitoneal anesthesia, animals were randomly assigned to the control group or to receive 6 or 18 hours of CMV or PSV. After sacrifice and incubation with 14C-phenylalanine, in vitro proteolysis and protein synthesis were measured on the costal region of the diaphragm. We also measured myofibrillar protein carbonyl levels and the activity of 20S proteasome and tripeptidylpeptidase II.ResultsCompared with control animals, diaphragmatic protein catabolism was significantly increased after 18 hours of CMV (33%, P = 0.0001) but not after 6 hours. CMV also decreased protein synthesis by 50% (P = 0.0012) after 6 hours and by 65% (P < 0.0001) after 18 hours of mechanical ventilation. Both 20S proteasome activity levels were increased by CMV. Compared with CMV, 6 and 18 hours of PSV showed no significant increase in proteolysis. PSV did not significantly increase protein synthesis versus controls. Both CMV and PSV increased protein carbonyl levels after 18 hours of mechanical ventilation from +63% (P < 0.001) and +82% (P < 0.0005), respectively.ConclusionsPSV is efficient at reducing mechanical ventilation-induced proteolysis and inhibition of protein synthesis without modifications in the level of oxidative injury compared with continuous mechanical ventilation. PSV could be an interesting alternative to limit ventilator-induced diaphragmatic dysfunction.

Mechanisms of skeletal muscle atrophy.

Purpose of reviewRecent clinical and mechanistic studies have shown that increased proteolysis is a major determinant of muscle wasting in numerous catabolic states and of alterations in myopathies or dystrophies. The implications of these observations for improving muscle mass and function are discussed. Recent findingsSeveral proteolytic systems (i.e. the ubiquitin–proteasome system, the lysosomal, the Ca2+-dependent, and the caspase systems) are responsible for muscle wasting. The Ca2+-dependent and caspase systems may initiate myofibrillar proteolysis. The ubiquitin–proteasome system is believed to degrade actin and myosin heavy chain and, consequently, plays a major role in muscle wasting. Multiple steps in the ubiquitin–proteasome system (ubiquitination, deubiquitination, proteasome activities) are upregulated in muscle wasting diseases. Few key components of the ubiquitin–proteasome system that are strictly necessary for muscle wasting have been so far characterized. Recent studies have led to the elucidation of various signaling pathways of the ubiquitin–proteasome system that are activated in muscle wasting conditions. SummaryAlthough the precise role of the different muscle proteolytic machineries is still largely unknown, current studies are leading to new pharmacologic approaches that can be useful in blocking or partially preventing muscle wasting or improving muscle function in human patients.

Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1

Muscle atrophy prevails in numerous diseases (cancer cachexia, renal failure, infections, etc.), mainly results from elevated proteolysis, and is accelerated by bed rest. This largely contributes to increased health costs. Devising new strategies to prevent muscle wasting is a major clinical challenge. The ubiquitin proteasome system (UPS) degrades myofibrillar proteins, but the precise mechanisms responsible for actin breakdown are surprisingly poorly characterized. We report that chimeric flag‐actin was destabilized and polyubiquitinylated in stably transfected C2C12 myotubes treated with the catabolic agent dexa‐methasone (1 μM) and that only proteasome inhibitors blocked its breakdown. Actin polyubiquitinylation was also detected in wild‐type C2C12 myotubes and human muscle biopsies from control participants and patients with cancer. The muscle‐specific E3 ubiquitin ligase MuRF1 is up‐regulated in catabolic conditions and polyubiquitinylates components of the thick filament. We also demonstrate that recombinant GST‐MuRF1 physically interacted and polyubiquitinylated actin in vitro and that MuRF1 is a critical component for actin breakdown, since MuRF1 siRNA stabilized flag‐actin. These data identify unambiguously the abundant contractile protein actin as a target of the UPS in skeletal muscle both in vitro and in vivo, further supporting the need for new strategies blocking specifically the activation of this pathway in muscle wasting conditions.—Polge, C., Heng, A.‐E., Jarzaguet, M., Ventadour, S., Claustre, A., Combaret, L., Béchet, D., Matondo, M., Uttenweiler‐Joseph, S., Monsarrat, B., Attaix, D., Taillandier, D. Muscle actin is polyubiquitinylated in vitro and in vivo and targeted for breakdown by the E3 ligase MuRF1. FASEB J. 25, 3790–3802 (2011). www.fasebj.org

Regulation of proteolysis.

The mechanisms of proteolysis remain to be fully defined. This review focuses on recent advances in our understanding of the ubiquitin-proteasome-dependent pathway, which is involved in the control of many major biological functions. The ubiquitinylation/deubiquitinylation system is a complex machinery responsible for the specific tagging and proof-reading of substrates degraded by the 26S proteasome, as well as having other functions. The formation of a polyubiquitin degradation signal is required for proteasome-dependent proteolysis. Several families of enzymes, which may comprise hundreds of members to achieve high selectivity, control this process. The substrates tagged by ubiquitin are then recognized by the 26S proteasome and degraded into peptides. In addition, the 26S proteasome also recognizes and degrades some non-ubiquitinylated proteins. In fact, there are multiple ubiquitin- or proteasome-dependent pathways. These systems presumably degrade specific classes of substrates and single proteins by alternative mechanisms and could be interconnected. They may also interfere or cooperate with other proteolytic pathways.

Manipulation of the ubiquitin-proteasome pathway in cachexia: pentoxifylline suppresses the activation of 20S and 26S proteasomes in muscles from tumor-bearing rats

The development of pharmacological approaches for preventing the loss of muscle proteins would be extremely valuable for cachectic patients. For example, severe wasting in cancer patients correlates with a reduced efficacy of chemotherapy and radiotherapy. Pentoxifylline (PTX) is a very inexpensive xanthine derivative, which is widely used in humans as a haemorheological agent, and inhibits tumor necrosis factor transcription. We have shown here that a daily administration of PTX prevents muscle atrophy and suppresses increased protein breakdown in Yoshida sarcoma-bearing rats by inhibiting the activation of a nonlysosomal, Ca2+-independent proteolytic pathway. PTX blocked the ubiquitin pathway, apparently by suppressing the enhanced expression of ubiquitin, the 14-kDa ubiquitin conjugating enzyme E2, and the C2 20S proteasome subunit in muscle from cancer rats. The 19S complex and 11S regulator associate with the 20S proteasome and regulate its peptidase activities. The mRNA levels for the ATPase subunit MSS1 of the 19S complex increased in cancer cachexia, in contrast with mRNAs of other regulatory subunits. This adaptation was suppressed by PTX, suggesting that the drug inhibited the activation of the 26S proteasome. This is the first demonstration of a pharmacological manipulation of the ubiquitin-proteasome pathway in cachexia with a drug which is well tolerated in humans. Overall, the data suggest that PTX can prevent muscle wasting in situations where tumor necrosis factor production rises, including cancer, sepsis, AIDS and trauma.