benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Muscular-Atrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

Topics: Animals; Disease Models, Animal; Drosophila melanogaster; Gene Expression Regulation; Genetic Testing; Humans; Leupeptins; Muscular Atrophy; Muscular Dystrophy, Oculopharyngeal; Mutation; Poly(A)-Binding Protein I; Proof of Concept Study; Proteasome Endopeptidase Complex; Protein Aggregates; Ubiquitin

Skeletal muscle is adversely affected in type-1 diabetes, and excessively stimulated ubiquitin-proteasome system (UPS) was found to be a leading cause of muscle wasting or atrophy. The role of endoplasmic reticulum (ER) stress in muscle atrophy of type-1 diabetes is not known. Hence, we investigated the role of UPS and ER stress in the muscle atrophy of chronic diabetes rat model.. Diabetes was induced with streptozotocin (STZ) in male Sprague-Dawley rats and were sacrificed 2- and 4-months thereafter to collect gastrocnemius muscle. In another experiment, 2-months post-STZ-injection diabetic rats were treated with MG132, a proteasome inhibitor, for the next 2-months and gastrocnemius muscle was collected.. The muscle fiber cross-sectional area was diminished in diabetic rats. The expression of UPS components: E1, MURF1, TRIM72, UCHL1, UCHL5, ubiquitinated proteins, and proteasome activity were elevated in the diabetic rats indicating activated UPS. Altered expression of ER-associated degradation (ERAD) components and increased ER stress markers were detected in 4-months diabetic rats. Proteasome inhibition by MG132 alleviated alterations in the UPS and ER stress in diabetic rat muscle.. Increased UPS activity and ER stress were implicated in the muscle atrophy of diabetic rats and proteasome inhibition exhibited beneficiary outcome.

Topics: Animals; Diabetes Mellitus, Experimental; Endoplasmic Reticulum Stress; Leupeptins; Male; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Tripartite Motif Proteins; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases; Ubiquitinated Proteins; Vesicular Transport Proteins

Oxidative stress is thought to be one of the most important mechanisms implicated in the muscle wasting of chronic obstructive pulmonary disease (COPD) patients, but its role has never been demonstrated. We therefore assessed the effects of both pro-oxidant and antioxidant treatments on the oxidative stress levels and atrophic signaling pathway of cultured COPD myotubes. Treatment of cultured COPD myotubes with the pro-oxidant molecule H2O2 resulted in increased ROS production (P = 0.002) and protein carbonylation (P = 0.050), in association with a more pronounced atrophy of the myotubes, as reflected by a reduced diameter (P = 0.003), and the activated expression of atrophic markers MuRF1 and FoxO1 (P = 0.022 and P = 0.030, respectively). Conversely, the antioxidant molecule ascorbic acid induced a reduction in ROS production (P<0.001) and protein carbonylation (P = 0.019), and an increase in the myotube diameter (P<0.001) to a level similar to the diameter of healthy subject myotubes, in association with decreased expression levels of MuRF1, atrogin-1 and FoxO1 (P<0.001, P = 0.002 and P = 0.042, respectively). A significant negative correlation was observed between the variations in myotube diameter and the variations in the expression of MuRF1 after antioxidant treatment (P = 0.047). Moreover, ascorbic acid was able to prevent the H2O2-induced atrophy of COPD myotubes. Last, the proteasome inhibitor MG132 restored the basal atrophy level of the COPD myotubes and also suppressed the H2O2-induced myotube atrophy. These findings demonstrate for the first time the involvement of oxidative stress in the atrophy of COPD peripheral muscle cells in vitro, via the FoxO1/MuRF1/atrogin-1 signaling pathway of the ubiquitin/proteasome system.

Topics: Aged; Antioxidants; Ascorbic Acid; Female; Forkhead Box Protein O1; Humans; Hydrogen Peroxide; Leupeptins; Male; Middle Aged; Muscle Fibers, Skeletal; Muscle Proteins; Muscular Atrophy; Oxidative Stress; Pulmonary Disease, Chronic Obstructive; Signal Transduction; SKP Cullin F-Box Protein Ligases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transmembrane transporter expressed in many cell types, including neurons. Mutations that inactivate transport activity of MCT8 cause severe X-linked psychomotor retardation in male patients, a syndrome originally described as the Allan-Herndon-Dudley syndrome. Treatment options currently explored the focus on finding thyroid hormone-like compounds that bypass MCT8 and enter cells through different transporters. Because MCT8 is a multipass transmembrane protein, some pathogenic mutations affect membrane trafficking while potentially retaining some transporter activity. We explore here the effects of chemical and pharmacological chaperones on the expression and transport activity of the MCT8 mutant ΔPhe501. Dimethylsulfoxide, 4-phenylbutyric acid as well as its sodium salt, and the isoflavone genistein increase T3 uptake into MDCK1 cells stably transfected with mutant MCT8-ΔPhe501. We show that ΔPhe501 represents a temperature-sensitive mutant protein that is stabilized by the proteasome inhibitor MG132. 4-Phenylbutyrate has been used to stabilize ΔPhe508 mutant cystic fibrosis transmembrane conductance regulator protein and is in clinical use in patients with urea cycle defects. Genistein is enriched in soy and available as a nutritional supplement. It is effective in stabilizing MCT8-ΔPhe501 at 100 nM concentration. Expression of the L471P mutant is increased in response to phenylbutyrate, but T3 uptake activity is not induced, supporting the notion that the chaperone specifically increases membrane expression. Our findings suggest that certain pathogenic MCT8 mutants may be responsive to (co-)treatment with readily available compounds, which increase endogenous protein function.

Topics: Animals; Cell Membrane; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Dogs; Genistein; Iodine Radioisotopes; Leupeptins; Madin Darby Canine Kidney Cells; Mental Retardation, X-Linked; Microscopy, Confocal; Monocarboxylic Acid Transporters; Muscle Hypotonia; Muscular Atrophy; Mutation; Oocytes; Phenylbutyrates; Protein Transport; Symporters; Thyroxine; Triiodothyronine; Xenopus

Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although the mechanisms have yet to be defined. Here we report that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of MyoD. Muscle injury induced by sciatic nerve transection up-regulated RFP and RFP physically interacted with both Pax7 and MyoD. RFP and Pax7 synergistically reduced the protein amounts of MyoD but not the mRNA. RFP-induced reduction of MyoD protein was blocked by proteasome inhibitors. The Pax7-induced reduction MyoD was attenuated by RFP siRNA and by MG132, a proteasome inhibitor. RFPΔR, an RFP construct that lacks the RING domain, failed to reduce MyoD amounts. RFP ubiquitinated MyoD, but RFPΔR failed to do so. Forced expression of RFP, but not RFPΔR, enhanced Pax7-induced ubiquitination of MyoD, whereas RFP siRNA blocked the ubiquitination. Sciatic nerve injury-induced muscle atrophy as well the reduction in MyoD was attenuated in RFP knockout mice. Taken together, our results show that RFP works as a novel E3 ligase in the Pax7-mediated degradation of MyoD in response to skeletal muscle atrophy.

Topics: Animals; Cell Line; Disease Models, Animal; DNA-Binding Proteins; HEK293 Cells; Humans; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Skeletal; Muscular Atrophy; MyoD Protein; Nuclear Proteins; PAX7 Transcription Factor; Protease Inhibitors; Protein Binding; Proteolysis; Regeneration; RNA Interference; RNA, Small Interfering; Ubiquitin-Protein Ligases; Ubiquitination

Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

Topics: Animals; Apoptosis; Blotting, Western; Cysteine Proteinase Inhibitors; Disease Models, Animal; Fluorescent Antibody Technique; Laminin; Leupeptins; Mice; Mice, Knockout; Mice, Transgenic; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Muscular Dystrophy, Animal; Phenotype; Phosphorylation; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Ubiquitin; Ubiquitination

Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU).. Twenty-seven NMRI mice were assigned to a weight-bearing control, a 6-day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group.. Gastrocnemius wasting was significantly less in HU+MG132 mice (-6.7 ± 2.0%) compared with HU animals (-12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF-1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF-1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin-conjugated proteins were changed by HU.. Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin-ligases.

Topics: Animals; Cysteine Proteinase Inhibitors; Hindlimb Suspension; Leupeptins; Male; Mice; Muscular Atrophy; Muscular Disorders, Atrophic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Random Allocation

Skeletal muscle atrophy is a serious concern for the rehabilitation of patients afflicted by prolonged limb restriction. This debilitating condition is associated with a marked activation of NFκB activity. The ubiquitin-proteasome pathway degrades the NFκB inhibitor IκBα, enabling NFκB to translocate to the nucleus and bind to the target genes that promote muscle atrophy. Although several studies showed that proteasome inhibitors are efficient to reduce atrophy, no studies have demonstrated the ability of these inhibitors to preserve muscle function under catabolic condition.. We recently developed a new hindlimb immobilization procedure that induces significant skeletal muscle atrophy and used it to show that an inflammatory process characterized by the up-regulation of TNFα, a known activator of the canonical NFκB pathway, is associated with the atrophy. Here, we used this model to investigate the effect of in vivo proteasome inhibition on the muscle integrity by histological approach. TNFα, IL-1, IL-6, MuRF-1 and Atrogin/MAFbx mRNA level were determined by qPCR. Also, a functional measurement of locomotors activity was performed to determine if the treatment can shorten the rehabilitation period following immobilization.. In the present study, we showed that the proteasome inhibitor MG132 significantly inhibited IκBα degradation thus preventing NFκB activation in vitro. MG132 preserved muscle and myofiber cross-sectional area by downregulating the muscle-specific ubiquitin ligases atrogin-1/MAFbx and MuRF-1 mRNA in vivo. This effect resulted in a diminished rehabilitation period.. These finding demonstrate that proteasome inhibitors show potential for the development of pharmacological therapies to prevent muscle atrophy and thus favor muscle rehabilitation.

Topics: Animals; Cell Line; Cysteine Proteinase Inhibitors; Disease Models, Animal; Hindlimb Suspension; Leupeptins; Male; Mice; Mice, Inbred Strains; Muscle, Skeletal; Muscular Atrophy; Proteasome Endopeptidase Complex; Proteasome Inhibitors

Congenital muscular dystrophy caused by laminin α2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease, characterized by massive muscle wasting. The ubiquitin-proteasome system plays a major role in muscle wasting and we recently demonstrated that increased proteasomal activity is a feature of MDC1A. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy-lysosome pathway is dysregulated in muscular dystrophies, including MDC1A. Using the dy(3K)/dy(3K) mouse model of laminin α2 chain deficiency and MDC1A patient muscle, we show here that expression of autophagy-related genes is upregulated in laminin α2 chain-deficient muscle. Moreover, we found that autophagy inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. In particular, we show that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and muscle mass. Importantly, lifespan and locomotive behavior were also greatly improved. These findings indicate that enhanced autophagic activity is pathogenic and that autophagy inhibition holds a promising therapeutic potential in the treatment of MDC1A.

Topics: Adenine; Animals; Apoptosis; Autophagy; Behavior, Animal; Disease Models, Animal; Drug Therapy, Combination; Fibrosis; Gene Expression Regulation; Injections; Laminin; Leupeptins; Mice; Motor Activity; Muscles; Muscular Atrophy; Muscular Dystrophies; Peripheral Nervous System Diseases; Phenotype; Phosphorylation; Proto-Oncogene Proteins c-akt; Regeneration; Survival Analysis

Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome.

Topics: Adenosine Triphosphatases; Animals; Chymotrypsin; Culture Techniques; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dexamethasone; Endopeptidases; Gene Expression Regulation; Glucocorticoids; Leupeptins; Male; Multienzyme Complexes; Muscle Fibers, Fast-Twitch; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein Subunits; Rats; Rats, Wistar; RNA, Messenger; Ubiquitins