Inhibition of Atrogin-1/MAFbx Mediated MyoD Proteolysis Prevents Skeletal Muscle Atrophy In Vivo

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed

Exclusion of NFAT5 from Mitotic Chromatin Resets Its Nucleo-Cytoplasmic Distribution in Interphase

The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5.Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD). NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin.Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export signals acting in interphase, resets the cytoplasmic localization of NFAT5 and prevents its nuclear accumulation and association with DNA in the absence of hypertonic stress

Myofibrillar Myopathies: New Perspectives from Animal Models to Potential Therapeutic Approaches

International audienceMyofibrillar myopathies (MFMs) are muscular disorders involving proteins that play a role in the structure, maintenance processes and protein quality control mechanisms closely related to the Z-disc in the muscular fibers. MFMs share common histological characteristics including progressive disorganization of the interfibrillar network and protein aggregation. Currently no treatment is available. In this review, we describe first clinical symptoms associated with mutations of the six genes (DES, CRYAB, MYOT, ZASP, FLNC and BAG3) primary involved in MFM and defining the origin of this pathology. As mechanisms determining the aetiology of the disease remain unclear yet, several research teams have developed animal models from invertebrates to mammalians species. Thus we describe here these different models that often recapitulate human clinical symptoms. Therefore they are very useful for deeper studies to understand early molecular and progressive mechanisms determining the pathology. Finally in the last part, we emphasize on the potential therapeutic approaches for MFM that could be conducted in the future. In conclusion, this review offers a link from patients to future therapy through the use of MFMs animal models

Impact of environmental stress and new models for pathophysiological and therapeutic studies of desminopathies

20th International Congress of the World-Muscle-Society, Brighton, ENGLAND, SEP 30-OCT 04, 2015International audienceno abstrac

Myofibrillar Myopathies: New Perspectives from Animal Models to Potential Therapeutic Approaches

International audienceMyofibrillar myopathies (MFMs) are muscular disorders involving proteins that play a role in the structure, maintenance processes and protein quality control mechanisms closely related to the Z-disc in the muscular fibers. MFMs share common histological characteristics including progressive disorganization of the interfibrillar network and protein aggregation. Currently no treatment is available. In this review, we describe first clinical symptoms associated with mutations of the six genes (DES, CRYAB, MYOT, ZASP, FLNC and BAG3) primary involved in MFM and defining the origin of this pathology. As mechanisms determining the aetiology of the disease remain unclear yet, several research teams have developed animal models from invertebrates to mammalians species. Thus we describe here these different models that often recapitulate human clinical symptoms. Therefore they are very useful for deeper studies to understand early molecular and progressive mechanisms determining the pathology. Finally in the last part, we emphasize on the potential therapeutic approaches for MFM that could be conducted in the future. In conclusion, this review offers a link from patients to future therapy through the use of MFMs animal models

Inhibiting de novo ceramide synthesis restores mitochondrial and protein homeostasis in muscle aging.

Disruption of mitochondrial function and protein homeostasis plays a central role in aging. However, how these processes interact and what governs their failure in aging remain poorly understood. Here, we showed that ceramide biosynthesis controls the decline in mitochondrial and protein homeostasis during muscle aging. Analysis of transcriptome datasets derived from muscle biopsies obtained from both aged individuals and patients with a diverse range of muscle disorders revealed that changes in ceramide biosynthesis, as well as disturbances in mitochondrial and protein homeostasis pathways, are prevalent features in these conditions. By performing targeted lipidomics analyses, we found that ceramides accumulated in skeletal muscle with increasing age across Caenorhabditis elegans, mice, and humans. Inhibition of serine palmitoyltransferase (SPT), the rate-limiting enzyme of the ceramide de novo synthesis, by gene silencing or by treatment with myriocin restored proteostasis and mitochondrial function in human myoblasts, in C. elegans, and in the skeletal muscles of mice during aging. Restoration of these age-related processes improved health and life span in the nematode and muscle health and fitness in mice. Collectively, our data implicate pharmacological and genetic suppression of ceramide biosynthesis as potential therapeutic approaches to delay muscle aging and to manage related proteinopathies via mitochondrial and proteostasis remodeling

Cardiac arrhythmia and late-onset muscle weakness caused by a myofibrillar myopathy with unusual histopathological features due to a novel missense mutation in FLNC

Myofibrillar myopathies (MFM) are mostly adult-onset diseases characterized by progressive morphological alterations of the muscle fibers beginning in the Z-disk and the presence of protein aggregates in the sarcoplasm. They are mostly caused by mutations in different genes that encode Z-disk proteins, including DES, CRYAB, LDB3, MYOT, FLNC and BAG3. A large family of French origin, presenting an autosomal dominant pattern, characterized by cardiac arrhythmia associated to late-onset muscle weakness, was evaluated to clarify clinical, morphological and genetic diagnosis. Muscle weakness began during adult life (over 30 years of age), and had a proximal distribution. Histology showed clear signs of a myofibrillar myopathy, but with unusual, large inclusions. Subsequently, genetic testing was performed in MFM genes available for screening at the time of clinical/histological diagnosis, and desmin (DES), αB-crystallin (CRYAB), myotilin (MYOT) and ZASP (LDB3), were excluded. LMNA gene screening found the p.R296C variant which did not co-segregate with the disease. Genome wide scan revealed linkage to 7q.32, containing the FLNC gene. FLNC direct sequencing revealed a heterozygous c.3646T>A p.Tyr1216Asn change, co-segregating with the disease, in a highly conserved amino acid of the protein. Normal filamin C levels were detected by Western-blot analysis in patient muscle biopsies and expression of the mutant protein in NIH3T3 showed filamin C aggregates. This is an original FLNC mutation in a MFM family with an atypical clinical and histopathological presentation, given the presence of significantly focal lesions and prominent sarcoplasmic masses in muscle biopsies and the constant heart involvement preceding significantly the onset of the myopathy. Though a rare etiology, FLNC gene should not be excluded in early-onset arrhythmia, even in the absence of myopathy, which occurs later in the disease course.publisher: Elsevier articletitle: Cardiac arrhythmia and late-onset muscle weakness caused by a myofibrillar myopathy with unusual histopathological features due to a novel missense mutation in FLNC journaltitle: Revue Neurologique articlelink: http://dx.doi.org/10.1016/j.neurol.2016.07.017 content_type: article copyright: © 2016 Elsevier Masson SAS. All rights reserved.status: publishe

Dispersed Sites of HIV Vif-Dependent Polyubiquitination in the DNA Deaminase APOBEC3F

APOBEC3F and APOBEC3G are DNA cytosine deaminases that potently restrict Human Immunodeficiency Virus-type 1 replication when the virus is deprived of its accessory protein Vif. Vif counteracts these restriction factors by recruiting APOBEC3F and APOBEC3G to an E3 ubiquitin ligase complex that mediates their polyubiquitination and proteasomal degradation. While previous efforts have identified single amino acid residues in APOBEC3 proteins required for Vif recognition, less is known about the downstream ubiquitin acceptor sites that are targeted. One prior report identified a cluster of polyubiquitinated residues in APOBEC3G and proposed an antiparallel model of APOBEC3G interaction with the Vif-E3 ubiquitin ligase complex wherein Vif binding at one terminus of APOBEC3G orients the opposite terminus for polyubiquitination [Iwatani Y, et al. (2009) PNAS 106(46):19539–19544]. To test the generalizability of this model, we carried out a complete mutagenesis of the lysine residues in APOBEC3F and used a complementary, unbiased proteomic approach to identify ubiquitin acceptor sites targeted by Vif. Our data indicate that internal lysines are the dominant ubiquitin acceptor sites in both APOBEC3F and APOBEC3G. In contrast with the proposed antiparallel model, however, we find that the Vif-dependent polyubiquitination of APOBEC3F and APOBEC3G can occur at multiple acceptor sites dispersed along predicted lysine-enriched surfaces of both the N- and C-terminal deaminase domains. These data suggest an alternative model for binding of APOBEC3 proteins to the Vif-E3 ubiquitin ligase complex and diminish enthusiasm for the amenability of APOBEC3 ubiquitin acceptor sites to therapeutic intervention