A novel 2D-electrophoresis method for the simultaneous visualization of phosphorylated and O-GlcNAcylated proteoforms of a protein.

Post-translational modifications (PTMs), such as phosphorylation and O-N-acetyl-ß-d-glucosaminylation (O-GlcNAcylation), are involved in the fine spatiotemporal regulation of protein functions, and their dynamic interplay is at the heart of protein language. The coexistence of phosphorylation and O-GlcNAcylation on a protein leads to the diversification of proteoforms. It is therefore essential to decipher the phosphorylation/O-GlcNAcylation interplay on protein species that orchestrates cellular processes in a specific physiological or pathophysiological context. However, simultaneous visualization of phosphorylation and O-GlcNAcylation patterns on a protein of interest remains a challenge. To map the proteoforms of a protein, we have developed an easy-to-use two-dimensional electrophoresis method with a single sample processing permitting simultaneous visualization of the phosphorylated and the O-GlcNAcylated forms of the protein of interest. This method, we termed 2D-WGA-Phos-tag-PAGE relies on proteoforms retardation by affinity gel electrophoresis. With this novel approach, we established the cartography of phospho- and glycoforms of αB-crystallin and desmin in the whole extract and the cytoskeleton protein subfraction in skeletal muscle cells. Interestingly, we have shown that the pattern of phosphorylation and O-GlcNAcylation depends of the subcellular subfraction. Moreover, we have also shown that proteotoxic stress condition increased the complexity of the pattern of PTMs on αB-crystallin.

Oxygen supplementation to limit hypoxia-induced muscle atrophy in C2C12 myotubes: comparison with amino acid supplement and electrical stimulation.

In skeletal muscle, chronic oxygen depletion induces a disturbance leading to muscle atrophy. Mechanical stress (physical exercise) and nutritional supplement therapy are commonly used against loss of muscle mass and undernutrition in hypoxia, while oxygenation therapy is preferentially used to counteract muscle fatigue and exercise intolerance. However, the impact of oxygenation on skeletal muscle cells remains poorly understood, in particular on signalling pathways regulating protein balance. Thus, we investigated the effects of each separated treatment (mechanical stress, nutritional supplementation and oxygenation therapy) on intracellular pathways involved in protein synthesis and degradation that are imbalanced in skeletal muscle cells atrophy resulting from hypoxia. Myotubes under hypoxia were treated by electrical stimulation, amino acids supplement or oxygenation period. Signalling pathways involved in protein synthesis (PI3K-Akt-mTOR) and degradation (FoxO1 and FoxO3a) were investigated, so as autophagy, ubiquitin-proteasome system and myotube morphology. Electrical stimulation and oxygenation treatment resulted in higher myotube diameter, myogenic fusion index and myotubes density until 48 h post-treatment compared to untreated hypoxic myotubes. Both treatments also induced inhibition of FoxO3a and decreased activity of ubiquitin-proteasome system; however, their impact on protein synthesis pathway was specific for each one. Indeed, electrical stimulation impacted upstream proteins to mTOR (i.e., Akt) while oxygenation treatment activated downstream targets of mTOR (i.e., 4E-BP1 and P70S6K). In contrast, amino acid supplementation had very few effects on myotube morphology nor on protein homeostasis. This study demonstrated that electrical stimulation or oxygenation period are two effective treatments to fight against hypoxia-induced muscle atrophy, acting through different molecular adaptations.

Synaptic protein changes after a chronic period of sensorimotor perturbation in adult rats: a potential role of phosphorylation/O-GlcNAcylation interplay.

In human, a chronic sensorimotor perturbation (SMP) through prolonged body immobilization alters motor task performance through a combination of peripheral and central factors. Studies performed on a rat model of SMP have shown biomolecular changes and a reorganization of sensorimotor cortex through events such as morphological modifications of dendritic spines (number, length, functionality). However, underlying mechanisms are still unclear. It is well known that phosphorylation regulates a wide field of synaptic activity leading to neuroplasticity. Another post-translational modification that interplays with phosphorylation is O-GlcNAcylation. This atypical glycosylation, reversible, and dynamic, is involved in essential cellular and physiological processes such as synaptic activity, neuronal morphogenesis, learning, and memory. We examined potential roles of phosphorylation/O-GlcNAcylation interplay in synaptic plasticity within rat sensorimotor cortex after a SMP period. For this purpose, sensorimotor cortex synaptosomes were separated by sucrose gradient, in order to isolate a subcellular compartment enriched in proteins involved in synaptic functions. A period of SMP induced plastic changes at the pre- and post-synaptic levels, characterized by a reduction in phosphorylation (synapsin1, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors (AMPAR) GluA2) and expression (synaptophysin, PSD-95, AMPAR GluA2) of synaptic proteins, as well as a decrease in MAPK/ERK42 activation. Expression levels of O-GlcNAc transferase/O-GlcNAcase enzymes was unchanged but we observed a specific reduction of synapsin1 O-GlcNAcylation in sensorimotor cortex synaptosomes. The synergistic regulation of synapsin1 phosphorylation/O-GlcNAcylation could affect pre-synaptic neurotransmitter release. Associated with other pre- and post-synaptic changes, synaptic efficacy could be impaired in somatosensory cortex of SMP rat. Thus, phosphorylation/O-GlcNAcylation interplay appears to be involved in synaptic plasticity by finely regulating neural activity.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

BACKGROUND: The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. METHODS: C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. RESULTS: Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. CONCLUSIONS: For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. GENERAL SIGNIFICANCE: In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.

The lacdiNAc-specific adhesin LabA mediates adhesion of Helicobacter pylori to human gastric mucosa.

Adhesion of Helicobacter pylori to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases. In this study, we investigated the GalNAcß1-4GlcNAc motif (also known as N,N'-diacetyllactosediamine [lacdiNAc]) carried by MUC5AC gastric mucins as the target for bacterial binding to the human gastric mucosa. The expression of LacdiNAc carried by gastric mucins was correlated with H. pylori localization, and all strains tested adhered significantly to this motif. Proteomic analysis and mutant construction allowed the identification of a yet uncharacterized bacterial adhesin, LabA, which specifically recognizes lacdiNAc. These findings unravel a target of adhesion for H. pylori in addition to moieties recognized by the well-characterized adhesins BabA and SabA. Localization of the LabA target, restricted to the gastric mucosa, suggests a plausible explanation for the tissue tropism of these bacteria. These results pave the way for the development of alternative strategies against H. pylori infection, using adherence inhibitors.

Phospho-GlcNAc modulation of slow MLC2 during soleus atrophy through a multienzymatic and sarcomeric complex.

Although calcium is the major regulator of excitation-contraction coupling, myofilament function can also be modulated through post-translational modifications. In particular, phosphorylation and O-GlcNAcylation are key modulators of calcium activation parameters. Among the regulatory proteins of skeletal muscle contraction, the myosin light chain 2 (MLC2) can undergo both types of post-translational modification. During aging or physical inactivity, the phosphorylation status of the slow isoform of MLC2 (sMLC2) does not correlate with calcium sensitivity, suggesting that the O-GlcNAcylation might modulate sMLC2 activity. To increase understanding of the contractile dysfunction associated with muscle atrophy, we studied the phosphorylation/O-GlcNAcylation interplay on the sMLC2. We demonstrate a two-fold decrease of O-GlcNAcylation level on sMLC2 in a rat model of skeletal muscle atrophy (hindlimb unloading), while phosphorylation increased. Both post-translational modifications were mutually exclusive. Their interplay reversed during reloading. The expression of enzymes involved in the phosphorylation and O-GlcNAcylation interplay on sMLC2 was modified on whole protein pattern as well as on myofilament, and was load-dependent. All enzymes were colocalized on the contractile apparatus. Finally, we describe a multienzymatic complex which might finely modulate the phosphorylation/dephosphorylation and O-GlcNAcylation/de-O-GlcNAcylation of sMLC2 that could be involved in the contractile dysfunction of atrophied muscle. Importantly, this complex was localized at the Z-disk, a nodal point of signalling in skeletal muscle.

Impact of MG132 induced-proteotoxic stress on αB-crystallin and desmin phosphorylation and O-GlcNAcylation and their partition towards cytoskeleton.

Small Heat Shock Proteins are considered as the first line of defense when proteostasis fails. Among them, αB-crystallin is expressed in striated muscles in which it interacts with desmin intermediate filaments to stabilize them, maintaining cytoskeleton's integrity and muscular functionalities. Desmin is a key actor for muscle health; its targeting by αB-crystallin is thus crucial, especially in stress conditions. αB-crystallin is phosphorylated and O-GlcNAcylated. Its phosphorylation increases consecutively to various stresses, correlated with its recruitment for cytoskeleton's safeguarding. However, phosphorylation as unique signal for cytoskeleton translocation remains controversial; indeed, O-GlcNAcylation was also proposed to be involved. Thus, there are still some gaps for a deeper comprehension of how αB-crystallin functions are finely regulated by post-translational modifications. Furthermore, desmin also bears both post-translational modifications; while desmin phosphorylation is closely linked to desmin intermediates filaments turnover, it is unclear whereas its O-GlcNAcylation could impact its proper function. In the herein paper, we aim at identifying whether phosphorylation and/or O-GlcNAcylation are involved in αB-crystallin targeting towards cytoskeleton in proteotoxic stress induced by proteasome inhibition in C2C12 myotubes. We demonstrated that proteotoxicity led to αB-crystallin's phosphorylation and O-GlcNAcylation patterns changes, both presenting a dynamic interplay depending on protein subfraction. Importantly, both post-translational modifications showed a spatio-temporal variation correlated with αB-crystallin translocation towards cytoskeleton. In contrast, we did not detect any change of desmin phosphorylation and O-GlcNAcylation. All together, these data strongly support that αB-crystallin phosphorylation/O-GlcNAcylation interplay rather than changes on desmin is a key regulator for its cytoskeleton translocation, preserving it towards stress.

Desmin and its molecular chaperone, the αB-crystallin: How post-translational modifications modulate their functions in heart and skeletal muscles?

Maintenance of the highly organized striated muscle tissue requires a cell-wide dynamic network through protein-protein interactions providing an effective mechanochemical integrator of morphology and function. Through a continuous and complex trans-cytoplasmic network, desmin intermediate filaments ensure this essential role in heart and in skeletal muscle. Besides their role in the maintenance of cell shape and architecture (permitting contractile activity efficiency and conferring resistance towards mechanical stress), desmin intermediate filaments are also key actors of cell and tissue homeostasis. Desmin participates to several cellular processes such as differentiation, apoptosis, intracellular signalisation, mechanotransduction, vesicle trafficking, organelle biogenesis and/or positioning, calcium homeostasis, protein homeostasis, cell adhesion, metabolism and gene expression. Desmin intermediate filaments assembly requires αB-crystallin, a small heat shock protein. Over its chaperone activity, αB-crystallin is involved in several cellular functions such as cell integrity, cytoskeleton stabilization, apoptosis, autophagy, differentiation, mitochondria function or aggresome formation. Importantly, both proteins are known to be strongly associated to the aetiology of several cardiac and skeletal muscles pathologies related to desmin filaments disorganization and a strong disturbance of desmin interactome. Note that these key proteins of cytoskeleton architecture are extensively modified by post-translational modifications that could affect their functional properties. Therefore, we reviewed in the herein paper the impact of post-translational modifications on the modulation of cellular functions of desmin and its molecular chaperone, the αB-crystallin.

Potential regulation of human muscle plasticity by MLC2 post-translational modifications during bed rest and countermeasures.

This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR+Ex subjects were submitted to combined aerobic and resistive exercises, and BR+Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS-PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR+Nut while BR+Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosylation observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atrophy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading.

Global O-GlcNAcylation changes impact desmin phosphorylation and its partition toward cytoskeleton in C2C12 skeletal muscle cells differentiated into myotubes.

Desmin is the guardian of striated muscle integrity, permitting the maintenance of muscle shape and the efficiency of contractile activity. It is also a key mediator of cell homeostasis and survival. To ensure the fine regulation of skeletal muscle processes, desmin is regulated by post-translational modifications (PTMs). It is more precisely phosphorylated by several kinases connecting desmin to intracellular processes. Desmin is also modified by O-GlcNAcylation, an atypical glycosylation. However, the functional consequence of O-GlcNAcylation on desmin is still unknown, nor its impact on desmin phosphorylation. In a model of C2C12 myotubes, we modulated the global O-GlcNAcylation level, and we determined whether the expression, the PTMs and the partition of desmin toward insoluble material or cytoskeleton were impacted or not. We have demonstrated in the herein paper that O-GlcNAcylation variations led to changes in desmin behaviour. In particular, our data clearly showed that O-GlcNAcylation increase led to a decrease of phosphorylation level on desmin that seems to involve CamKII correlated to a decrease of its partition toward cytoskeleton. Our data showed that phosphorylation/O-GlcNAcylation interplay is highly complex on desmin, supporting that a PTMs signature could occur on desmin to finely regulate its partition (i.e. distribution) with a spatio-temporal regulation.

Electrostimulation during hindlimb unloading modulates PI3K-AKT downstream targets without preventing soleus atrophy and restores slow phenotype through ERK.

Our aim was to analyze the role of phosphatidylinositol 3-kinase (PI3K)-AKT and MAPK signaling pathways in the regulation of muscle mass and slow-to-fast phenotype transition during hindlimb unloading (HU). For that purpose, we studied, in rat slow soleus and fast extensor digitorum longus muscles, the time course of anabolic PI3K-AKT-mammalian target of rapamycin, catabolic PI3K-AKT-forkhead box O (FOXO), and MAPK signaling pathway activation after 7, 14, and 28 days of HU. Moreover, we performed chronic low-frequency soleus electrostimulation during HU to maintain exclusively contractile phenotype and so to determine more precisely the role of these signaling pathways in the modulation of muscle mass. HU induced a downregulation of the anabolic AKT, mammalian target of rapamycin, 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, and glycogen synthase kinase-3ß targets, and an upregulation of the catabolic FOXO1 and muscle-specific RING finger protein-1 targets correlated with soleus muscle atrophy. Unexpectedly, soleus electrostimulation maintained 70-kDa ribosomal protein S6 kinase, 4E-binding protein 1, FOXO1, and muscle-specific RING finger protein-1 to control levels, but failed to reduce muscle atrophy. HU decreased ERK phosphorylation, while electrostimulation enabled the maintenance of ERK phosphorylation similar to control level. Moreover, slow-to-fast myosin heavy chain phenotype transition and upregulated glycolytic metabolism were prevented by soleus electrostimulation during HU. Taken together, our data demonstrated that the processes responsible for gradual disuse muscle plasticity in HU conditions involved both PI3-AKT and MAPK pathways. Moreover, electrostimulation during HU restored PI3K-AKT activation without counteracting soleus atrophy, suggesting the involvement of other signaling pathways. Finally, electrostimulation maintained initial contractile and metabolism properties in parallel to ERK activation, reinforcing the idea of a predominant role of ERK in the regulation of muscle slow phenotype.

O-GlcNAcylation as a regulator of the functional and structural properties of the sarcomere in skeletal muscle: An update review.

Although the O-GlcNAcylation process was discovered in 1984, its potential role in the physiology and physiopathology of skeletal muscle only emerged 20 years later. An increasing number of publications strongly support a key role of O-GlcNAcylation in the modulation of important cellular processes which are essential for skeletal muscle functions. Indeed, over a thousand of O-GlcNAcylated proteins have been identified within skeletal muscle since 2004, which belong to various classes of proteins, including sarcomeric proteins. In this review, we focused on these myofibrillar proteins, including contractile and structural proteins. Because of the modification of motor and regulatory proteins, the regulatory myosin light chain (MLC2) is related to several reports that support a key role of O-GlcNAcylation in the fine modulation of calcium activation parameters of skeletal muscle fibres, depending on muscle phenotype and muscle work. In addition, another key function of O-GlcNAcylation has recently emerged in the regulation of organization and reorganization of the sarcomere. Altogether, this data support a key role of O-GlcNAcylation in the homeostasis of sarcomeric cytoskeleton, known to be disturbed in many related muscle disorders.

Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of synapse-associated proteins within the rat sensorimotor cortex.

BACKGROUND: Several studies have shown the importance of phosphorylation, O-GlcNAcylation and their interplay in neuronal processes. NEW METHOD: To get understanding about molecular mechanisms of synaptic plasticity, we performed a preparation of synaptic protein-enriched fraction on a small sample of rat sensorimotor cortex. We then optimized a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. We compared different protocols (solubilisation buffer, reticulation and composition of the gel, migration buffer) to optimize separating conditions for 2D-gel electrophoresis of synaptic proteins. The O-GlcNAcome was revealed using Click chemistry and the azide-alkyne cycloaddition of a fluorophore on O-GlcNAc moieties. The phosphoproteome was detected by Phospho-Tag staining, while the whole proteome was visualized through SYPRORuby staining. RESULTS: This method permitted, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome of synapse-associated proteins. CONCLUSION: This original method of differential proteomic analysis will permit to identify key markers of synaptic plasticity that are O-GlcNAcylated and/or phosphorylated, and their molecular regulations in neuronal processes.

Impact of different methods of induction of cellular hypoxia: focus on protein homeostasis signaling pathways and morphology of C2C12 skeletal muscle cells differentiated into myotubes.

Hypoxia, occurring in several pathologies, has deleterious effects on skeletal muscle, in particular on protein homeostasis. Different induction methods of hypoxia are commonly used in cellular models to investigate the alterations of muscular function consecutive to hypoxic stress. However, a consensus is not clearly established concerning hypoxia induction methodology. Our aim was to compare oxygen deprivation with chemically induced hypoxia using cobalt chloride (CoCl2) or desferrioxamine (DFO) on C2C12 myotubes which were either cultured in hypoxia chamber at an oxygen level of 4% or treated with CoCl2 or DFO. For each method of hypoxia induction, we determined their impact on muscle cell morphology and on expression or activation status of key signaling proteins of synthesis and degradation pathways. The expression of HIF-1α increased whatever the method of hypoxia induction. Myotube diameter and protein content decreased exclusively for C2C12 myotubes submitted to physiological hypoxia (4% O2) or treated with CoCl2. Results were correlated with a hypophosphorylation of key proteins regulated synthesis pathway (Akt, GSK3-ß and P70S6K). Similarly, the phosphorylation of FoxO1 decreased and the autophagy-related LC3-II was overexpressed with 4% O2 and CoCl2 conditions. Our results demonstrated that in vitro oxygen deprivation and the use of mimetic agent such as CoCl2, unlike DFO, induced similar responses on myotube morphology and atrophy/hypertrophy markers. Thus, physiological hypoxia or its artificial induction using CoCl2 can be used to understand finely the molecular changes in skeletal muscle cells and to evaluate new therapeutics for hypoxia-related muscle disorders.

Interplay between hypoactivity, muscle properties and motor command: How to escape the vicious deconditioning circle?

Activity-dependent processes addressing the central nervous system (CNS) and musculoskeletal structures are critical for maintaining motor performance. Chronic reduction in activity, whether due to a sedentary lifestyle or extended bed rest, results in impaired performance in motor tasks and thus decreased quality of life. In the first part of this paper, we give a narrative review of the effects of hypoactivity on the neuromuscular system and behavioral outcomes. Motor impairments arise from a combination of factors including altered muscle properties, impaired afferent input, and plastic changes in neural structure and function throughout the nervous system. There is a reciprocal interplay between the CNS and muscle properties, and these sensorimotor loops are essential for controlling posture and movement. As a result, patients under hypoactivity experience a self-perpetuating cycle, in with sedentarity leading to decreased motor activity and thus a progressive worsening of a situation, and finally deconditioning. Various rehabilitation strategies have been studied to slow down or reverse muscle alteration and altered motor performance. In the second part of the paper, we review representative protocols directed toward the muscle, the sensory input and/or the cerebral cortex. Improving an understanding of the loss of motor function under conditions of disuse (such as extended bed rest) as well as identifying means to slow this decline may lead to therapeutic strategies to preserve quality of life for a range of individuals. The most efficient strategies seem multifactorial, using a combination of approaches targeting different levels of the neuromuscular system.

Predicting left ventricular remodeling after a first myocardial infarction by plasma proteome analysis.

Recent improvements in therapeutic strategies did not prevent left ventricular remodeling (LVR), which remains a common event (30%) after acute myocardial infarction (AMI). We report the use of a systematic approach, based on comparative proteomics, to select circulating biomarkers that may be associated with LVR. We selected 93 patients enrolled in a prospective study. These patients with anterior wall Q-wave AMI underwent echocardiographic follow-up at hospitalization, 3 months and 1 year after AMI. They were divided into three groups (no, low, or high remodeling). Plasma samples of these patients (day 5 of hospitalization) were processed and stored at -80 degrees C within 2 h and analyzed using SELDI-TOF protein chip technology. This systematic approach allowed to select candidate proteins modulated by LVR: post-translational variants of alpha1-chain of haptoglobin (Hpalpha1) corresponding to m/z 9493, 9565, and 9623, which were more elevated in remodeling patients. The peak 9493 m/z was shown having a receiving-operating characteristic (ROC) value of 0.71 between non- and remodeling patients. SELDI-TOF approach may lead to the identification of circulating proteins associated with LVR. Whether these candidate proteins will help to identify patients who are at high risk of heart failure after AMI will have to be tested in future studies.

Involvement of O-GlcNAcylation in the Skeletal Muscle Physiology and Physiopathology: Focus on Muscle Metabolism.

Skeletal muscle represents around 40% of whole body mass. The principal function of skeletal muscle is the conversion of chemical energy toward mechanic energy to ensure the development of force, provide movement and locomotion, and maintain posture. This crucial energy dependence is maintained by the faculty of the skeletal muscle for being a central place as a "reservoir" of amino acids and carbohydrates in the whole body. A fundamental post-translational modification, named O-GlcNAcylation, depends, inter alia, on these nutrients; it consists to the transfer or the removal of a unique monosaccharide (N-acetyl-D-glucosamine) to a serine or threonine hydroxyl group of nucleocytoplasmic and mitochondrial proteins in a dynamic process by the O-GlcNAc Transferase (OGT) and the O-GlcNAcase (OGA), respectively. O-GlcNAcylation has been shown to be strongly involved in crucial intracellular mechanisms through the modulation of signaling pathways, gene expression, or cytoskeletal functions in various organs and tissues, such as the brain, liver, kidney or pancreas, and linked to the etiology of associated diseases. In recent years, several studies were also focused on the role of O-GlcNAcylation in the physiology and the physiopathology of skeletal muscle. These studies were mostly interested in O-GlcNAcylation during muscle exercise or muscle-wasting conditions. Major findings pointed out a different "O-GlcNAc signature" depending on muscle type metabolism at resting, wasting and exercise conditions, as well as depending on acute or long-term exhausting exercise protocol. First insights showed some differential OGT/OGA expression and/or activity associated with some differential stress cellular responses through Reactive Oxygen Species and/or Heat-Shock Proteins. Robust data displayed that these O-GlcNAc changes could lead to (i) a differential modulation of the carbohydrates metabolism, since the majority of enzymes are known to be O-GlcNAcylated, and to (ii) a differential modulation of the protein synthesis/degradation balance since O-GlcNAcylation regulates some key signaling pathways such as Akt/GSK3ß, Akt/mTOR, Myogenin/Atrogin-1, Myogenin/Mef2D, Mrf4 and PGC-1α in the skeletal muscle. Finally, such involvement of O-GlcNAcylation in some metabolic processes of the skeletal muscle might be linked to some associated diseases such as type 2 diabetes or neuromuscular diseases showing a critical increase of the global O-GlcNAcylation level.

O-GlcNAcylation site mapping by (azide-alkyne) click chemistry and mass spectrometry following intensive fractionation of skeletal muscle cells proteins.

The O-linked-N-acetyl-d-glucosaminylation (O-GlcNAcylation) modulates numerous aspects of cellular processes. Akin to phosphorylation, O-GlcNAcylation is highly dynamic, reversible, and responds rapidly to extracellular demand. Despite the absolute necessity to determine post-translational sites to fully understand the role of O-GlcNAcylation, it remains a high challenge for the major reason that unmodified proteins are in excess comparing to the O-GlcNAcylated ones. Based on a click chemistry approach, O-GlcNAcylated proteins were labelled with azido-GalNAc and coupled to agarose beads. The proteome extracted from C2C12 myotubes was submitted to an intensive fractionation prior to azide-alkyne click chemistry. This combination of fractionation and click chemistry is a powerful methodology to map O-GlcNAc sites; indeed, 342 proteins were identified through the identification of 620 peptides containing one or more O-GlcNAc sites. We localized O-GlcNAc sites on proteins involved in signalling pathways or in protein modification, as well as structural proteins. Considering the recent role of O-GlcNAcylation in the modulation of sarcomere morphometry and interaction between key structural protein, we focused on proteins involved in the cytoarchitecture of skeletal muscle cells. In particular, several O-GlcNAc sites were located into protein-protein interaction domains, suggesting that O-GlcNAcylation could be strongly involved in the organization and reorganization of sarcomere and myofibrils. SIGNIFICANCE: O-GlcNAcylation is an atypical glycosylation involved in the regulation of almost all if not all cellular processes, but its precise role remains sometimes obscure because of the ignorance of the O-GlcNAc site localization; thus, it remains indispensable to precisely map the O-GlcNAcylated sites to fully understand the role of O-GlcNAcylation on a given protein. For this purpose, we combined extensive fractionation of skeletal muscle cells proteome with click chemistry to map O-GlcNAc sites without an a priori consideration. A total of 620 peptides containing one or more O-GlcNAc sites were identified; interestingly, several of them belong to low expressed proteins, in particular proteins involved in signalling pathways. We also focused on structural proteins in view of recent data supporting the role of O-GlcNAcylation in the modulation of sarcomere cytoarchitecture; importantly, some of the O-GlcNAc sites were mapped into protein-protein interaction domains, reinforcing the involvement of O-GlcNAcylation in the organization and reorganization of sarcomere, and in larger extent, of myofibrils.

O-GlcNAc level variations are associated with the development of skeletal muscle atrophy.

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory posttranslational modification of nucleocytoplasmic proteins, which consists of the attachment of N-acetylglucosamine to serine or threonine residues of a protein. This glycosylation is a ubiquitous posttranslational modification, which probably plays important roles in many aspects of protein function. Our laboratory has previously reported that, in skeletal muscle, proteins of the glycolytic pathway and energetic metabolism and contractile proteins were O-GlcNAc modified (Cieniewski-Bernard C, Bastide B, Lefebvre T, Lemoine J, Mounier Y, and Michalski JC. Mol Cell Proteomics 3: 577-585, 2004). O-GlcNAc has been recently demonstrated to play a role in modulating cellular function in response to nutrition and also in stress conditions. Therefore, we have investigated here the implication of the glycosylation/deglycosylation process in the development of atrophy in rat skeletal muscle after hindlimb unloading. The high O-GlcNAc level found in control soleus [compared with control extensor digitorum longus (EDL)] becomes lower in atrophied soleus. On the opposite side, the low rate of O-GlcNAc in control EDL reaches higher levels in EDL, not atrophied after hindlimb unloading. These variations in O-GlcNAc level are correlated with a variation of the O-GlcNAc process enzyme activities and could be associated with a differential expression of heat shock proteins. Our results suggest that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.

Exercise training and high-fat diet elicit endocannabinoid system modifications in the rat hypothalamus and hippocampus.

The purpose of the present study was to examine the effect of chronic exercise on the hypothalamus and hippocampus levels of the endocannabinoids (eCBs) anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and of two AEA congeners and on the expression of genes coding for CB1, CB2 receptors (Cnr1 and Cnr2, respectively), and the enzymes responsible for eCB biosynthesis and degradation, in rats fed with a standard or high-fat diet. Male Wistar rats (n = 28) were placed on a 12-week high-fat (HFD) or standard diet period, followed by 12 weeks of exercise training for half of each group. Tissue levels of eCBs and related lipids were measured by liquid chromatography mass spectrometry, and expression of genes coding for CB1 and CB2 receptors and eCB metabolic enzymes was measured by quantitative real-time polymerase chain reaction (qPCR). HFD induced a significant increase in 2-AG (p < 0.01) in hypothalamus. High-fat diet paired with exercise training had no effect on AEA, 2-AG, and AEA congener levels in the hypothalamus and hippocampus. Cnr1 expression levels were significantly increased in the hippocampus in response to HFD, exercise, and the combination of both (p < 0.05). Our results indicate that eCB signaling in the CNS is sensitive to diet and/or exercise.