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.

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.

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.

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.