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THE INTRA-MITOCHONDRIAL O-GLCNACYLATION SYSTEM ACUTELY REGULATES OXPHOS CAPACITY AND ROS DYNAMICS IN THE HEART

ABSTRACT: O-linked N-acetylglucosamination (O-GlcNAcylation) of proteins is increasingly recognized as an important cellular regulatory mechanism. In the heart, nucleocytoplasmic O-GlcNAcylation is involved in the modulation of multiple pathophysiological processes. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc cycling system without potential confounding effects of O-GlcNAcylation in other cellular compartments. We performed a comparative analysis of the O-GlcNAcylome of isolated cardiac mitochondria acutely (30 min) exposed to UDP-GlcNAc in presence/absence of NButGT, a specific O-GlcNAcylation inducer, and evaluated the impact on mitochondrial function. 412 O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry analysis, with 189 (Q<0.05) displaying increased O-GlcNAcylation in response to NButGT. Among the O-GlcNAcylated pathways identified, oxidative phosphorylation was the main affected. These acute changes in protein O-GlcNAcylation were associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Heart, Cardiocyte

SUBMITTER: Didier Vertommen

LAB HEAD: Luc Bertrand

PROVIDER: PXD026495 | Pride | 2022-02-11

REPOSITORIES: Pride

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Project description:The O-N-acetyl-β-D-glucosaminylation, termed O-GlcNAcylation, is an atypical glycosylation corresponding to the transfer of a unique saccharide, the N-acetyl-β-D-glucosamine, on the hydroxyl group of serine and threonine amino acids of nuclear, cytosolic and mitochondrial proteins. Because of its dynamism, its reversibility and the swiftness of GlcNAc moieties transfer, O-GlcNAcylation is akin to phosphorylation and it is able to respond rapidly to both extracellular and intracellular demands. This post-translational modification is highly represented on intracellular proteins (in particular cytosolic, nuclear and mitochondrial proteins), and is involved in almost if not all cellular processes. O-GlcNAcylation is nowadays clearly associated with the etiology of several acquired diseases, in particular diabetes, neuro-degenerative disorders, cardiovascular diseases or cancer. The O-GlcNAcylation rapidly emerged as a major cellular mechanism which compete with phosphorylation in terms of modified proteins and the importance in cellular physiology. The O-GlcNAcylated sites could also correspond to phosphorylated ones; many proteins are modified by both O-GlcNAc and phosphates groups, and these two post-translational modifications could compete to the same or at neighboring sites. However, despite the undeniable role of O-GlcNAcylation in the modulation of almost all cellular processes, its precise function remains still misunderstood, mainly because of the unknown position of O-GlcNAcylation on specific domains of proteins. Thus, the precise localization of O-GlcNAcylated sites remains an indispensable prerequisite for the fine understanding of its biological function. However, mapping the O-GlcNAcylated sites remains laborious, but challenging, because of (i) the low stoichiometry of O-GlcNAcylation; (ii) the ion suppression of the modified peptide by the unmodified peptide present in larger excess; and (iii) the labile β bound between serine or threonine and the O-GlcNAc moiety which is broken during the CID (Collision-Induced Dissociation) fragmentation process, leading to loss of site information. To respond to this biological demand and to overcome these difficulties, enrichment of O-GlcNAc modified proteins and the use of other fragmentation processes like ECD (Electron Capture Dissociation), ETD (Electron Transfer Dissociation), or HCD (High-energy Collisional Dissociation), able to limit the O-GlcNAc loss during the fragmentation, are crucial. Our strategy was an alternative, specific, efficient and selective purification of O-GlcNAc bearing proteins from skeletal muscle cells by the use of the Click chemistry methodology. To extensively map O-GlcNAc sites on proteins, we proposed herein an intensive fractionation of the muscle cell proteome according to solubility, hydrophobicity and isoelectric point of proteins. The method of Click chemistry was achieved on each enriched-fraction containing proteins of interest.

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Dataset Information

THE INTRA-MITOCHONDRIAL O-GLCNACYLATION SYSTEM ACUTELY REGULATES OXPHOS CAPACITY AND ROS DYNAMICS IN THE HEART

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