The intra-mitochondrial O-GlcNAcylation system acutely regulates OXPHOS capacity and ROS dynamics in the heart Justine Dontaine Asma Bouali Frederic Daussin Laurent Bultot https://orcid.org/0000-0002-5088-0101 Didier Vertommen Manon Martin Rahulan Rathagirishnan Alexanne Cuillerier University of Ottawa Sandrine Horman Christophe Beauloye Université catholique de Louvain, Institut de Recherche Expérimentale et Clinique Laurent Gatto UCLouvain https://orcid.org/0000-0002-1520-2268 Benjamin Lauzier Luc Bertrand https://orcid.org/0000-0003-0655-7099 Yan Burelle ( [email protected] ) University of Ottawa https://orcid.org/0000-0001-9379-146X Article Keywords: O-GlcNAcylation, OXPHOS capacity, cellular regulatory mechanism Posted Date: July 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-690671/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 THE INTRA-MITOCHONDRIAL O-GLCNACYLATION SYSTEM ACUTELY 2 REGULATES OXPHOS CAPACITY AND ROS DYNAMICS IN THE HEART. 3 4 Justine Dontaine1, A. Bouali2, F. Daussin3, L. Bultot1, D. Vertommen4, M. Martin5, R. 5 Rathagirishnan2, A. Cuillerier6, S. Horman1, C. Beauloye1,7, L. Gatto5, B. Lauzier8, L. Bertrand1,9*, 6 Y. Burelle2,6* 7 1Pole of cardiovascular research (CARD), Institute of Experimental and Clinical Research (IREC), UCLouvain, 8 Brussels, Belgium 9 2Interdisciplinary School of Health Sciences, Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada 10 3Univ. Lille, Univ. Artois, Univ. Littoral Côte d’Opale, ULR 7369 - URePSSS - Unité de Recherche Pluridisciplinaire 11 Sport Santé Société, F-59000 Lille, France. 12 4Pole of Protein phosphorylation (PHOS), de Duve Institute (DDUV), UCLouvain, Brussels, Belgium 13 5Pole of Computational biology and bioinformatics (CBIO), de Duve Institute (DDUV), UCLouvain, Brussels, 14 Belgium 15 6Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada 16 7Division of Cardiology, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium 17 8Institute of Thorax, INSERM, CNRS, University of Nantes, Nantes, France 18 9WELBIO, Walloon Excellence in Life Sciences and BIOtechnology, Belgium 19 *: Co-senior authors 20 21 Corresponding author: 22 Yan Burelle, Ph.D. 23 Professor 24 University Research Chair in Integrative Mitochondrial Biology 25 Interdisciplinary School of Health Sciences, Faculty of Health Sciences 26 University of Ottawa 27 RGN building, 28 451 Smyth Road, Ottawa Ontario 29 K1N 8M5 30 Email: [email protected] 31 32 Word count: 4156 33 References: 36 1 ABSTRACT 2 3 Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory 4 mechanism, in multiple organs including the heart. However, the mechanisms leading to O- 5 GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. 6 In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- 7 GlcNAc system without potential cytoplasmic confounding effects. We compared the O- 8 GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to 9 NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial 10 proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was 11 associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI 12 substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- 13 GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our 14 work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of 15 rapidly modifying mitochondrial function. 16 WORD COUNT: 149 17 DATA ACCESSIBILITY TO THE REVIEWERS: 18 The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 19 via the PRIDE partner repository with the following dataset identifier. 20 21 Reviewer Account Details: 22 Website: http://www.ebi.ac.uk/pride 23 Username: [email protected] 24 Password: MSf4tVMi 1 1. INTRODUCTION 2 O-linked N-acetylglucosamination (O-GlcNAcylation) is a dynamic post-translational 3 modification of proteins characterized by the addition of a single acetylated hexosamine moiety to 4 certain Ser/Thr residues through O-linkage on their hydroxyl (Bond 2015, Mailleux 2016). Protein 5 O-GlcNAcylation requires uridine disphosphate N-acetylglucosamine (UDP-GlcNAc) as 6 substrate, which is mainly derived from glucose through the hexosamine biosynthesis pathway 7 (HBP), driven by its rate limiting enzyme the glutamine:fructose-6-phosphate amidotransferase 8 (GFAT). O-GlcNAcylation is regulated by the uridine diphospho-N-acetylglucosamine transferase 9 (OGT), and the O-GlcNAcase (OGA), which respectively add and remove O-GlcNAc moieties 10 (Bond 2015, Mailleux 2016). These highly conserved and ubiquitously expressed enzymes are 11 present in various cellular locations where they regulate several protein properties such as their 12 activation state, localization, stability and/or degradation (Joiner 2019). Over the past several years, 13 protein O-GlcNAcylation has emerged as both pathogenic factor (Nie 2019) and important 14 mechanism involved in physiological processes such as development and protection against 15 cellular stress (Ong 2018, Jensen 2019). Such dual action can be found in the heart. Indeed, chronic 16 elevation of protein O-GlcNAcylation is believed to participate in the development of metabolic 17 and contractile dysfunctions associated with diabetes (Ma 2016) and cardiac hypertrophy (Mailleux 18 2016, Gelinas 2018). Conversely, acute hyper O-GlcNAcylation is known to confer protection 19 against ischemic damage (Jensen 2019) and sepsis-induced contractile dysfunction (Ferron 2019, 20 Silva 2019), which has contributed to position protein O-GlcNAcylation as a potential therapeutic 21 target for the management of both chronic and acute cardiovascular conditions. 22 While OGT and OGA are mainly localized to the nuclear and cytosolic compartments, several 23 studies have shown that mitochondria are major targets for O-GlcNAcylation (Bond 2015, Zhao 24 2016). Furthermore, growing evidence suggest that mitochondrial protein O-GlcNAcylation in fact 1 plays a role not only in the development of diabetic cardiomyopathy (Hu 2009, Banerjee 2015), 2 but also in the cardio-protective effect of acute hyper O-GlcNAcylation (Ngoh 2011, Jensen 2019). 3 This notion has been recently reinforced by results showing the presence of an UDP-GlcNAc 4 carrier along with OGA and a 103 kDa isoform of OGT (mOGT) in the mitochondrial compartment 5 (Hanover 2003, Love 2003, Banerjee 2015). Some controversies nevertheless exist regarding the 6 presence of these enzymes in the mitochondrial compartment (Trapannone 2016). More 7 importantly, since mitochondrial O-GlcNAcylation has mainly been investigated in cell culture 8 models or in vivo, the direct effects of the putative mitochondrial O-GlcNAc cycling system have 9 been difficult to distinguish from the indirect effects mediated by the nucleocytoplasmic O- 10 GlcNAcylation system. 11 In this study, we therefore took advantage of an in vitro reconstitution assay to characterize 12 the intra-mitochondrial O-GlcNAcylation system in isolated cardiac mitochondria. Our results 13 confirm the presence of a fully functional and dynamic O-GlcNAc cycling system in these 14 organelles. Using comparative O-GlcNAc proteomics (O-GlcNAcylomics), we provide evidence 15 that the local mitochondrial O-GlcNAcylation system can trigger broad and rapid changes in 16 protein O-GlcNAcylation, which are highly reminiscent of the mitochondrial O-GlcNAcylation 17 profile observed in vivo. Importantly, we also reveal that acute hyper-O-GlcNAcylation increases 18 maximal respiratory capacity, and drastically reduces ROS release though a complex-I mediated 19 mechanism, illustrating the capacity of this system to rapidly modify mitochondrial function. 1 2. RESULTS 2 2.1. In vitro reconstitution assay allows to target the intra-mitochondrial O-GlcNAcylation 3 system. 4 In order to characterize the mitochondrial O-GlcNAcylation system without the potential 5 confounding effects of the O-GlcNAcylation in other cellular compartments, we devised an in vitro 6 reconstitution assay in which isolated cardiac mitochondria were acutely exposed (i.e. 30 min) to 7 the OGT substrate UDP-GlcNAc in presence or absence of the OGA inhibitor NButGT, with the 8 goal of inducing rapid changes in protein O-GlcNAcylation levels. 9 As some controversy exists regarding the expression of the mOGT isoform in murine tissues 10 (Trapannone 2016), and the presence of sufficient OGA in the mitochondrial compartment 11 (Banerjee 2015), the presence of these enzymes was first verified by immunoblotting in whole 12 lysates from crude and Percoll-purified mitochondria (Fig 1A). Specific protein markers were 13 firstly assessed to confirm the purity of the different purified fractions (Fig 1B). As expected, the 14 mitochondrial marker TOM20 was highly enriched in the crude and Percoll-purified mitochondrial 15 fractions, while histone H3 and alpha tubulin were mostly recovered in the nuclear and cytosolic 16 fractions, respectively. Small amounts of histone H3 and alpha-tubulin remained present in the 17 crude mitochondrial preparation, but were largely removed by the Percoll purification step. As 18 represented in