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THÈMES DE RECHERCHE

 

Molecular and Systems Bioenergetics (leader: Uwe Schlattner, PR)

Main efforts of team have been dedicated to molecular bioenergetics, in particular structure and function of key kinases and their role in cellular microcompartments and energy homeostasis. This yielded over 70 publications since 2009, including Science, Nature, Nat Struct Mol Biol, and Mol Cell Biology. This research was funded by the EU FP6 (EXGENESIS) and FP7 programs, Erasmus Mundus BioHealth Computing, ANR (SYBECAR), ANRS, Région Rhône-Alpes (CIBLE and MIRA), French Ministry of Foreign Affairs (H.Curien program), and FRM.

  • AMP-activated protein kinase (AMPK) has emerged as the central cellular energy sensor and regulator, but the full extent of its signaling network and its precise activation mechanism(s) were not yet fully understood. We have performed three different screens to identify new AMPK interactors and/or substrates to extend the AMPK signaling network: in vitro phosphorylation (kinase assay), in vitro interaction (by surface plasmon resonance assays - Biacore) and in vivo interaction (by novel cytosolic yeast-two-hybrid assays). From these, three interactors/substrates have been published so far (fumarate hydratase, glutathion-S-transferase, B-type creatine kinase - BCK) while characterization of others is still ongoing (Klaus et al 2012 J Proteomics, 2013 Plos ONE; Ramirez Rios et al 2014 BBA Bioenerg). Importantly, phosphorylation of BCK seems to affect not its enzyme activity but BCK localization within the cell, which is novel for an AMPK substrate. For allosteric AMPK activation by AMP (and ADP), we were the first to publish a conformational switch model as central part of its mechanism (Riek et al 2008 JBC). This remained controversial due to the absence of conformational changes between inactive and active (AMP-bound) states in the first X-ray structures of AMPK core complex. However, we could fully confirm a conformational switch and correct some errors in the initial studies in collaboration with JW Wu (Beijing, China) by using X-ray crystallography, NMR and mutational studies (Chen et al 2012 Nat Struct Mol Biol; Chen et al 2013 Nature; Riek et al 2009 Biotechniques). Based on these data, we have performed extensive molecular engineering to obtain a fluorescent, genetically encoded FRET sensor that reports allosteric activation of AMPK and thus also of intracellular AMP and ADP levels (patent EP14173018).
  • Nucleoside diphosphate kinase (NDPK or NM23) is essential to generate nucleoside triphosphates (NTPs) other than ATP by using ATP and NDPs. These enzymes were considered as simple house-keeping enzymes without regulatory importance. We could show in recent years for the mitochondrial isoform (NDPK-D or NM23-H4) that the enzyme has indeed multiple regulatory functions (Boissan et al. 2014 Science; Schlattner et al. 2013 JBC). First, as a kinase, it directly channels its product GTP to mitochondrial dynamin-like GTPases such as OPA1 to maintain inner membrane dynamics, and ADP to the adenylate translocator (ANT) and mitochondrial ATPase to regenerate ATP. As a second function, NM23-H4 binds and cross-links the two mitochondrial membranes to facilitate inter-membrane lipid transfer. NM23-H4-induced transfer of cardiolipin from the inner to the outer mitochondrial membrane can then serve as a trigger for apoptosis.
  • Mitochondrial interactosome containing among others creatine kinase (CK), ANT, and voltage-dependent anion channel (VDAC) induces contacts between inner and outer mitochondrial membrane and is involved in energy fluxes at the cytosol/mitochondria interface. Detailed respiratory and metabolic control analysis provided further evidence that these structures are key regulators of muscle cell respiration (Guzun et al 2009 BBA Bioenerg; Tepp et al 2011, BBA Bioenerg; Kuznetsov et al 2012 Biochem J). Following-up our earlier studies (Rostovtseva et al 2008 PNAS), we substantiated a role of tubulin/VDAC interactions for this type of regulation, as well as for the regular arrangement of mitochondria in cardiomyocytes (Guzun et al. 2011 BBA Bioenerg; Varikmaa et al 2014 BBA Bioenerg). Also creatine itself binds membranes, affects membrane properties, and may alter functions of the above membrane proteins (Tokarska-Schlattner et al 2012 PLOS One). As an international leader in CK research, we were involved in a number of collaborations addressing the role of mitochondrial CK in human pathologies: myocardial inflammation (Ebermann et al 2009 Basic Res Cardiol), McArdle disease (Kitaoka et al 2013 Mol Genet Metab), and HIV infection (Schmid et al 2013 Antivir Ther).
  • ATPase family AAA Domain-containing protein 3 (ATAD3) is an abundant mitochondrial inner membrane protein of unknown function. We have successfully initiated studies to understand its molecular topology and functions in mitochondria and for cell physiology. ATAD3 protein was recombinantly expressed in yeast, its expression pattern analyzed, and functions for mitochondrial biogenesis and remodeling was evidenced during adipocyte differentiation (Li et al. 2012 Prot Purif Expr, 2013 Gene, 2014 Mol Cell Biol).
  • Tyrosine nitration plays an important role in the regulation of kinases, mitochondrial metabolism and cell homeostasis. We showed that it is required for MEK1 and ERK ½ phosphorylation/activation and Akt inhibition (Csibi et al 2010 PLoS One) as well as for other enzymes (Daiber et al 2013 Int J Mol Sci). As a marker of nitro-oxidative stress, we developed an assay for plasma nitroalbumin (Wayenberg et al. 2009 FRBM  and 2010 Neonatology; patents Botari et al PCT/EP2009/000036 and USA 12/812 498).

Team was further active in studying perturbation of energy and metabolic homeostasis in cellular model systems or clinical patients, induced by exercise, drugs or chronic disease. As it was our intention, systems biology approaches were progressively integrated into this type of study, and tools were developed to support such integrated analysis. However, some parts of our ambitions in this field have been delayed, since Prof. V Saks had to leave the unit in 2011 due to health issues (he was thought to continue on an emeritus status), and a new recruitment has been impossible due to UJF budget restrictions. A large part of the mentioned studies is still ongoing, but already yielded some important publications (Angew Chem Int Edit, Anal Chem, Cardiovasc Res) or patents (EP14173018, EP2432800). This research was funded by EU FP7 (ANTHRAPLUS, MeDALL, SysCLAD) and ANR (NANOMITO, CADMIDIA).

  • Biosensors allow cellular in vivo analysis with high temporal and spatial resolution as needed for systems biology approaches. The genetically encoded AMPK FRET sensor (see above) is an example that allows studying cellular energy state at subcellular resolution (patent EP14173018). Novel miniaturized electro­chemical biosensors based on peroxidase-redox polymer-modified electrodes provide selective detection of H2O2 with nanomolar sensitivity, large linear response, and fast response time at single mitochondria resolution. This allows e.g. detection of H2O2 fluxes and bursts during and immediately after complexIII inhibition (Suraniti et al 2013 Anal Chem; Suraniti et al 2014, Angew Chem Int Edit).        
  • Anthracycline-induced cardiotoxicity is an ideal model system to study cell signaling under energy stress. The toxic side-effect of these efficient anti-cancer drugs limits their use, but is still poorly understood, and preventive or therapeutic treatments are not available. Using rat isolated perfused heart and animal models treated with clinically relevant drug doses to mimic acute and chronic toxicity, transcriptomics and phosphoproteomics revealed widespread suppression of stress signaling, but specifically altered phosphorylation levels of some proteins causing energy imbalance and myofibrillar disorganization (Tokarska-Schlattner et al 2010 Am J Physiol Integr Comp Physiol; Gratia et al 2012 J Proteomics). In particular AMPK signaling was inhibited by the drugs, at least in part because of negative crosstalk with Akt and MAPK pathways, largely triggered by DNA damage signaling (Gratia et al 2012 Cardiovasc Res). We also settled the fundamental differences in bioenergetic regulation of mitochondria in normal versus cancer cells (Kaambre et al 2013 Front Physiol).
  • Chronic obstructive pulmonary disease (COPD) leads to severe respiratory insufficiency, associated with unexplained loss of peripheral muscle mass, and often requires lung transplantation. We further studied the beneficial effects of mild exercise observed in COPD patients (Guzun et al 2012 Acta Physiol). In rats, docosahexaenoic acid (DHA)-enriched diet, combined or not with endurance training, led to enhanced muscle function and exercise tolerance. DHA alone could mimic the exercise effect, including the activation of AMPK (Le Guen 2014, submitted). By a series of conceptional papers, we participated in setting the scene for systems medicine projects combating chronic non-communicable diseases (Bousquet et al 2011 Genome Med), namely predicting chronic lung allograph dysfunction (SysCLAD; Auffray et al 2010 Chest; Bousquet et al 2014 Curr Pharm Design; Pison et al 2014 Eur Respir J), and deciphering mechanisms in the development of allergy (MeDALL; Bousquet 2011 Allergy).

Bioenergetics and metabolism (leader: Eric Fontaine, PUPH)

Team has two main fields of interest in bioenergetics, namely: (i) mitochondrial physiology and cell death, and (ii) energy metabolism, exercise and nutrition. The former has been one of the strong points of the unit, yielding several important publications (Cell Death Dis, JBC), and being supported by public, association and industry funding, including ANR (CADMIDIA), AGIR à dom, and POXEL (Lyon, France). However, several aspects of our original project were affected by the premature death of Prof. Xavier Leverve (PUPH) in 2010, former director of our unit, and to lesser degree by the retirement of Dr Christiane Chauvin (CR INSERM) and the relocation of Dr Luc Demaison (CR INRA) for private reasons to Clermont-Ferrand in 2012. New recruitments at UJF have been impossible so far due to UJF budget restrictions.

The mitochondrial permeability transition pore (PTP) is a mitochondrial inner membrane channel involved in cell death induced by oxidative stress. Even if its molecular nature is still an issue of controversy, its regulation has been studied in quite some detail in hepatic tissue, with the implicit assumption that it is identical in all other tissues. Although a technique for measuring PTP opening in isolated mitochondria has been known for long, its direct visualization in intact cells remained a challenge for a long time, only solved in 1999. However, this initial technique (the so-called calcein-cobalt-method) was unable to differentiate between a transient and persistent PTP opening, with only the latter triggering cell death. This led to inconsistencies between the number of cells where PTP was (or had been) in an open state, and the number of cells undergoing apoptosis. In this context, we have been at the origin of two major advances:

  • First, we have established a novel PTP detection technique based on the simultaneous measurement of mitochondrial membrane potential and cellular repartition of NADH. It is capable to differentiate between transient and permanent PTP opening (Dumas et al 2009 J Biol Chem). This technique is now routinely used in our laboratory and allowed to demonstrate that PTP opening is involved in glucose, fructose (Lablanche et al 2011 Cell Death Dis), and alcohol toxicity (Lamarche et al 2013 Chem Res Toxicol) and ischemia-reperfusion (Lablanche et al in revision).
  • Second, we demonstrated that regulation of PTP is partially tissue-dependent. This makes the study of PTP even more complex and suggests that its molecular nature is considerably different, depending on the tissue considered. In turn, this discovery allowed us to develop a strategy to induce cell death selectively in certain cancerous cells but preserving the healthy parental cells (Devun et al 2010 PLoS One). Back ten years ago, we have been at the origin of the observation that inhibitors of respiratory complex I also inhibit PTP opening, but only in certain tissues. In the present contracting, we have up-dated the molecular mechanism at the origin of this observation (Li et al 2012 BBA Bioenergetics).

Mitochondria and the adaptation to the metabolic environment, in particular in liver, has been another topic in mitochondrial physiology that has been pursued despite the premature death of the scientific leader.

  • Exposure to high fat diet, a key feature of contemporary western diet, affects regulation of oxidative phosphorylation in a rat model by (i) lowering the mitochondrial quinone pool, (ii) increasing its degree of reduction, (iii) affecting lipid composition of mitochondrial membranes, and (iv) increasing production of reactive oxygen species (Vial et al 2011 J Hepatol).
  • Preclinical tests of a novel oral antidiabetic drug (Imeglimin, Poxel) have been under embargo until recently and will be published shortly.

We are very much interested to continue this topic for which we acquired a recognized expertise, among others by industry contracts. Nevertheless, in the context of our limited human resources, this topic requires to be reinforced by the appointment of a competent scientist in this domain.

Effects of exercise and nutrition on energy metabolism

  • The study of polyphenols of nutritional origin is another important topic of the team. It is at the origin of numerous scientific collaborations and industry contracts. During the past 5-year period, have shown that cinnamon extract modifies the liver metabolism of glycogen (Couturier et al 2011, Metabolism) and prevents the deleterious effects of a high fat/high fructose diet in the brain (Anderson et al 2013, PLoS One). More recently, we have been interested in the effects of aleurone and we could show that this polyphenol modifies the composition of omega-3 fatty acids within membrane phospholipids (2014, Food and Nutrition Research, in press).
  • The importance of physical exercise and nutrition on muscle metabolism, but also on other tissues, is an emerging topic of our team. This topic has not yet yielded publications coming from our team, but very promising results have been obtained in our laboratory that motivate us to develop this subject. For this reason, C Moinard (MCF Univ. Paris Descartes), an expert in this field, has joined us in 2013 in the frame of a “delegation universitaire”, with the explicit ambition to recruit him on a PR position. A summary of his research during the last 5 years is given below.