Etude des mécanismes de régulation de l'équipement enzymatique mitochondrial lors de la croissance de la levure S. cerevisiae (rôle des protéines kinases dépendantes de l'AMPc)

La voie AMPc contribue à l'ajustement des mitochondries à la demande énergétique lors de la croissance sur substrat non fermentescible. La sous-unité catalytique de la protéine kinase AMPc dépendante de la levure S. cerevisiae existe sous 3 isoformes codées par les gènes TPK. Nous avons montré que la protéine Tpk3p joue un rôle majeur dans la maintenance de l'équipement enzymatique mitochondrial lors de la croissance. Nous avons étudié les mécanismes moléculaires de cette signalisation et nous avons montré que l'absence de la protéine Tpk3p entraîne une augmentation de la production mitochondriale de ROS. Celle-ci semble affecter la biogenèse mitochondriale que nous avons estimé en mesurant l'activité des facteurs de transcription HAP2,3,4,5 impliqués dans la transcription de nombreux gènes mitochondriaux. Cette altération provoque une diminution de l'équipement enzymatique, réversible lors de l'ajout d'antioxydant. La diminution de l'activité du complexe HAP entraîne des modifications mitochondriales à l'origine d'une production de ROS encore plus importante. Afin de déterminer l'origine du processus d'ajustement des mitochondries à la demande énergétique, nous avons utilisé un autre modèle en incubant dans un milieu de "resting-cell" des levures prélevées lors d'une croissance. L'étude réalisée en absence de croissance suggère une modification de l'état stationnaire du fonctionnement des oxydations phosphorylantes sans une modification quantitative des mitochondries. A partir de ces données préliminaires nous pouvons émettre l'hypothèse que l'ajustement quantitatif des mitochondries lors de la croissance nécessite prolifération cellulaire.In yeast, cAMP pathway contributes to the mitochondrial adjustment to energy demand during growth on non-fermentable carbon source. cAMP-dependent protein kinase catalytic subunits are encoded by 3 different TPK genes. We showed that the Tpk3p plays a major role in the maintenance of optimal mitochondrial amount in response to energy demand. We investigated the molecular mechanisms and we have shown that lack of Tpk3p leads to an increase in mitochondrial ROS production. In order to demonstrate whether the decrease in amount of mitochondria was linked to a decrease in mitochondrial biogenesis, we assessed the activity of the Hap2/3/4/5p transcription factor which is known to be necessary for transcriptional activation of many mitochondrial genes. We show that ROS should be responsible for a decrease in Hap activity, which in turn leads to mitochondrial modifications. This generates a vicious circle which can be restored by using antioxidant, since modifications are responsible for more ROS production. cAMP pathway contributes to the adjustment of mitochondria to the energy demand by modulating the amount of mitochondria. In order to determine the origin of this adjustment we use another approach, we take cells in proliferation state and we arrest proliferation by transferring cells into a resting medium. Using various strains, our preliminary data show that regulation is identical regardless of the strain, involves a modification of the mitochondrial steady state respiration and there is no mitochondrial amount modification. We can hypothesis on these facts that mitochondrial amount adjustment during growth, needs cell proliferation in order to drops mitochondrial amount.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Kv1.3 inhibitors have differential effects on glucose uptake and AMPK activity in skeletal muscle cell lines and mouse ex vivo skeletal muscle

Knockout of Kv1.3 improves glucose homeostasis and confers resistance to obesity. Additionally, Kv1.3 inhibition enhances glucose uptake. This is thought to occur through calcium release. Kv1.3 inhibition in T-lymphocytes alters mitochondrial membrane potential, and, as many agents that induce Ca2+release or inhibit mitochondrial function activate AMPK, we hypothesised that Kv1.3 inhibition would activate AMPK and increase glucose uptake. We screened cultured muscle with a range of Kv1.3 inhibitors for their ability to alter glucose uptake. Only Psora4 increased glucose uptake in C2C12myotubes. None of the inhibitors had any impact on L6 myotubes. Magratoxin activated AMPK in C2C12myotubes and only Pap1 activated AMPK in the SOL. Kv1.3 inhibitors did not alter cellular respiration, indicating a lack of effect on mitochondrial function. In conclusion, AMPK does not mediate the effects of Kv1.3 inhibitors and they display differential effects in different skeletal muscle cell lines without impairing mitochondrial function

Reactive Oxygen Species-mediated Regulation of Mitochondrial Biogenesis in the Yeast Saccharomyces cerevisiae*

Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated, and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of reactive oxygen species that signal to the HAP2/3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We establish that an increase in mitochondrial reactive oxygen species production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria quality control process

Trypanosoma vivax GM6 antigen: a candidate antigen for diagnosis of African animal trypanosomosis in cattle.

International audienceBackground: Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km(2) of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases. Methodology/Principle Findings: the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4). Conclusion/Significance: this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle

Representative TvGM6 and TcoGM6 ELISA analysis of longitudinal experimental infection sera with (A, B) <i>T. vivax</i> and (C,D) <i>T. congolense</i> in individual animals.

<p>TvGM6 ELISA using sera from infections with (A) <i>T. vivax</i> IL2172 (drug-sensitive) and (B) <i>T. vivax</i> Komborodougou (drug-resistant). TcoGM6 ELISA using sera from infections with (C) <i>T. congolense</i> O2J (drug-sensitive) and (D) <i>T. congolense</i> KONT2/133 (drug-resistant). All animals were treated with 3.5mg/kg of diminazene diaceturate at the dates indicated by the arrows, (D) was treated a second time with 1 mg/kg isometamidium chloride. For figures (A), (C) and (D) parasitaemia score can be related to approximate amounts of parasites as follows: 2 (1-10/preparation), 3(1-2/field), 4 (1-10/field), 5 (10-50/>50 field), 6 (>100/field). TvGM6 or TcoGM6 ELISA OD (£), whole trypanosome lysate ELISA OD () and parasitaemia () are indicated. Arrows indicate trypanocidal treatment, and the x-axis is the threshold for positivity. </p

Use of Cells Expressing gamma Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) γ2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca2+ ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca2+, and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration