Flavine adénine dinucléotide

La flavine adénine dinucléotide (FAD) est une coenzyme d'oxydo-réduction dérivant de la riboflavine (vitamine B2). Il est associé aux enzymes de la classe des oxydo-réductases auxquelles il est lié par une liaison covalente : c'est un groupement prosthétique. Ce coenzyme est notamment utilisé par les flavoprotéines du complexe II de la chaîne respiratoire mitochondriale : glycérol 3-P déshydrogénase, acylCoA déshydrogénase, succinate déshydrogénase. Au niveau de cette chaine respiratoire mitochondriale, le FAD permet la formation de 2ATP grâce au pompage de 6 protons dans l'espace inter-membranaire. Le couple rédox est le suivant : \text{FAD} \leftrightarrow \text{FADH}_{2} Le FAD intervient également dans la β-oxydation des acides gras lors de l'oxydation de l'acylCoA en TransΔ2-3 énoylCoA via l'acylCoA déshydrogénase. Cette réaction est essentielle à la progression de la β-oxydation mais peut être shunté dans certains cas : acide gras insaturé avec une double liaiso

Characterization of iron reduction in Clostridium acetobutylicum mutants obtained by targeted and transposon mutagenesis

Matthieu Jonas Valentin Amos

Iron reduction in Gram-positive bacteria is not well understood yet, even if it has been investigated in some extent for Gram-positive bacteria. The mechanisms involved in the delivery of electrons to a solid terminal electron acceptor like iron oxides have not been defined. Clostridium acetobutylicum is an appropriate Gram-positive bacterium to study those mechanisms as genetic tools have been developed due to its industrial interest, allowing easy targeted and random mutagenesis. In this Master’s project, phenotype of two mutants, whose dihydroorotate dehydrogenase 1B (pyrD) or ferredoxin hydrogenase (hydA) gene have been knocked out through ClosTron mutagenesis, have been characterized and no phenotype diverging from the wild strain has been detected. However, evidences of flavin presence in the spent growth medium have been observed during experiment. An attempt to measure their redox state, either by direct measurement or through the addition of AQDS, has been done but results are non-conclusive so far. A screening of mutant issued from untargeted mutagenesis has been tried, but the protocol could not furnish stable results during the test phase using the wild type strain. The protocol has been improved, but there still are problems regarding growth and iron reduction.

2017

Histone methyl transferases and demethylases; can they link metabolism and transcription?

Johan Auwerx, Kristina Schoonjans

Heritable changes to the transcriptome that are independent to changes in the genome are defined as epigenetics. DNA methylation and posttranslational modifications of histones, such as acetylation/deacetylation and methylation/demethylation of lysine residues, underlie these epigenetic phenomena, which impact on many physiological processes. This perspective focuses on the emerging biology of histone methylation and demethylation, highlighting how these reactions depend on metabolic coenzymes like S-adenosylmethionine, flavin adenine dinucleotide, and α-ketoglutarate. Furthermore, we illustrate that methyltranferases and demethylases affect many metabolic pathways. Despite the preliminary evidence that methyltranferases and demethylases could link metabolic signals to chromatin and alter transcription, further research is indispensable to consolidate these enticing observations.

Elsevier2010

Semisynthetic Biosensors for the Quantification of Nicotinamide Adenine Dinucleotides in Live Cells

Olivier Vincent Sallin

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are crucial redox cofactors regulating energy metabolism, reductive biosynthesis and the antioxidant defense system. Besides their role as redox cofactors, NAD(P)s have more recently also been described as important signaling molecules, acting for example as cosubstrate of different enzymes such as the sirtuins and poly(ADP-ribose) polymerases (PARPs), involved in the control of important cellular functions. Highlighting their important role, an imbalance of their intracellular concentration has been associated with aging and different pathologies. Therefore, the development of methods enabling the accurate subcellular quantification of NAD(P) in live cells is of great importance. This thesis describes the development and application of two FRET-based semisynthetic biosensors for NADPH/NADP+ and NAD+ in live cells. These sensors, named NAD(P)-Snifits, possess a high dynamic range, high specificity, are pH insensitive and can be excited at long wavelenghts. By using ratiometric imaging and fluorescence lifetime imaging, we quantified free NADPH/NADP+ and NAD+ in different subcellular compartments in live cells. We report here, for the first time, that free NADPH/NADP+ is significantly higher in the mitochondria than in the cytosol and observed moderately higher NAD+ level in the mitochondrial matrix consistent with previous report. We have also demonstrated that NADP-Snifit can be used to monitor the dynamics of NADPH/NADP+ caused by oxidative stress using conventional widefield fluorescence microscopy. The easy handing and use of these sensors make it possible to study not only the kinetics of cellular H2O2 scavenging and NADPH regeneration rate, but also to monitor drug-related side effects. Finally, we measured the effect of different conditions and drug treatment mimicking calorie restriction on free cytosolic and mitochondrial NADPH/NADP+ and NAD+ by flow cytometry. We observed that all these treatments increased free cytosolic and mitochondrial NAD+ concentrations with various levels of efficiency, but had divergent effects on the different subcellular NADPH/NADP+. NAD(P)-Snifits have the potential of becoming valuable tools to study the NAD(P) metabolism in physiological relevant conditions. Their wide applicability, in particular with high-throughput methods such as flow cytometry opens the possibilities for their use in metabolic and drug screening of large libraries.

EPFL2018

Semisynthetic Biosensors for the Quantification of Nicotinamide Adenine Dinucleotides in Live Cells

Olivier Vincent Sallin

EPFL2018