Correction to: Use of MFM-20 to monitor SMA types 1 and 2 patients treated with nusinersen.

peer reviewe

Caractérisation moléculaire et biochimique des préphénate et arogénate déshydrogénases d'Arabidopsis thaliana, de synechocystis et de levure

L'arogénate deshydrogénase catalyse la réaction de décarboxylation oxydative de l'arogénate en tyrosine. Malgré l'importance de cette activité enzymatique chez les plantes, aucune arogénate déshydrogénase de plante n'avait été clonée ou purifiée au début de nos travaux. Notre étude a permis l'identification de deux gènes, notés tyrAAT1 et tyrAAT2, codant des arogénate deshydrogènase chez la plante modèle Arabidopsis thaliana. Une étude moléculaire et biochimique a montré que le produit du gène tyrAAT1 est constitué de domaines catalytiques très similaires et actifs séparément. Au contraire, le produit du gène tyrAAT2, comme toutes les préphénate et/ou arogénate deshydrogénases bactériennes actuellement identifiées n'est composé que par un seul domaine catalytique. L'étude de la localisation de ces deux isoformes chez Arabidopsis montre que ces deux gènes codent pour des enzymes plastidiales. Une étude biochimique réalisée à partir des protéines reombinantes TyrAAT1 et TyrAAT2 purifiées montre que les deux protéines possèdent des propriétés biochimiques proches et une étude in planta montre que les gènes tyrAAT1 et tyrAAT2 sont exprimés avec des abondances relatives proches dans tous les tissus étudiés.Une étude biochimique et structurale comparée entre les arogénate deshydrogénases d'Arabidopsis et de Synechocystis et la préphénate deshydrogénase de levure a été entreprise. Cette étude montre des mécanismes de régulation des enzymes différents permettant à ces organismes de mieux répondre aux besoins des cellules en acides aminés aromatiques. L'exploitation des connaissances acquises sur les activités préphénate et arogénate deshydrogénases a permis l'utilisation en transgenèse végétale de la préphénate deshydrogénase de levure afin de rendre les plantes tolérantes à des doses plus élevées en DKN.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Engineering Plant Shikimate Pathway for Production of Tocotrienol and Improving Herbicide Resistance

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants

Tyrosine and Phenylalanine Are Synthesized within the Plastids in Arabidopsis1[W]

While the presence of a complete shikimate pathway within plant plastids is definitively established, the existence of a cytosolic postchorismate portion of the pathway is still debated. This question is alimented by the presence of a chorismate mutase (CM) within the cytosol. Until now, the only known destiny of prephenate, the product of CM, is incorporation into tyrosine (Tyr) and/or phenylalanine (Phe). Therefore, the presence of a cytosolic CM suggests that enzymes involved downstream of CM in Tyr or Phe biosynthesis could be present within the cytosol of plant cells. It was thus of particular interest to clarify the subcellular localization of arogenate dehydrogenases (TYRAs) and arogenate dehydratases (ADTs), which catalyze the ultimate steps in Tyr and Phe biosynthesis, respectively. The aim of this study was to address this question in Arabidopsis (Arabidopsis thaliana) by analysis of the subcellular localization of the two TYRAAts and the six AtADTs. This article excludes the occurrence of a spliced TYRAAt1 transcript encoding a cytosolic TYRA protein. Transient expression analyses of TYRA- and ADT-green fluorescent protein fusions reveal that the two Arabidopsis TYRA proteins and the six ADT proteins are all targeted within the plastid. Accordingly, TYRA and ADT proteins were both immunodetected in the chloroplast soluble protein fraction (stroma) of Arabidopsis. No TYRA or ADT proteins were immunodetected in the cytosol of Arabidopsis cells. Taken together, all our data exclude the possibility of Tyr and/or Phe synthesis within the cytosol, at least in green leaves and Arabidopsis cultured cells

Distal motor function assessments of children with spinal muscular atrophy: the use of a tablets as a part of the proposed kinect-MFM study

International audienceIntroduction: Given the progress of research and management in the neuromuscular diseases, particularly in Spinal Muscular Atrophy (SMA), validated tools are needed to assess patients' motor function. These tools are fundamental in order to improve the understanding of the natural history and to quantify the impact of new therapeutics in these populations. The Motor Function Measure (MFM) is a validated scale for the measurement of functional motor capacities usable in all neuromuscular diseases. Purpose: Clinicians from a neuromuscular diseases reference center (Hospices Civils de Lyon, France) and G-SCOP research team (INP Grenoble, France) were developing the instrumented Kinect-MFM, an automated system to assess SMA patients' motor function using new and low cost technology. By using, these technologies, our objectives were to improve the quality and reproducibility of the MFM by suppressing subjectivity linked to heteroevaluation. Method: The first step of this work was to assess the relevance of the tablet to capture and measure distal motor functions during a MFM test. The second was to compare the scoring of MFM items provided by a therapist with the scoring provided by the system. Results: Three applications were developed on this system to allow the comparison. They show difficulties to reproduce exactly the same conditions than in the current MFM. The size, the sensitivity, the multipoint control and the accuracy of the tablet constitute some challenge we have to take up. Conclusions: the proposed tablet was initially user to control the complete system by the therapist. The complexity of measuring distal functions by the Kinect led us to use this technology to complete the MFM instrumented protocol researchers proposed

L’évaluation automatisée et ludique de la fonction motrice : le projet Kinect MFM

National audienc

L’évaluation automatisée et ludique de la fonction motrice : le projet Kinect MFM

National audienc

Methionine metabolism in plants. Chloroplasts are autonomous for de novo methionine synthesis and can import s-adenosylmethionine from the cytosol

International audienceThe subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B12-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism

Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction

Influence of a two-year steroid treatment on body composition as measured by dual X-ray absorptiometry in boys with Duchenne muscular dystrophy

Abstract Steroids are nowadays routinely used as a long-term treatment in Duchenne muscular dystrophy (DMD). Their effects on body composition were assessed using dual X-ray absorptiometry. The study followed over 2 years 29 genetically confirmed DMD patients: 21 in the steroid-treated group and 8 in the steroid-naïve group. After 2 years of steroid treatment, the lean tissue mass values increased significantly (p < 0.0001), the percentage of body fat mass remained practically constant (p = 0.94) in comparison with the initial visit. In the steroid-naïve patients, there were no significant increases in the lean tissue mass but deterioration in body composition confirmed by a significant increase in the percentage of body fat mass. Besides, significant negative correlations were found between the percentage of body fat mass and the MFM total score (R = À0.79, n = 76, p < 0.0001). A 2-year steroid treatment improves significantly body composition of boys with DMD through a significant increase in lean tissue mass. We suggest that a thorough check of body composition should be carried out before steroid treatment discontinuation in case of overweight gain