RÉSUMÉ
Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter ß-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.
RÉSUMÉ
The phosphorylated pathway of serine biosynthesis represents an important pathway in plants. The pathway consist of three reactions catalyzed by the phosphoglycerate dehydrogenase, the phosphoserine aminotransferase and the phosphoserine phosphatase, and the genes encoding for all enzymes of the pathway have been identified. Previously, the importance of the phosphoglycerate dehydrogenase and phosphoserine phosphatase for plant metabolism and development has been shown, but due to the lack of T-DNA insertion mutants, a physiological characterization of the phosphoserine aminotransferase is still missing. Hence, we generated silencing lines specifically down-regulated in the expression of the major PSAT1 gene. The morphological characterization of the obtained PSAT1-silenced lines revealed a strong inhibition of shoot and root growth. In addition, these lines are hypersensitive to the inhibition of the photorespiratory serine biosynthesis, when growing the plants at elevated CO2. Metabolic analysis of PSAT1-silenced lines, showed a strong accumulation of certain amino acids, most likely due to an enhanced ammonium assimilation. Furthermore, phenotypic analysis under low and high-light conditions and in the presence of sucrose revealed, that the phosphorylated pathway of serine biosynthesis is essential for light and sugar-dependent growth promotion in plants.
RÉSUMÉ
Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.
Sujet(s)
Arabidopsis/métabolisme , Dioxyde de carbone/métabolisme , Consommation d'oxygène/physiologie , Photosynthèse/physiologie , Feuilles de plante/métabolisme , Sérine/biosynthèse , Arabidopsis/génétique , Arabidopsis/croissance et développement , Protéines d'Arabidopsis/génétique , Bases de données génétiques , Expression des gènes , Glycolates/métabolisme , Voies et réseaux métaboliques , Mutation , Oxygène/métabolisme , Phénotype , Phosphoglycerate dehydrogenase/déficit , Phosphoglycerate dehydrogenase/génétique , Phosphoric monoester hydrolases/déficit , Phosphoric monoester hydrolases/génétique , Phosphorylation , Feuilles de plante/génétique , Feuilles de plante/croissance et développement , Végétaux génétiquement modifiés , Pollen/génétique , Pollen/croissance et développement , Pollen/métabolisme , Graines/génétique , Graines/croissance et développement , Graines/métabolismeRÉSUMÉ
In plants, two independent serine biosynthetic pathways, the photorespiratory and glycolytic phosphoserine (PS) pathways, have been postulated. Although the photorespiratory pathway is well characterized, little information is available on the function of the PS pathway in plants. Here, we present a detailed characterization of phosphoglycerate dehydrogenases (PGDHs) as components of the PS pathway in Arabidopsis thaliana. All PGDHs localize to plastids and possess similar kinetic properties, but they differ with respect to their sensitivity to serine feedback inhibition. Furthermore, analysis of pgdh1 and phosphoserine phosphatase mutants revealed an embryo-lethal phenotype and PGDH1-silenced lines were inhibited in growth. Metabolic analyses of PGDH1-silenced lines grown under ambient and high CO2 conditions indicate a direct link between PS biosynthesis and ammonium assimilation. In addition, we obtained several lines of evidence for an interconnection between PS and tryptophan biosynthesis, because the expression of PGDH1 and phosphoserine aminotransferase1 is regulated by MYB51 and MYB34, two activators of tryptophan biosynthesis. Moreover, the concentration of tryptophan-derived glucosinolates and auxin were reduced in PGDH1-silenced plants. In essence, our results provide evidence for a vital function of PS biosynthesis for plant development and metabolism.
Sujet(s)
Composés d'ammonium/métabolisme , Protéines d'Arabidopsis/physiologie , Arabidopsis/enzymologie , Phosphoglycerate dehydrogenase/physiologie , Tryptophane/biosynthèse , Arabidopsis/croissance et développement , Arabidopsis/métabolisme , Protéines d'Arabidopsis/génétique , Protéines d'Arabidopsis/métabolisme , Voies de biosynthèse , Rétrocontrôle physiologique , Régulation de l'expression des gènes végétaux , Isoenzymes/génétique , Isoenzymes/métabolisme , Phosphoglycerate dehydrogenase/génétique , Phosphoglycerate dehydrogenase/métabolisme , Phosphosérine/métabolisme , Plastes/métabolismeRÉSUMÉ
3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the high-energy sulfate donor for sulfation reactions. Plants produce some PAPS in the cytosol, but it is predominantly produced in plastids. Accordingly, PAPS has to be provided by plastids to serve as a substrate for sulfotransferase reactions in the cytosol and the Golgi apparatus. We present several lines of evidence that the recently described Arabidopsis thaliana thylakoid ADP/ATP carrier TAAC transports PAPS across the plastid envelope and thus fulfills an additional function of high physiological relevance. Transport studies using the recombinant protein revealed that it favors PAPS, 3'-phosphoadenosine 5'-phosphate, and ATP as substrates; thus, we named it PAPST1. The protein could be detected both in the plastid envelope membrane and in thylakoids, and it is present in plastids of autotrophic and heterotrophic tissues. TAAC/PAPST1 belongs to the mitochondrial carrier family in contrast with the known animal PAPS transporters, which are members of the nucleotide-sugar transporter family. The expression of the PAPST1 gene is regulated by the same MYB transcription factors also regulating the biosynthesis of sulfated secondary metabolites, glucosinolates. Molecular and physiological analyses of papst1 mutant plants indicate that PAPST1 is involved in several aspects of sulfur metabolism, including the biosynthesis of thiols, glucosinolates, and phytosulfokines.
Sujet(s)
Antiports/physiologie , Protéines d'Arabidopsis/physiologie , Arabidopsis/métabolisme , Cytosol/métabolisme , PAPS/métabolisme , Thylacoïdes/métabolisme , Antiports/génétique , Antiports/métabolisme , Protéines d'Arabidopsis/génétique , Protéines d'Arabidopsis/métabolisme , Transport biologique , PAPS/biosynthèse , Plastes/métabolismeRÉSUMÉ
Targeting protein kinases with small organic molecules is a promising strategy to regulate unwanted kinase activity in both chemical biology and medicinal chemistry research. Traditionally, kinase inhibitors are identified in activity-based screening assays using enzymatically active kinase preparations to measure the perturbation of substrate phosphorylation, often resulting in the enrichment of classical ATP competitive (Type I) inhibitors. However, addressing enzymatically incompetent kinase conformations offers new opportunities for targeted therapies and is moving to the forefront of kinase inhibitor research. Here we report the development of a new FLiK (Fluorescent Labels in Kinases) binding assay to detect small molecules that induce changes in the conformation of the glycine-rich loop. Due to cross-talk between the glycine-rich loop and the activation loop in kinases, this alternative labeling approach can also detect ligands that stabilize inactive kinase conformations, including slow-binding Type II and Type III kinase inhibitors. Protein X-ray crystallography validated the assay results and identified a novel DFG-out binding mode for a quinazoline-based inhibitor in p38alpha kinase. We also detected the high-affinity binding of a clinically relevant and specific VEGFR2 inhibitor, and we provide structural details of its binding mode in p38alpha, in which it stabilizes the DFG-out conformation. Last, we demonstrate the power of this new FLiK labeling strategy to detect the binding of Type I ligands that induce conformational changes in the glycine-rich loop as a means of gaining affinity for the target kinase. This approach may be a useful alternative to develop direct binding assays for kinases that do not adopt the DFG-out conformation while also avoiding the use of expensive kits, detection reagents, or radioactivity frequently employed with activity-based assays.
Sujet(s)
Colorants fluorescents/composition chimique , Glycine/composition chimique , Inhibiteurs de protéines kinases , Cristallographie aux rayons X , Glycine/antagonistes et inhibiteurs , Glycine/métabolisme , Modèles moléculaires , Conformation moléculaire , Structure moléculaire , Inhibiteurs de protéines kinases/composition chimique , Inhibiteurs de protéines kinases/pharmacologieRÉSUMÉ
Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.
Sujet(s)
Évaluation préclinique de médicament/méthodes , Mitogen-Activated Protein Kinase 14/antagonistes et inhibiteurs , Inhibiteurs de protéines kinases/composition chimique , Cristallographie aux rayons X , Stabilité enzymatique , Humains , Ligands , Mitogen-Activated Protein Kinase 14/composition chimique , Liaison aux protéines , Conformation des protéines , Bibliothèques de petites molécules , Relation structure-activitéRÉSUMÉ
Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less conserved allosteric sites would be expected to offer new opportunities for scaffold development. Because no high-throughput method was previously available, we developed a fluorescence-based kinase binding assay for identifying and characterizing ligands which stabilize the inactive kinase conformation. Here, we present a description of the development and validation of this assay using the serine/threonine kinase p38alpha. By covalently attaching fluorophores to the activation loop of the kinase, we were able to detect conformational changes and measure the K(d), k(on), and k(off) associated with the binding and dissociation of ligands to the allosteric pocket. We report the SAR of a synthesized focused library of pyrazolourea derivatives, a scaffold known to bind with high affinity to the allosteric pocket of p38alpha. Additionally, we used protein X-ray crystallography together with our assay to examine the binding and dissociation kinetics to characterize potent quinazoline- and quinoline-based type II inhibitors, which also utilize this binding pocket in p38alpha. Last, we identified the b-Raf inhibitor sorafenib as a potent low nanomolar inhibitor of p38alpha and used protein X-ray crystallography to confirm a unique binding mode to the inactive kinase conformation.