New red-shifted fluorescent biosensor for monitoring intracellular redox changes.

Maintenance of intracellular redox homeostasis is critical for cell survival, proliferation, differentiation, and signaling. In this regard, major changes in the intracellular redox milieu may lead to cell death whereas subtle increases in the level of certain oxidizing species may act as signals that regulate a plethora of cellular processes. Redox-sensitive variants of green fluorescent proteins (roGFP2 and rxYFP) were developed and proved useful to monitor intracellular redox changes in a non-invasive and online manner. With the aim to extend the spectral range of the fluorescent redox biosensors, we here describe the generation, biochemical characterization and biological validation of a new redox reporter based on the red-shifted mRuby2 protein (rxmRuby2). Spectrofluorimetric analysis performed with the recombinant biosensor shows a reversible redox response produced by two redox-active cysteine residues predicted by molecular modeling. rxmRuby2 is highly selective for the couple glutathione/glutathione disulfide in the presence of the oxidoreductase glutaredoxin. The estimated redox potential of rxmRuby2 (E° -265 ±â€¯22 mV) makes it suitable for its use in reducing subcellular compartments. Titration assays demonstrated the capacity of rxmRuby2 to monitor redox changes within a physiological pH range. rxmRuby2 responded sensitively and reversibly to different redox stimuli applied to HeLa and HEK293 cells expressing transiently and/or stable the biosensor. Fusing rxmRuby2 to the Clover fluorescent protein allowed normalization of the redox signal to the expression level of the reporter protein and/or to other factors that may affect fluorescence. The new red-shifted redox biosensor show promises for deep-tissue and in vivo imaging applications.

New pieces to the carbon metabolism puzzle of Nitrosomonas europaea: Kinetic characterization of glyceraldehyde-3 phosphate and succinate semialdehyde dehydrogenases.

Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD+/NADH as cofactor. CbbG showed ∼6-fold higher Km value for 1,3bisPGA but ∼5-fold higher kcat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD+ or NADP+, thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed ∼6-fold higher Km value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP+. Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.