Novel mechanisms for PKC family enzymes to regulate PI3K signaling pathway

Phosphatidylinositol 3-kinase (PI3K) generates membrane phospholipids that serve as second messengers to recruit signaling proteins to plasma membrane consequently regulating cell growth and survival. PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory p85 subunit. Association of the p85 with other signal proteins is critical for induced PI3K activation. Activated PI3K, in turn, leads to signal flows through a variety of PI3K effectors including PDK1, AKT, GSK3, BAD, p70 S6K and NFκB. The PI3K pathway is under regulation by multiple signal proteins representing cross-talk between different signaling cascades. In this study, we have evaluated the role of protein kinase C family kinases on signaling through PI3K at multiple levels. Firstly, we observed that the action of PKC specific inhibitors like Ro-31-8220 and GF109203X was associated with an increased AKT phosphorylation and activity, suggesting that PKC kinases might play a negative role in the regulation of PI3K pathway. Then, we demonstrated the stimulation of AKT by PKC inhibition was dependent on functional PI3K enzyme and able to be transmitted to the AKT effector p70 S6K. Furthermore, we showed an inducible physical association between the PKCζ isotype and AKT, which was accompanied by an attenuated AKT activity. However, a kinase-dead form of PKC failed to affect AKT. In the second part of our research we revealed the ability of a different PKC family member, PKCδ to bind to the p85 subunit of PI3K in response to oxidative stress, a process requiring the activity of src tyrosine kinases. The interaction was demonstrated to be a direct and specific contact between the carboxyl terminal SH2 domain of p85 and tyrosine phosphorylated PKCδ. Several different types of agonists were capable to induce this association including tyrosine kinases and phorbol esters with PKCδ tyrosine phosphorylation being integral components. Finally, the PKCδ-PI3K complex was related to a reduction in the AKT phosphorylation induced by src. A kinase-deficient mutant of PKCδ was equally able to inhibit AKT signal as the wild type, indicative of a process independent of PKCδ catalytic activity. Altogether, our data illustrate different PKC isoforms regulating PI3K pathway at multiple levels, suggesting a mechanism to control signal flows through PI3K for normal cell activities. Although further investigation is required for full understanding of the regulatory mechanism, we propose that complex formation of signal proteins in PI3K pathway and specific PKC isoforms plays important role in their functional linkage. ^

Activity of ETS enzymes and phosphatase in the Sea of Okhotsk and Kuril region in summer 1992 and 1993

An estimate of rate of transformation of organic matter and regeneration of nutrients (in particular phosphorus) was calculated for different regions of the Sea of Okhotsk. The rate was estimated by means of rate of complete oxidation of organic matter to CO2 and H2O catalyzed by enzymes of the electron transport system (ETS) and rate of hydrolytic splitting of phosphate from organic phosphorus compounds catalyzed by alkaline phosphatase. Organic matter destruction rate was at its maximum on the shelf of Kamchatka and Sakhalin, as well as in the layer of maximum oxygen gradients in deep waters. It was found that zones of intensive primary production were characterized by high rates of phosphorus regeneration, which provided for 80% of primary production when concentration of mineral phosphorus was low.

Enzyme activities of digestive and metabolic enzymes of Calanus finmarchicus from Maria S. Merian cruise MSM26 in spring 2013

The present study aimed to contribute to the knowledge on the intraspecific variations of enzyme activities in populations of Calanus finmarchicus from different longitudes across the North Atlantic Ocean and their relation to changing environmental conditions. C. finmarchicus was sampled across the North Atlantic in basins with decreasing temperature regimes from east to west (Iceland Basin, Irminger Basin and Labrador Basin) in late March/early April 2013. Potential maximum enzyme activities of digestive (proteinases and lipases/esterases) and metabolic (citrate synthase) enzymes of copepods from all sampling stations were analysed and thermal profiles (5-50°C) of enzyme activities were determined. In order to investigate its acclimation potential, C. finmarchicus were acclimated to 4°C and 15°C for two weeks and thermal profiles of enzyme activities were compared afterwards.

In vitro evaluation of commercial fibrolytic enzymes for improving the nutritive value of low-quality forages.

The aim of this work was to assess the effects of four doses of three commercial fibrolytic enzymes on ruminal fermentation of rice straw, maize stover and Pennisetum purpureum clon Cuba CT115 hay in batch cultures of ruminal micro-organisms from sheep. One enzyme was produced by Penicillium funiculosum (PEN) and two were from Trichoderma longibrachiatum (TL1 and TL2). Each liquid enzyme was diluted 200 (D1), 100 (D2), 50 (D3) and 10 (D4) - fold and applied to each substrate in quadruplicate over time and incubated for 120 h in rumen fluid. The D4 dose of each enzyme increased (P<0.05) the fractional rate of gas production and organic matter effective degradability for all substrates, and TL2 had similar effects when applied at D3. In 9 h incubations, PEN at D4, TL1 at all tested doses, and TL2 at D2, D3 and D4 increased (P<0.05) volatile fatty acid production and dry matter degradability for all substrates. The commercial enzymes tested were effective at increasing in vitro ruminal fermentation of low-quality forages, although effective doses varied with the enzyme.

Treatment of tropical forages with exogenous fibrolytiic enzymes: effects on chemical composition and in vitro rumen fermentation

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism that perform a variety of essential functions in higher plants. Studies over the past 30 years have supported a model in which flavonoid metabolism is catalyzed by an enzyme complex localized to the endoplasmic reticulum [Hrazdina, G. & Wagner, G. J. (1985) Arch. Biochem. Biophys. 237, 88–100]. To test this model further we assayed for direct interactions between several key flavonoid biosynthetic enzymes in developing Arabidopsis seedlings. Two-hybrid assays indicated that chalcone synthase, chalcone isomerase (CHI), and dihydroflavonol 4-reductase interact in an orientation-dependent manner. Affinity chromatography and immunoprecipitation assays further demonstrated interactions between chalcone synthase, CHI, and flavonol 3-hydroxylase in lysates from Arabidopsis seedlings. These results support the hypothesis that the flavonoid enzymes assemble as a macromolecular complex with contacts between multiple proteins. Evidence was also found for posttranslational modification of CHI. The importance of understanding the subcellular organization of elaborate enzyme systems is discussed in the context of metabolic engineering.