Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Nitrate reductase and glutamine synthetase activity in coffee leaves during fruit development

Nitrate reductase is the first enzyme in the pathway of nitrate reduction by plants, followed by glutamine synthetase, which incorporates ammonia to glutamine. The purpose of this study was to evaluate the nitrate reductase and glutamine synthetase activity, total soluble protein content, N and Ni content in coffee leaves during fruit development under field conditions to establish new informations to help assess the N nutritional status and fertilizer management. The experimental design was in randomized complete blocks, arranged in a 3 x 6 factorial design, with five replications. The treatments consisted of 3 N rates (0 - control, 150 and 300 kg ha-1) and six evaluation periods (January, February, March, April, May, and June) in six-year-old coffee (Coffea arabica L.) plants of Catuaí Vermelho IAC 44 cv. The nitrate reductase and glutamine synthetase activities, leaf soluble protein, and N concentrations increased linearly with the N rates. During fruit development, the enzyme activity, leaf soluble protein and N content decreased, due to the leaf senescence process caused by nutrient mobilization to other organs, e.g, to the berries. Leaf Ni increased during fruit development. Beans and raisin-fruits of plants well-supplied with N had higher Ni contents. Enzyme activities, total leaf N and leaf soluble protein, evaluated during the green fruit stage in March, were significantly correlated with coffee yield. These variables can therefore be useful for an early assessment of the coffee N nutritional status as well as coffee yield and N fertilization management.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize

In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient) and Catetão (inefficient) were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction) and nitrate reductase did not differ among the varieties.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.

Thioredoxin-interacting protein links oxidative stress to inflammasome activation.

The NLRP3 inflammasome has a major role in regulating innate immunity. Deregulated inflammasome activity is associated with several inflammatory diseases, yet little is known about the signaling pathways that lead to its activation. Here we show that NLRP3 interacted with thioredoxin (TRX)-interacting protein (TXNIP), a protein linked to insulin resistance. Inflammasome activators such as uric acid crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner and allowed it to bind NLRP3. TXNIP deficiency impaired activation of the NLRP3 inflammasome and subsequent secretion of interleukin 1beta (IL-1beta). Akin to Txnip(-/-) mice, Nlrp3(-/-) mice showed improved glucose tolerance and insulin sensitivity. The participation of TXNIP in the NLRP3 inflammasome activation may provide a mechanistic link to the observed involvement of IL-1beta in the pathogenesis of type 2 diabetes.

Modulation of plant HMG-CoA reductase by protein phosphatase 2A: Positive and negative control at a key node of metabolism

The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B" regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR.

Design, synthesis, and biological and crystallographic evaluation of novel inhibitors of Plasmodium falciparum enoyl-ACP-reductase (PfFabI).

Malaria, a disease of worldwide significance, is responsible for over one million deaths annually. The liver-stage of Plasmodium's life cycle is the first, obligatory, but clinically silent step in malaria infection. The P. falciparum type II fatty acid biosynthesis pathway (PfFAS-II) has been found to be essential for complete liver-stage development and has been regarded as a potential antimalarial target for the development of drugs for malaria prophylaxis and liver-stage eradication. In this paper, new coumarin-based triclosan analogues are reported and their biological profile is explored in terms of inhibitory potency against enzymes of the PfFAS-II pathway. Among the tested compounds, 7 and 8 showed the highest inhibitory potency against Pf enoyl-ACP-reductase (PfFabI), followed by 15 and 3. Finally, we determined the crystal structures of compounds 7 and 11 in complex with PfFabI to identify their mode of binding and to confirm outcomes of docking simulations.

HMG CoA reductase inhibitors (statins) for preventing acute kidney injury after surgical procedures requiring cardiac bypass.

BACKGROUND: Acute kidney injury (AKI) is common in patients undergoing cardiac surgery among whom it is associated with poor outcomes, prolonged hospital stays and increased mortality. Statin drugs can produce more than one effect independent of their lipid lowering effect, and may improve kidney injury through inhibition of postoperative inflammatory responses. OBJECTIVES: This review aimed to look at the evidence supporting the benefits of perioperative statins for AKI prevention in hospitalised adults after surgery who require cardiac bypass. The main objectives were to 1) determine whether use of statins was associated with preventing AKI development; 2) determine whether use of statins was associated with reductions in in-hospital mortality; 3) determine whether use of statins was associated with reduced need for RRT; and 4) determine any adverse effects associated with the use of statins. SEARCH METHODS: We searched the Cochrane Renal Group's Specialised Register to 13 January 2015 through contact with the Trials' Search Co-ordinator using search terms relevant to this review. SELECTION CRITERIA: Randomised controlled trials (RCTs) that compared administration of statin therapy with placebo or standard clinical care in adult patients undergoing surgery requiring cardiopulmonary bypass and reporting AKI, serum creatinine (SCr) or need for renal replacement therapy (RRT) as an outcome were eligible for inclusion. All forms and dosages of statins in conjunction with any duration of pre-operative therapy were considered for inclusion in this review. DATA COLLECTION AND ANALYSIS: All authors extracted data independently and assessments were cross-checked by a second author. Likewise, assessment of study risk of bias was initially conducted by one author and then by a second author to ensure accuracy. Disagreements were arbitrated among authors until consensus was reached. Authors from two of the included studies provided additional data surrounding post-operative SCr as well as need for RRT. Meta-analyses were used to assess the outcomes of AKI, SCr and mortality rate. Data for the outcomes of RRT and adverse effects were not pooled. Adverse effects taken into account were those reported by the authors of included studies. MAIN RESULTS: We included seven studies (662 participants) in this review. All except one study was assessed as being at high risk of bias. Three studies assessed atorvastatin, three assessed simvastatin and one investigated rosuvastatin. All studies collected data during the immediate perioperative period only; data collection to hospital discharge and postoperative biochemical data collection ranged from 24 hours to 7 days. Overall, pre-operative statin treatment was not associated with a reduction in postoperative AKI, need for RRT, or mortality. Only two studies (195 participants) reported postoperative SCr level. In those studies, patients allocated to receive statins had lower postoperative SCr concentrations compared with those allocated to no drug treatment/placebo (MD 21.2 µmol/L, 95% CI -31.1 to -11.1). Adverse effects were adequately reported in only one study; no difference was found between the statin group compared to placebo. AUTHORS' CONCLUSIONS: Analysis of currently available data did not suggest that preoperative statin use is associated with decreased incidence of AKI in adults after surgery who required cardiac bypass. Although a significant reduction in SCr was seen postoperatively in people treated with statins, this result was driven by results from a single study, where SCr was considered as a secondary outcome. The results of the meta-analysis should be interpreted with caution; few studies were included in subgroup analyses, and significant differences in methodology exist among the included studies. Large high quality RCTs are required to establish the safety and efficacy of statins to prevent AKI after cardiac surgery.

Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass

Background: Wine Saccharomyces cerevisiae strains, adapted to anaerobic must fermentations, suffer oxidative stress when they are grown under aerobic conditions for biomass propagation in the industrial process of active dry yeast production. Oxidative metabolism of sugars favors high biomass yields but also causes increased oxidation damage of cell components. The overexpression of the TRX2 gene, coding for a thioredoxin, enhances oxidative stress resistance in a wine yeast strain model. The thioredoxin and also the glutathione/glutaredoxin system constitute the most important defense against oxidation. Trx2p is also involved in the regulation of Yap1p-driven transcriptional response against some reactive oxygen species. Results: Laboratory scale simulations of the industrial active dry biomass production process demonstrate that TRX2 overexpression increases the wine yeast final biomass yield and also its fermentative capacity both after the batch and fed-batch phases. Microvinifications carried out with the modified strain show a fast start phenotype derived from its enhanced fermentative capacity and also increased content of beneficial aroma compounds. The modified strain displays an increased transcriptional response of Yap1p regulated genes and other oxidative stress related genes. Activities of antioxidant enzymes like Sod1p, Sod2p and catalase are also enhanced. Consequently, diminished oxidation of lipids and proteins is observed in the modified strain, which can explain the improved performance of the thioredoxin overexpressing strain. Conclusions: We report several beneficial effects of overexpressing the thioredoxin gene TRX2 in a wine yeast strain. We show that this strain presents an enhanced redox defense. Increased yield of biomass production process in TRX2 overexpressing strain can be of special interest for several industrial applications.

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of a factor (Doa10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: a case-control study in Spain.

Background: Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) gene mutation have been postulated as a possible cause of recurrent miscarriage (RM). There is a wide variation in the prevalence of MTHFR polymorphisms and homocysteine (Hcy) plasma levels among populations around the world. The present study was undertaken to investigate the possible association between hyperhomocysteinemia and its causative genetic or acquired factors and RM in Catalonia, a Mediterranean region in Spain. Methods: Sixty consecutive patients with ≥ 3 unexplained RM and 30 healthy control women having at least one child but no previous miscarriage were included. Plasma Hcy levels, MTHFR gene mutation, red blood cell (RBC) folate and vitamin B12 serum levels were measured in all subjects. Results: No significant differences were observed neither in plasma Hcy levels, RBC folate and vitamin B12 serum levels nor in the prevalence of homozygous and heterozygous MTHFR gene mutation between the two groups studied. Conclusions: In the present study RM is not associated with hyperhomocysteinemia, and/or the MTHFR gene mutation.

Multilevel control of arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A

Plants synthesize a myriad of isoprenoid products that are required both for essential constitutive processes and for adaptive responses to the environment. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes a key regulatory step of the mevalonate pathway for isoprenoid biosynthesis and is modulated by many endogenous and external stimuli. In spite of that, no protein factor interacting with and regulating plant HMGR in vivo has been described so far. Here, we report the identification of two B99 regulatory subunits of protein phosphatase 2A (PP2A), designated B99a and B99b, that interact with HMGR1S and HMGR1L, the major isoforms of Arabidopsis thaliana HMGR. B99a and B99b are Ca2+ binding proteins of the EF-hand type. We show that HMGR transcript, protein, and activity levels are modulated by PP2A in Arabidopsis. When seedlings are transferred to salt-containing medium, B99a and PP2A mediate the decrease and subsequent increase of HMGR activity, which results from a steady rise of HMGR1-encoding transcript levels and an initial sharper reduction of HMGR protein level. In unchallenged plants, PP2A is a posttranslational negative regulator of HMGR activity with the participation of B99b. Our data indicate that PP2A exerts multilevel control on HMGR through the fivemember B99 protein family during normal development and in response to a variety of stress conditions.

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.