SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation

B-cell activating factor (BAFF) is a key regulator of B-lymphocyte development. Its biological role is mediated by the specific receptors BCMA, TACI and BAFF-R. We have determined the crystal structure of the extracellular domain of BAFF-R bound to BAFF at a resolution of 3.3 A. The cysteine-rich domain (CRD) of the BAFF-R extracellular domain adopts a beta-hairpin structure and binds to the virus-like BAFF cage in a 1:1 molar ratio. The conserved DxL motif of BAFF-R is located on the tip of the beta-turn and is indispensable in the binding of BAFF. The crystal structure shows that a unique dimeric contact occurs between the BAFF-R monomers in the virus-like cage complex. The extracellular domain of TACI contains two CRDs, both of which contain the DxL motif. Modeling of TACI-BAFF complex suggests that both CDRs simultaneously interact with the BAFF dimer in the virus-like cage.     

Crystal structure of the MAPK phosphatase Pyst1 catalytic domain and implications for regulated activation

The crystal structure of the catalytic domain from the MAPK phosphatase Pyst1 (Pyst1-CD) has been determined at 2.35 A. The structure adopts a protein tyrosine phosphatase (PTPase) fold with a shallow active site that displays a distorted geometry in the absence of its substrate with some similarity to the dual-specificity phosphatase cdc25. Functional characterization of Pyst1-CD indicates it is sufficient to dephosphorylate activated ERK2 in vitro. Kinetic analysis of Pyst1 and Pyst1-CD using the substrate p-nitrophenyl phosphate (pNPP) reveals that both molecules undergo catalytic activation in the presence of recombinant inactive ERK2, switching from a low- to high-activity form. Mutation of Asp 262, located 5.5 A distal to the active site, demonstrates it is essential for catalysis in the high-activity ERK2-dependent conformation of Pyst1 but not for the low-activity ERK2-independent form, suggesting that ERK2 induces closure of the Asp 262 loop over the active site, thereby enhancing Pyst1 catalytic efficiency.     

Crystal structure of human micro-crystallin complexed with NADPH

Human cytosolic 3,5,3'-triiodo-L-thyronine-binding protein, also called mu-crystallin or CRYM, plays important physiological roles in transporting 3,5,3'-triiodo-L-thyronine (T(3)) into nuclei and regulating thyroid-hormone-related gene expression. The crystal structure of human CRYM's bacterial homolog Pseudomonas putida ornithine cyclodeaminase and Archaeoglobus fulgidus alanine dehydrogenase have been available, but no CRYM structure has been reported. Here, we report the crystal structure of human CRYM bound with NADPH refined to 2.6 A, and there is one dimer in the asymmetric unit. The structure contains two domains: a Rossmann fold-like NADPH-binding domain and a dimerization domain. Different conformations of the loop Arg83-His92 have been observed in two monomers of human CRYM in the same asymmetric unit. The peptide bond of Val89-Pro90 is a trans-configuration in one monomer but a cis-configuration in the other. A detailed comparison of the human mu-crystallin structure with its structurally characterized homologs including the overall comparison and superposition of active sites was conducted. Finally, a putative T(3)-binding site in human CRYM is proposed based on comparison with structural homologs.     

Crystal structure and fluorescence studies reveal the role of helical dimeric interface of staphylococcal FabG1 in positive cooperativity for NADPH

Crystal structure of Staphylococcal β‐ketoacyl‐ACP reductase 1 (SaFabG1) complexed with NADPH is determined at 2.5 Å resolution. The enzyme is essential in FAS‐II pathway and utilizes NADPH to reduce β‐ketoacyl‐ACP to (S)‐β‐hydroxyacyl‐ACP. Unlike the tetrameric FabGs, dimeric SaFabG1 shows positive homotropic cooperativity towards NADPH. Analysis of FabG:NADPH binary crystal structure endorses that NADPH interacts directly with the helices α4 and α5 those are present on a dimerization interface. A steady shift in tryptophan (of α4 helix) emission peak upon steady increment of NADPH concentration reveals that the dimeric interface is formed by α4‐α4′ and α5‐α5′ helices. This dimeric interface imparts positive homotropic cooperativity towards NADPH. PEG, a substrate mimicking molecule is also found near the active site of the enzyme. Proteins 2012; © 2011 Wiley Periodicals, Inc.     

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue

Summary Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - GR Glutathione Reductase - ME Malic Enzyme - tBHP t-Butyl Hydroperoxide - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide     

Molecular cloning and characterization of rice 6-phosphogluconate dehydrogenase gene that is up-regulated by salt stressa

The pentose phosphate pathway (PPP) is the important metabolism pathway in plant. In the present study, a cDNA encoding one of the key enzymes of PPP, 6-phosphogluconate dehydrogenase(6PGDH), was isolated from rice and designated as Os6PGDH. The Os6PGDH encoding protein is a cytosolic isoenzyme according to the absence of plastid transit peptide at the N-terminus. The full-length cDNA of 1751 bp encodes 480 amino acids and its putative protein sequence is 94%, 84% and 83% identical to maize, spinach and alfalfa 6PGDHs respectively. Comparison of the cloned mRNA sequence with that of the genomic sequence from the Rice Genome Project showed a simple genomic organization devoid of introns in the translated region of the gene. RT-PCR experiments revealed that Os6PGDH expression was high in inflorescence, low in root and embryos but almost absent in leaves. Furthermore, Os6PGDH was up-regulated in the shoots under salt stress. It is suggested that 6PGDH in plant may play an important role in cell division and salt response.     

Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications

We report here the crystal structure of a trehalose-6-phosphate phosphatase-related protein (T6PP) from Thermoplasma acidophilum, TA1209, determined by the dual-wavelength anomalous diffraction (DAD) method. T6PP is a member of the haloacid dehalogenase (HAD) superfamily with significant sequence homology with trehalose-6-phosphate phosphatase, phosphoserine phosphatase, P-type ATPases and other members of the family. T6PP possesses a core domain of known alpha/beta-hydrolase fold, characteristic of the HAD family, and a cap domain, with a tertiary fold consisting of a four-stranded beta-sheet with two alpha-helices on one side of the sheet. An active-site magnesium ion and a glycerol molecule bound at the interface between the two domains provide insight into the mode of substrate binding by T6PP. A trehalose-6-phosphate molecule modeled into a cage formed by the two domains makes favorable interactions with the protein molecule. We have confirmed that T6PP is a trehalose phosphatase from amino acid sequence, three-dimensional structure, and biochemical assays.     

The control of the urokinase-catalyzed activation of human glutamic acid 1-plasminogen by positive and negative effectors

The urokinase-catalyzed activation of human Glu1-plasminogen (Glu1Pg) has been found to be inhibited by monovalent anions in the following order of effectiveness: I- greater than SCN- greater than Cl- greater than IO3- greater than HCOO- greater than F- greater than OAc-. The inhibition is reversed by epsilon-aminocaproic acid, with its effectiveness in this capacity generally inversely proportional to the strength of the binding of the anion. The physical basis for the anion inhibition and epsilon-aminocaproic acid stimulation lies in the ability of these effectors to cause measurable opposite alterations in the conformation of Glu1Pg, which are revealed through study of the sedimentation velocity of the protein under various conditions. The kinetic mechanism of the chloride inhibition of Glu1Pg activation has been examined in detail. It has been found that the Glu1Pg.Cl complex serves as an alternate substrate to Glu1Pg for urokinase, with a greatly increased Km (25 +/- 3 and 2.2 +/- 0.3 microM, respectively) for activation. The kcat for the urokinase.Glu1Pg.Cl complex is approximately the same as that for urokinase.Glu1Pg (1.6 +/- 0.2 - 2.0 +/- 0.2/s). Similarly, the stimulation by epsilon-aminocaproic acid also results from effects on the Km of the activation, which is reduced to 1.8 +/- 0.2 microM for the Glu1Pg.Cl.epsilon-aminocaproic acid complex. The kcat for the urokinase.Glu1Pg.Cl.epsilon-aminocaproic acid of 2.4 +/- 0.3/s complex is not greatly different from that for urokinase.Glu1Pg.Cl. Nuclear magnetic resonance studies of the Glu1Pg-induced line broadening of the 35Cl- spectra in the presence and absence of epsilon-aminocaproic acid suggest that Cl- and epsilon-aminocaproic acid simultaneously bind to the protein and that each of these effectors displays its effects through separate binding sites.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

Mitochondrial glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase abrogate p53 induced apoptosis in a yeast model: Possible implications for apoptosis resistance in cancer cells

BackgroundEscape from apoptosis is an important hallmark of tumor progression and drug resistance in cancer cells. It is well demonstrated that over-expression of human wtp53 in Saccharomyces cerevisiae induces apoptosis by directly targeting the mitochondria. In this study, we showed that how S.cerevisiae escaped from p53 induced apoptosis in the presence of a fermentable carbon source (sucrose), but not on non-fermentable carbon source (glycerol).MethodsMitochondrial fractions from yeast cultures grown in the presence of sucrose or glycerol with and without p53 expression were fractionated and analyzed by LC-MS/MS. Differentially expressed proteins were studied and detailed biochemical analysis for selected proteins was performed.The effect of mitochondrial HXK-2 over-expression induced by p53 in sucrose grown cells on cell survival was evaluated using gene deletion/tagging, co-localisation and mitochondrial ROS detection.ResultsWe observe that mitochondria isolated from p53 over-expressing cells accumulate Pentose phosphate Pathway (PPP) enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) which led to enhanced mitochondrial NADPH production only when cells are cultured in sucrose but not glycerol. In contrast, mitochondria isolated from Δhxk2 p53 over-expressing cells grown in sucrose did not accumulate G6PDH and 6PGDH and resulted in defective growth.ConclusionsEnhanced association of HXK2 with the mitochondria with the concomitant accumulation of G6PDG and 6PGDH results in increased NADPH that scavenges ROS and provides resistance to apoptosis.General significanceGiven the extensive similarity of aerobic glycolysis between humans and yeast, the phenomena described here could as well be responsible for the escape of apoptosis in cancer cells.     

Reactive oxygen species production and activation mechanism of the rice NADPH oxidase OsRbohB

Reactive oxygen species (ROS) produced by plant NADPH oxidases (NOXes) are important in plant innate immunity. The Oryza sativa respiratory burst oxidase homologue B (OsRbohB) gene encodes a NOX the regulatory mechanisms of which are largely unknown. Here, we used a heterologous expression system to demonstrate that OsRbohB shows ROS-producing activity. Treatment with ionomycin, a Ca(2+) ionophore, and calyculin A, a protein phosphatase inhibitor, activated ROS-producing activity; it was thus OsRbohB activated by both Ca(2+) and protein phosphorylation. Mutation analyses revealed that not only the first EF-hand motif but also the upstream amino-terminal region were necessary for Ca(2+)-dependent activation, while these regions are not required for phosphorylation-induced ROS production.     

Crystal structure of CD14 and its implications for lipopolysaccharide signaling

Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.