Crystal structure of the human dual specificity phosphatase 1 catalytic domain

The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38‐alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.     

Influence of Feeding Inorganic Vanadium on Growth Performance,Endocrine Variables and Biomarkers of Bone Health in Crossbred Calves

The nutritional essentialities of transition element vanadium (V) as micro-nutrient in farm animals have not yet been established, though in rat model, vanadium as vanadate has been reported to exert insulin-mimetic effect and shown to be needed for proper development of bones. The objective of this study was to determine the effect of V supplementation on growth performance, plasma hormones and bone health status in calves. Twenty-four crossbred calves (body weight 72.83 ± 2.5 kg; age 3–9 months) were blocked in four groups and randomly assigned to four treatment groups (n = 6) on body weight and age basis. Experimental animals were kept on similar feeding regimen except that different groups were supplemented with either 0, 3, 6 or 9 ppm inorganic V/kg DM. Effect of supplementation during 150-day experimental period was observed on feed intake, body weight gain, feed efficiency, body measures, endocrine variables, plasma glucose and biomarkers of bone health status. Supplementation of V did not change average daily gain (ADG), dry matter intake (DMI), feed efficiency and body measures during the experimental period. During the post-V supplementation period plasma insulin-like growth factor-1 (IGF-1), triiodothyronine (T₃) and thyroxin (T₄) concentrations were increased and observed highest in 9 mg V/kg DM fed calves; however, levels of insulin, glucose, parathyroid hormone (PTH) and calcitonin hormones remained similar among calves fed on basal or V-supplemented diets. Bone alkaline phosphatase (Bone-ALP) concentration was increased (P < 0.05); however, plasma protein tyrosine phosphatase (PTP) level decreased (P < 0.05) in 6 and 9 mg V/kg DM supplemented groups. Plasma hydroxyproline (Hyp) and tartrate-resistant acid phosphatase (TRAP) concentration were unchanged by V supplementation. Blood V concentration showed positive correlation with supplemental V levels. These results suggest that V may play a role in modulation of the action of certain endocrine variables and biomarkers of bone health status in growing crossbred calves.     

Expansion and diversification of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) family in land plants

Key message

Here we uncover the major evolutionary events shaping the evolution of the GID1 family of gibberellin receptors in land plants at the sequence, structure and gene expression levels.

Abstract

Gibberellic acid (gibberellin, GA) controls key developmental processes in the life cycle of land plants. By interacting with the GIBBERELLIN INSENSITIVE DWARF1 (GID1) receptor, GA regulates the expression of a wide range of genes through different pathways. Here we report the systematic identification and classification of GID1s in 54 plants genomes, encompassing from bryophytes and lycophytes, to several monocots and eudicots. We investigated the evolutionary relationship of GID1s using a comparative genomics framework and found strong support for a previously proposed phylogenetic classification of this family in land plants. We identified lineage-specific expansions of particular subfamilies (i.e. GID1ac and GID1b) in different eudicot lineages (e.g. GID1b in legumes). Further, we found both, shared and divergent structural features between GID1ac and GID1b subgroups in eudicots that provide mechanistic insights on their functions. Gene expression data from several species show that at least one GID1 gene is expressed in every sampled tissue, with a strong bias of GID1b expression towards underground tissues and dry legume seeds (which typically have low GA levels). Taken together, our results indicate that GID1ac retained canonical GA signaling roles, whereas GID1b specialized in conditions of low GA concentrations. We propose that this functional specialization occurred initially at the gene expression level and was later fine-tuned by mutations that conferred greater GA affinity to GID1b, including a Phe residue in the GA-binding pocket. Finally, we discuss the importance of our findings to understand the diversification of GA perception mechanisms in land plants.

     

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN(-) in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN(-). Further kinetic studies indicate that SCN(-) itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN(-) blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H(2)O(2) to O(2). Binding studies by optical difference spectroscopy indicate that SCN(-) binds LmAPX (Kd = 100 +/- 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN(-) acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T(m)) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T(m) to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T(m) value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.