Critical role for polar residues in coupling leukotriene B4 binding to signal transduction in BLT1

Leukotriene B(4) (LTB(4)) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB(4) binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB(4) binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.     

Probing of contacts between <Emphasis Type="Italic">Eco</Emphasis>RII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.     

An <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain Producing a Leaderless mRNA from the Chromosomal <Emphasis Type="Italic">lac</Emphasis> Promoter

A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of the leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens the possibilities of investigation of the specific conditions for the leaderless mRNA translation in vivo using the molecular genetic approaches.     

Physical mapping of the blue-grained gene from <Emphasis Type="Italic">Thinopyrum ponticum</Emphasis> chromosome 4Ag and development of blue-grain-related molecular markers and a FISH probe based on SLAF-seq technology

Key message

A Thinopyrum ponticum chromosome 4Ag physical map was constructed, the blue-grained gene was localized, and related specific markers and a FISH probe were developed by SLAF-seq.

Abstract

Decaploid Thinopyrum ponticum (2n?=?10x?=?70) serves as an important gene pool for wheat improvement. The wheat-Th. ponticum 4Ag (4D) disomic substitution line Blue 58, derived from a distant hybridization between Th. ponticum and common wheat (Triticum aestivum L.), bears blue coloration in the aleurone layer. To map the blue-grained gene, eight wheat-Th. ponticum 4Ag translocation lines with different chromosomal segment sizes were obtained from Blue 58 using ⁶⁰Co-γ ray irradiation and were characterized using cytogenetic and molecular marker analysis. A small-segment blue-grained wheat translocation line L13, accounting for one-fifth of 4AgL, was obtained. A physical map of chromosome 4Ag was constructed containing 573 specific-locus amplified fragment sequencing (SLAF-seq) markers, including three bins with 223 markers on 4AgS and eight bins with 350 markers on 4AgL. The blue-grained gene in three blue-grained translocation lines L5, L9, and L13, was located on bin 4AgL-6 with FL 0.75–0.89. Moreover, 89 blue-grain-related molecular markers and one fluorescence in situ hybridization (FISH) probe, pThp12.19, were identified in this bin. The newly developed translocation lines and the molecular markers and FISH probe will facilitate the application of the Th. ponticum-origin blue-grained characteristic in wheat breeding.

     

Molecular docking of <Emphasis Type="Italic">Glycine max</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis> ureases with urea; bioinformatics approaches

Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme catalyzing the hydrolysis of urea into ammonia and carbon dioxide. It is present in many bacteria, fungi, yeasts and plants. Most species, with few exceptions, use nickel metalloenzyme urease to hydrolyze urea, which is one of the commonly used nitrogen fertilizer in plant growth thus its enzymatic hydrolysis possesses vital importance in agricultural practices. Considering the essentiality and importance of urea and urease activity in most plants, this study aimed to comparatively investigate the ureases of two important legume species such as Glycine max (soybean) and Medicago truncatula (barrel medic) from Fabaceae family. With additional plant species, primary and secondary structures of 37 plant ureases were comparatively analyzed using various bioinformatics tools. A structure based phylogeny was constructed using predicted 3D models of G. max and M. truncatula, whose crystallographic structures are not available, along with three additional solved urease structures from Canavalia ensiformis (PDB: 4GY7), Bacillus pasteurii (PDB: 4UBP) and Klebsiella aerogenes (PDB: 1FWJ). In addition, urease structures of these species were docked with urea to analyze the binding affinities, interacting amino acids and atom distances in urease-urea complexes. Furthermore, mutable amino acids which could potentially affect the protein active site, stability and flexibility as well as overall protein stability were analyzed in urease structures of G. max and M. truncatula. Plant ureases demonstrated similar physico-chemical properties with 833–878 amino acid residues and 89.39–90.91 kDa molecular weight with mainly acidic (5.15–6.10 pI) nature. Four protein domain structures such as urease gamma, urease beta, urease alpha and amidohydro 1 characterized the plant ureases. Secondary structure of plant ureases also demonstrated conserved protein architecture, with predominantly α-helix and random coil structures. In structure-based phylogeny, plant ureases from G. max, M. truncatula and C. ensiformis were clearly diverged from bacterial ureases of B. pasteurii and K. aerogenes. Glu, Thr, His and Gly were commonly found as interacting residues in most urease-urea docking complexes while Glu was available in all docked structures. Besides, Ala and Arg residues, which are reported in active-site architecture of plant and bacterial ureases were present in G. max urea-urease complex but not present in others. Moreover, Arg435 and Arg437 in M. truncatula and G. max, respectively were identified as highly mutable hotspot residues residing in amidohydro 1 domain of enzyme. In addition, a number of stabilizing residues were predicted upon mutation of these hotspot residues however Cys and Thr made strong implications since they were also found in codon-aligned sequences as substitutions of hotspot residues. Comparative analyses of primary sequence and secondary structure in 37 different plants demonstrated quite conserved natures of ureases in plant kingdom. Structure-based phylogeny indicated the presence of a possible prokaryote-eukaryote split and implicated the subjection of bacterial ureases to heavy selection in prokaryotic evolution compared to plants. Urea-urease docking complexes suggested that different species could share common interacting residues as well as may have some other uncommon residues at species-dependent way. In silico mutation analyses identified mutable amino acids, which were predicted to reside in catalytic site of enzyme therefore mutagenesis at these sites seemed to have adverse effects on enzyme efficiency or function. This study findings will become valuable preliminary resource for future studies to further understand the primary, secondary and tertiary structures of urease sequences in plants as well as it will provide insights about various binding features of urea-urease complexes.     

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X, X ^m and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B₄ receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G_i2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α₅ integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB₄ binding in the presence of the purified G protein Gα_i2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gα_i2β₁γ₂ protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.     

Insights into a key sulfite scavenger enzyme sulfite oxidase (<Emphasis Type="Italic">SOX</Emphasis>) gene in plants

Sulfite oxidase (SOX) is a crucial molybdenum cofactor-containing enzyme in plants that re-oxidizes the sulfite back to sulfate in sulfite assimilation pathway. However, studies of this crucial enzyme are quite limited hence this work was attempted to understand the SOXs in four plant species namely, Arabidopsis thaliana, Solanum lycopersicum, Populus trichocarpa and Brachypodium distachyon. Herein studied SOX enzyme was characterized with both oxidoreductase molybdopterin binding and Mo-co oxidoreductase dimerization domains. The alignment and motif analyses revealed the highly conserved primary structure of SOXs. The phylogeny constructed with additional species demonstrated a clear divergence of monocots, dicots and lower plants. In addition, to further understand the phylogenetic relationship and make a functional inference, a structure-based phylogeny was constructed using normalized RMSD values in five superposed models from four modelled plant SOXs herein and one previously characterized chicken SOX structure. The plant and animal SOXs showed a clear divergence and also implicated their functional divergences. Based on tree topology, monocot B. distachyon appeared to be diverged from other dicots, pointing out a possible monocot–dicot split. The expression patterns of sulfite scavengers including SOX were differentially modulated under cold, heat, salt and high light stresses. Particularly, they tend to be up-regulated under high light and heat while being down-regulated under cold and salt stresses. The presence of cis-regulatory motifs associated with different stresses in upstream regions of SOX genes was thus justified. The protein–protein interaction network of AtSOX and network enrichment with gene ontology (GO) terms showed that most predicted proteins, including sulfite reductase, ATP sulfurylases and APS reductases were among prime enzymes involved in sulfite pathway. Finally, SOX–sulfite docked structures indicated that arginine residues particularly Arg374 is crucial for SOX–sulfite binding and additional two other residues such as Arg51 and Arg103 may be important for SOX–sulfite bindings in plants.     

Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.     

Contributions of <Emphasis Type="Italic">TaSUTs</Emphasis> to grain weight in wheat under drought

Key message

The homologous genes to OsSUT1-5 in wheat were identified and detailed analysed. TaSUT1 was the predominant sucrose transporter group and it illustrated the genotypic variations towards drought during grain filling.

Abstract

Sucrose transporters (SUT) play crucial roles in wheat stem water soluble carbohydrate (WSC) remobilization to grain. To determine the major functional SUT gene groups in shoot parts of wheat during grain development, drought tolerant varieties, Westonia and Kauz, were investigated in field drought experiments. Fourteen homologous genes to OsSUT1-5 were identified on five homeologous groups, namely TaSUT1_4A, TaSUT1_4B, TaSUT1_4D; TaSUT2_5A, TaSUT2_5B, TaSUT2_5D; TaSUT3_1A, TaSUT3_1D; TaSUT4_6A, TaSUT4_6B, TaSUT4_6D; TaSUT5_2A, TaSUT5_2B, and TaSUT5_2D, and their gene structures were analysed. Wheat plants above the ground were harvested from pre-anthesis to grain maturity and the stem, leaf sheath, rachis, lemma and developing grain were used for analysing TaSUT gene expression. Grain weight, thousand grain weight, kernel number per spike, biomass and stem WSC were characterized. The study showed that among the five TaSUT groups, TaSUT1 was the predominant sucrose transporting group in all organs sampled, and the expression was particularly high in the developing grain. In contrast to TaSUT1, the gene expression levels of TaSUT2, TaSUT3 and TaSUT4 were lower, except for TaSUT3 which showed preferential expression in the lemma before anthesis. The TaSUT5 gene group was very weakly expressed in all tissues. The upregulated gene expression of TaSUT1 Westonia type in stem and grain reveal a crucial role in stem WSC remobilization to grain under drought. The high TaSUT1 gene expression and the significant correlations with thousand grain weight (TGW) and kernel number per spike demonstrated the contribution in Kauz’s high grain yield in an irrigated environment and high TGW in Westonia under drought stress. Further molecular level identification is required for gene marker development.

     

Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase

Earlier, it has been shown that some amino acids and their derivatives are able to regulate activities of adenylyl cyclase (AC) and guanylyl cyclase (GC) in free-living infusoria Dileptus anser and Tetrahymena pyriformis. The goal of this work consisted in studying the molecular mechanisms of action of methionine, tyrosine, alanine, and neurohormone serotonin on the activity of enzyme-cyclases and in identification of their specific receptors in D. anser and T. pyriformis. Methionine and serotonin significantly increased the basal AC activity in both infusoria; the effect of serotonin on AC in T. pyriformis took place with participation of the Ca²⁺-dependent form of AC and of the heterotrimeric G-proteins. The AC-stimulating effect of tyrosine and alanine was expressed weakly and was revealed only in D. anser. Serotonin in both infusoria and alanine in D. anser stimulated GC activity, whereas methionine and tyrosine did not affect GC. Methionine and serotonin were bound with a high affinity to the surface receptors of infusoria. The K_D for [methyl-³H]methionine binding to D. anser and T. pyriformis were equal to 7.5 and 35.6 nM, and for [³H]serotonin binding, they were 2.7 and 4.7 nM, respectively. Alanine and tyrosine were bound to infusoria with low affinity. Thus, in the infusoria D. anser and T. pyriformis, there are chemosignal systems regulated by amino acids and their derivatives, including enzymes with cyclase activity. These systems are suggested to be similar to the hormonal signal systems of the higher eukaryotes and to be their predecessors.     

Mapping of novel powdery mildew resistance gene(s) from <Emphasis Type="Italic">Agropyron cristatum</Emphasis> chromosome 2P

Key message

A physical map of Agropyron cristatum 2P chromosome was constructed for the first time and the novel powdery mildew resistance gene(s) from chromosome 2P was(were) also mapped.

Abstract

Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a wild relative of common wheat, is highly resistant to powdery mildew. Previous studies showed that wheat-A. cristatum 2P disomic addition line II-9-3 displayed high resistance to powdery mildew, and the resistance was attributable to A. cristatum chromosome 2P. To utilize and physically map the powdery mildew resistance gene(s), 15 wheat-A. cristatum 2P translocation lines and three A. cristatum 2P deletion lines with different chromosomal segment sizes, obtained from II-9-3 using ⁶⁰Co-γ ray irradiation, were characterized using cytogenetic and molecular marker analysis. A. cristatum 2P chromosomal segments in the translocations were translocated to different wheat chromosomes, including 1A, 4A, 5A, 6A, 7A, 1B, 2B, 3B, 7B, 3D, 4D, and 6D. A physical map of the 2P chromosome was constructed with 82 STS markers, consisting of nine bins with 34 markers on 2PS and eight bins with 48 markers on 2PL. The BC₁F₂ populations of seven wheat-A. cristatum 2P translocation lines (2PT-3, 2PT-4, 2PT-5, 2PT-6, 2PT-8, 2PT-9, and 2PT-10) were developed by self-pollination, tested with powdery mildew and genotyped with 2P-specific STS markers. From these results, the gene(s) conferring powdery mildew resistance was(were) located on 2PL bin FL 0.66–0.86 and 19 2P-specific markers were identified in this bin. Moreover, two new powdery mildew-resistant translocation lines (2PT-4 and 2PT-5) with small 2PL chromosome segments were obtained. The newly developed wheat lines with powdery mildew resistance and the closely linked molecular markers will be valuable for wheat disease breeding in the future.

     

Mammaglobin in peripheral blood and the tumor of breast cancer patients

Currently, no molecular biological markers do exist for early diagnosis of breast cancer. One of the possible candidates for the marker of early breast cancer is mammaglobin (MGB1) or SCGB2A2 (secretoglobin, family 2A, member 2), characterized by the maximal expression level in early breast cancer. Using the RT-PCR method MGB1 mRNA expression was examined in 57 tumor tissue samples and 57 samples of morphologically non-malignant tissue (MNT) of breast cancer (BC) patients. Specificity and sensitivity of the MGB1 mRNA assay in peripheral blood of BC patients was evaluated by nested PCR. 169 blood samples (from 95 BC patients, 22 from patients with benign breast tumors, 28 from patients with tumors of other localizations, and 24 samples from healthy donors) have been analyzed. MGB1 expression was significantly higher in BC tissue samples compared to MNT (p = 0.0019). The maximal expression level was in the samples T1 (p = 0.013), stage I BC (p = 0.037), GI (p = 0.0019). MGB1 expression positively correlated with expression of estrogen (p = 0.034) and progesterone (p = 0.0004) receptors. Sensitivity and specificity of the MGB1 mRNA assay in peripheral blood were 60.6 and 92.3%, respectively. Expression of MGB1 was higher in BC than MNT and it decreased during BC progression. The sensitivity and specificity of the MGB1 mRNA assay may be used as an additional diagnostic method.