新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

A functional polymorphism in interleukin-1α (IL1A) gene is associated with risk of alopecia areata in Chinese populations

Alopecia areata (AA) is an inflammatory hair loss disorder with a major genetic component, which may cause great psychosocial distress for those affected. Studies have shown that interleukin-1 (IL-1) is a very potent inducer of hair loss and a significant human hair growth inhibitor. The 4-bp insertion/deletion (Indel) polymorphism (rs3783553) within the 3′ untranslated regions of IL1A gene has been suggested to be associated with risk of various types of cancers, possibly through regulating expression of IL-1α levels. In the current study, we estimated the susceptibility to AA associated with rs3783553 in two independent case–control panels of Eastern and Southern Chinese populations, totally containing 313 AA cases and 626 healthy controls. Logistic regression analysis showed that the heterozygote and the homozygote 4-bp ins/ins confer a significantly lower risk of AA in both panels and total subjects [odds ratio (OR) = 0.55, 95% confidence interval (C.I.) = 0.41–0.75, P = 6.24 × 10^− 5; OR = 0.47, 95% C.I. = 0.28–0.76, P = 0.001, respectively]. Stratification analysis based on age onset showed that the protective roles of ins/del and ins/ins genotype against developing AA was more obvious in AA patients with early age onset (< 30 years) under dominant model (OR = 0.48, 95% C.I. = 0.29–0.77, P = 0.001). The results of luciferase assay showed that rs3783553 could influence expression of IL-1α in a miR-122 dependant manner. Taken together, our results suggested that the IL1A 4-bp indel polymorphism may be a marker for genetic susceptibility to patchy (mild) AA in Chinese populations, likely through miR-122 mediated regulation.     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Are glutathione S-transferase polymorphisms (GSTM1, GSTT1) associated with primary open angle glaucoma? A meta-analysis

Background

Glutathione S-transferase (GST) variants have been considered as risk factors for the pathogenesis of primary open angle glaucoma (POAG). However, the results have been inconsistent. In this study, we performed a meta-analysis to assess the association between GSTM1 and GSTT1 null genotypes and the risk for POAG.

Methods

Published literature from PubMed and EMBASE databases was retrieved. All studies evaluating the association between GSTM1/GSTT1 variants and POAG were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using fixed- or random-effects model.

Results

14 studies (1711 POAG cases and 1537 controls) were included in the meta-analysis of GSTM1 genotypes and 10 studies (1306 POAG cases and 1114 controls) were included in the meta-analysis of GSTT1 genotypes. The overall result showed that the association between GSTM1 and GSTT1 null genotypes and risk for POAG was not statistically significant (GSTM1: OR = 1.19, 95% CI = 0.82–1.73, p = 0.361; GSTT1: OR = 1.26, 95% CI = 0.77–2.06, p = 0.365). The results by ethnicity showed that the association between the GSTM1 null genotype and risk for POAG is statistically significant in East Asians (OR = 1.41, 95% CI = 1.04–1.90, p = 0.026), but not in Caucasians (OR = 1.13, 95% CI = 0.69–1.84, p = 0.638) and Latin-American (OR = 1.09, 95% CI = 0.62–1.92, p = 0.767). In addition, there was no significant association of GSTT1 null genotype with risk for POAG in either ethnic population.

Conclusions

The present meta-analysis suggested that there might be a significant association of GSTM1 null genotype with POAG risk in East Asians.     

Relationship between GSTM1 and GSTT1 polymorphisms and schizophrenia: A case–control study in a Tunisian population

There is substantial evidence found in the literature that supports the fact that the presence of oxidative stress may play an important role in the pathophysiology of schizophrenia. The glutathione S-transferases (GSTs) forms one of the major detoxifying groups of enzymes responsible for eliminating products of oxidative stress. Interindividual differences observed in the metabolism of xenobiotics have been attributed to the genetic polymorphism of genes coding for enzymes involved in detoxification. Thus, in this study we investigated the association of glutathione S-transferase Mu-1 (GSTM1) and glutathione S-transferase theta-1 (GSTT1) gene deletion polymorphisms and schizophrenia in a Tunisian population. A case–control study including 138 schizophrenic patients and 123 healthy controls was enrolled. The GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction (PCR). No association was found between the GSTM1 genotype and schizophrenia, whereas the prevalence of the GSTT1 active genotype was significantly higher in the schizophrenic patients (57.2%) than in the controls (45.5%) with (OR = 0.6, IC 0.37–0.99, p = 0.039). Thus, we noted a significant association between schizophrenia and GSTT1 active genotype. Furthermore, the combination of the GSTM1 and GSTT1 null genotypes showed a non-significant trend to an increased risk of schizophrenia. The present finding indicated that GSTT1 seems to be a candidate gene for susceptibility to schizophrenia in at least Tunisian population.     

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3 × HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Visual learning and memory of Drosophila melanogaster wild type CS and the mutants dunce1, amnesiac, turnip and rutabaga

Dunce¹, amnesiac, turnip and rutabaga, mutants of Drosophila melanogaster deficient in olfactory learning and/or memory, were tested for visual learning ability and memory. All these mutants are able to learn conditioned visual information, but not as well as the corresponding wildtype CS. Memory of all four mutants is reduced during the first 30 min after training, but indistinguishable from that of the wildtype two hours after conditioning.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

N-acetylglucosamine kinase, HXK1 contributes to white–opaque morphological transition in Candida albicans

Morphological transition (yeast-hyphal and white–opaque) is an important biological process in the life cycle of pathogenic yeast, Candida albicans and is a major determinant of virulence. Earlier reports show that the amino sugar, N-acetylglucosamine (GlcNAc) induces white to opaque switching in this pathogen. We report here a new contributor to this switching phenomenon, namely N-acetylglucosamine kinase or HXK1, the first enzyme of the GlcNAc catabolic cascade. Microarray profile analysis of wild type vs. hxk1 mutant cells grown under switching inducing condition showed upregulation of opaque specific and cell wall specific genes along genes involved in the oxidative metabolism. Further, our qRT-PCR and immunoblot analysis revealed that the expression levels of Wor1, a master regulator of the white–opaque switching phenomenon remained unaltered during this HXK1 mediated transition. Thus the derepression of opaque specific gene expression observed in hxk1 mutant could be uncoupled to the expression of WOR1. Moreover, this regulation via HXK1 is independent of Ras1, a major regulator of morphogenetic transition and probably independent of MTL locus too. These results extend our understanding of multifarious roles of metabolic enzymes like Hxk1 and suggest an adaptive mechanism during host–pathogen interactions.     

Differences in β-strand Populations of Monomeric Aβ40 and Aβ42

Using homonuclear ¹H NOESY spectra, with chemical shifts, ³J_H^N_H^α scalar couplings, residual dipolar couplings, and ¹H-¹⁵N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1–40 (Aβ40) and amyloid-β 1–42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16–21) with residues 29–36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9–13, that sometimes forms a β-sheet by association with residues 35–37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

基于COⅢ和HNF-1α序列研究龟鳖类的系统进化特征

用PCR扩增和测序的方法,获得小鳄龟(Chelydra serpentina)的COⅢ和HNF-1α序列,并分别结合NCBI中其他龟鳖的同源性序列进行比对分析。比对后得到757 bp的COⅢ一致序列和769 bp的HNF-1α一致序列。其中,COⅢ一致序列含有可变位点324个,序列总变异率为42.8%,简约信息位点230个;T、C、A、G的平均含量分别为27.5%、26.6%、30.8%、15.1%,A+T含量(58.3%)高于G+C含量(41.7%),转换/颠换比率(R)为2.62。HNF-1α一致序列有变异位点112个,变异率为14.6%,简约信息位点67个;T、C、A、G的平均含量为26.1%、23.1%、28.3%、22.6%,A+T含量为54.4%,G+C含量为45.7%,转换/颠换比率(R)为1.42。基于Kimura双参数模型计算龟鳖类种间、属间、科间遗传距离,并采用邻接法、最大简约法和最大似然法构建分子系统进化树。结果显示:基于COⅢ序列的淡水龟科4个属间的遗传距离为0.090～0.153,平均遗传距离为0.129;曲颈龟亚目5个科之间的遗传距离为0.150～0.207,平均遗传距离为0.177;基于HNF-1α序列的龟科9属间的遗传距离为0.003～0.051,平均为0.016;鳄龟科、龟科、淡水龟科3科间的遗传距离为0.044～0.067,平均为0.053。由遗传距离和构建的系统进化树可知,淡水龟科与陆龟科具有较近的亲缘关系,而与龟科的亲缘关系较远;支持龟科重新划分为两个分支;鳄龟科和海龟科亲缘关系较近,大鳄龟(Macroclemys temminckii)和小鳄龟可能同为一属。     

Light Signal Transduction Pathway from Flavin Chromophore to the Jα Helix of Arabidopsis Phototropin1

In the plant blue-light sensor phototropin, illumination of the chromophoric LOV domains causes activation of the serine/threonine kinase domain. Flavin mononucleotide (FMN) is a chromophore molecule in the two LOV domains (LOV1 and LOV2), but only LOV2 is responsible for kinase activation. Previous studies reported an important role of an additional helix connected to the C-terminal of LOV2 (Jα helix) for the function of phototropin; however, it remains unclear how the Jα helix affects light-induced structural changes in LOV2. In this study we compared light-induced protein structural changes of the LOV2 domain of Arabidopsis phot1 in the absence (LOV2-core) and presence (LOV2-Jα) of the Jα helix by Fourier-transform infrared spectroscopy. Prominent peaks were observed only in the amide-I region (1650 (−)/1625 (+) cm⁻¹) of LOV2-Jα at physiological temperatures (≥260 K), corresponding to structural perturbation of the α-helix. The peaks were diminished by point mutation of functionally important amino acids such as Phe-556 between FMN and the β-sheet, Gln-575 being hydrogen-bonded with FMN, and Ile-608 on the Jα helix. We thus conclude that a light signal is relayed from FMN through these amino acids and eventually changes the interaction between LOV2-core and the Jα helix in Arabidopsis phot1.