PI4P and PI(4,5)P2 are essential but independent lipid determinants of membrane identity

The quantitatively minor phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] fulfills many cellular functions in the plasma membrane (PM), whereas its synthetic precursor, phosphatidylinositol 4-phosphate (PI4P), has no assigned PM roles apart from PI(4,5)P(2) synthesis. We used a combination of pharmacological and chemical genetic approaches to probe the function of PM PI4P, most of which was not required for the synthesis or functions of PI(4,5)P(2). However, depletion of both lipids was required to prevent PM targeting of proteins that interact with acidic lipids or activation of the transient receptor potential vanilloid 1 cation channel. Therefore, PI4P contributes to the pool of polyanionic lipids that define plasma membrane identity and to some functions previously attributed specifically to PI(4,5)P(2), which may be fulfilled by a more general polyanionic lipid requirement.     

Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.     

Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.     

PI3Kgamma is a key regulator of inflammatory responses and cardiovascular homeostasis

Class I phosphoinositide 3-kinase (PI3K) signaling pathways regulate several important cellular functions, including cellular growth, division, survival, and movement. Class IB PI3K (also known as PI3Kgamma) links heterotrimeric GTP-binding protein-coupled receptors to these pathways. Activation of class IB PI3K results in the rapid synthesis of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] and its dephosphorylation product PtdIns(3,4)P2 in the plasma membrane. These two lipid messengers bind to pleckstrin homology domain-containing effectors that regulate a complex signaling web downstream of receptor activation. Characteristic features of this pathway are the regulation of protein kinases and the regulation of small guanosine triphosphatases that control cellular movement, adhesion, contraction, and secretion. Most of the ligands that activate class IB PI3K are involved in coordinating the body's response to injury and infection, and recent studies suggest that small molecule inhibitors of this enzyme may represent a novel class of anti-inflammatory therapeutic agents.     

Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.     

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.     

Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation

The canonical Wnt-beta-catenin signaling pathway is initiated by inducing phosphorylation of one of the Wnt receptors, low-density lipoprotein receptor-related protein 6 (LRP6), at threonine residue 1479 (Thr1479) and serine residue 1490 (Ser1490). By screening a human kinase small interfering RNA library, we identified phosphatidylinositol 4-kinase type II alpha and phosphatidylinositol-4-phosphate 5-kinase type I (PIP5KI) as required for Wnt3a-induced LRP6 phosphorylation at Ser1490 in mammalian cells and confirmed that these kinases are important for Wnt signaling in Xenopus embryos. Wnt3a stimulates the formation of phosphatidylinositol 4,5-bisphosphates [PtdIns (4,5)P2] through frizzled and dishevelled, the latter of which directly interacted with and activated PIP5KI. In turn, PtdIns (4,5)P2 regulated phosphorylation of LRP6 at Thr1479 and Ser1490. Therefore, our study reveals a signaling mechanism for Wnt to regulate LRP6 phosphorylation.     

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.     

多效唑对绿豆黄化幼苗生长及肌醇磷脂代谢的抑制作用

多效唑抑制绿豆黄化幼苗生长和茎尖初生分生组织细胞分裂,降低内源ＩＡＡ、ＡＢＡ和甾醇水平。应用放射性标记及薄层层析技术,发现多效唑抑制肌醇磷脂代谢,ＰＩ、ＰＩＰ和ＰＩＰ２含量均高于对照,而ＤＡＧ含量低于对照。     

Structural mechanism of endosome docking by the FYVE domain

The recruitment of trafficking and signaling proteins to membranes containing phosphatidylinositol 3-phosphate [PtdIns(3)P] is mediated by FYVE domains. Here, the solution structure of the FYVE domain of the early endosome antigen 1 protein (EEA1) in the free state was compared with the structures of the domain complexed with PtdIns(3)P and mixed micelles. The multistep binding mechanism involved nonspecific insertion of a hydrophobic loop into the lipid bilayer, positioning and activating the binding pocket. Ligation of PtdIns(3)P then induced a global structural change, drawing the protein termini over the bound phosphoinositide by extension of a hinge. Specific recognition of the 3-phosphate was determined indirectly and directly by two clusters of conserved arginines.     

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation

Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.     

FB28对甜菜夜蛾围食膜蛋白影响的研究

[目的]研究荧光增白剂FB28对甜菜夜蛾围食膜蛋白的影响。[方法]用含有不同浓度的荧光增白剂FB28饲喂5龄甜菜夜蛾幼虫,解剖取其围食膜,SDS-PAGE电泳检测。[结果]FB28活体处理甜菜夜蛾围食膜在4h内高分子量蛋白条带逐渐消失,4-16h甜菜夜蛾围食膜蛋白逐渐恢复正常。[结论]FB28对甜菜夜蛾围食膜蛋白具有影响作用。     

基于绵羊胚胎骨骼肌蛋白质组学的PI3K-AKT信号通路分析

【目的】绵羊是重要的经济动物,其骨骼肌生长发育与产肉性能密切相关。胚胎期是绵羊骨骼肌生长发育的关键阶段,挖掘分析绵羊胚胎骨骼肌蛋白质组数据,为揭示绵羊肌肉发育重要时间节点、筛选绵羊胚胎骨骼肌生长发育调控蛋白质提供依据。【方法】本团队已对妊娠第85天、第105天和第135天的中国美利奴绵羊胚胎背最长肌进行串联质谱(tandem mass tag, TMT)蛋白质定量,鉴定到1316种差异丰度蛋白质。现利用GO、KEGG和R等方法对这些差异丰度蛋白质开展聚类、功能注释和通路分析等生物信息学分析。【结果】基于前期研究结果对差异丰度蛋白质进行R语言聚类,分析结果显示,cluster 5类蛋白在胚胎骨骼肌第105天具有较高丰度。对cluster 5 蛋白进行GO和KEGG富集分析发现,该类蛋白质参与胞内蛋白质代谢过程,显著富集于PI3K-AKT信号通路中,而在该信号通路中RAC-β丝氨酸/苏氨酸蛋白激酶X1(AKT2)具有较高表达丰度。蛋白质生物信息学结果表明,AKT2蛋白由481个氨基酸构成,AKT2蛋白理论分子量为55.58kD,由66个带正电荷的氨基酸残基和72个带负电荷的氨基酸残基组成,理论等电点为6.08,亲水性平均系数-0.454,属于亲水性蛋白。预测AKT2蛋白的481个氨基酸全部位于膜外,属于膜受体蛋白。AKT2蛋白有12个N-端糖基化位点,71个磷酸化位点,与蛋白酶K相似度为99%,属于蛋白酶催化亚基家族。【结论】绵羊胚胎骨骼肌蛋白质组数据发现,第105天是绵羊胚胎骨骼肌纤维由增殖分化到增大增粗的转折点,具有调控绵羊胚胎骨骼肌纤维生长发育作用的PI3K-AKT信号通路在该节点显著富集,AKT2是调控该信号通路的重要候选蛋白。综上,本研究结果对揭示胚胎骨骼肌生长发育及其调控分子机制具有重要理论指导意义。     

Membrane phosphatidylserine regulates surface charge and protein localization

Electrostatic interactions with negatively charged membranes contribute to the subcellular targeting of proteins with polybasic clusters or cationic domains. Although the anionic phospholipid phosphatidylserine is comparatively abundant, its contribution to the surface charge of individual cellular membranes is unknown, partly because of the lack of reagents to analyze its distribution in intact cells. We developed a biosensor to study the subcellular distribution of phosphatidylserine and found that it binds the cytosolic leaflets of the plasma membrane, as well as endosomes and lysosomes. The negative charge associated with the presence of phosphatidylserine directed proteins with moderately positive charge to the endocytic pathway. More strongly cationic proteins, normally associated with the plasma membrane, relocalized to endocytic compartments when the plasma membrane surface charge decreased on calcium influx.     

卵巢颗粒细胞自噬与PI3K/AKT/FOXO3a信号通路的相关性

细胞自噬是哺乳动物细胞物质代谢的一个重要机制,与细胞凋亡共同参与卵巢卵泡的发育和闭锁,并发挥重要的作用。近年研究发现,磷脂酰肌醇3-激酶/蛋白激酶B（phosphatidylinositol 3-kinase/protein kinase B,PI3K/AKT）信号通路参与卵巢疾病的发生。PI3K和AKT的过度激活可使原始卵泡过早发育以及卵泡过快凋亡,卵巢颗粒细胞作为卵泡发育重要的支持细胞,其功能的减退或凋亡很可能引发一系列女性内分泌方面的疾病。FOXO3a转录因子是PI3K/AKT信号通路下游的重要靶蛋白之一,参与抗增殖和凋亡。本文就关于卵巢颗粒细胞自噬与PI3K/AKT/FOXO3a信号通路的相关进展加以综述。     

无人机电控速度模糊PI双闭环控制仿真研究

【目的】针对农用无人机作业时,对速度的稳定恒速需求,研究无人机无刷直流电机的速度控制模糊PI闭环算法。【方法】分析无人机电控系统的结构原理,根据电控系统驱动无刷直流电机的速度控制要求,在Matlab/Simulink环境下,构建电控驱动无刷直流电机系统的仿真模型,采用速度电流双闭环控制策略,其中,速度环使用模糊PI控制器,电流环使用电流滞环控制。设置系统参数,进行仿真分析,搭建ARM电路仿真板,验证算法的有效性。【结果】采用模糊PI后,该系统加快了速度响应,减少了系统超调量,提高了系统的抗干扰能力,提高了系统的动态特性和鲁棒性。【结论】本研究提出的模糊PI控制策略是有效的,可为无人机实际电机控制系统设计和调试提供理论参考。     

磷脂酰肌醇3-磷酸对UV-B诱导蚕豆保卫细胞中过氧化氢产生和气孔关闭的影响

【目的】研究磷脂酰肌醇3-激酶（PI3K）催化产物磷脂酰肌醇3-磷酸（PI3P）对紫外线B（UV-B）诱导保卫细胞中过氧化氢（H2O2）产生和气孔关闭的影响,为进一步阐明植物细胞转导UV-B辐射信号的机制提供依据。【方法】以蚕豆（Vicia faba L.）表皮条为材料,采用PI3K的抑制剂沃曼青霉素（WM）和LY294002（LY）来抑制PI3P的形成,采用二苯基碘（DPI）和水杨基氧肟酸（SHAM）分别抑制H2O2形成的NADPH氧化酶途径和细胞壁过氧化物酶途径,通过气孔开度分析和激光扫描共聚焦显微镜技术,确定PI3P在0.8 W•m-2 UV-B辐射诱导蚕豆保卫细胞H2O2产生和气孔关闭中的作用。【结果】WM和LY能显著抑制UV-B诱导的保卫细胞H2O2产生和气孔关闭;外源H2O2处理能显著逆转WM和LY对UV-B诱导气孔关闭的抑制效应,但WM和LY不能抑制外源H2O2诱导的气孔关闭;UV-B诱导的保卫细胞H2O2产生和气孔关闭能被活性氧清除剂和SHAM显著抑制,但不能被DPI抑制。【结论】PI3P通过诱导蚕豆保卫细胞中产生于过氧化物酶途径的H2O2形成来介导UV-B辐射诱导的气孔关闭。     

西安市秋季大气细粒子（PM2．5）中化学元素的浓度特征和来源

[目的]研究西安市秋季大气细粒子（PM2.5）中化学元素的浓度特征及来源。[方法]于2009年10月利用微流量采样器采集西安大气中PM2.5样品,分析其元素浓度特征及来源。[结果]西安市秋季大气中PM2.5质量浓度的平均值为168.44μg/m3,最小值为53.29μg/m3,最大值达358.16μg/m3,高于北京、珠江三角洲;PM2.5中S、Zn、K、Cl、Ca、Fe的质量浓度均超过1.0μg/m3,处于较高污染水平;PM2.5中K与有机碳（OC）、元素碳（EC）的相关性较高,相关系数分别为0.76和0.75（P〈0.0001）,说明OC、EC与K具有相同的来源,生物质燃烧对OC、EC有一定的贡献;元素的富集因子分析表明,K、Ca、Fe、Ti、Mn和Cr主要来源于地壳或岩石风化等自然源,而S、Zn、Cl、Pb、Br、Mo、Cd和As主要受人为污染源的影响,而受土壤扬尘等自然源的影响较小,其中Cd的富集因子最大,主要来源于金属冶炼等人为污染;燃煤、生物质燃烧、机动车尾气排放、冶金化工、扬尘等是该区秋季PM2.5的主要来源。[结论]该研究为城市环境污染治理提供了理论依据。     

宁南干旱半干旱区不同施磷量对谷子产量及水分利用效率的影响

[目的]为宁南干旱半干旱区谷子（Setaria italica）生产中合理施磷、充分利用潜在的磷素资源提供科学依据,以期更好指导当地谷子科学施肥.[方法]设施纯磷0（CK） 、45、90、135、180、225 kg/hm^2 6个处理;一次性基施尿素300 kg/hm^2,硫酸钾150 kg/hm^2.[结果]在宁南干旱半干旱区,在基施定量的氮肥和钾肥的基础上,增施一定比例的磷肥,谷子产量发生明显变化,施纯磷0、45、90、135、180、225 kg/hm^2 6个处理产量分别为4 855.80、5 018.10、5 220.45、5 471.55、4 915.80、5 773.95 kg/hm^2,施纯磷135、225 kg/hm^2处理与0kg/hm^2处理差异极显著;6个处理间农艺性状差异不大;施纯磷45 kg/hm^2处理水分利用效率最低为16.61 kg/（hm^2.mm）,施纯磷225kg/hm^2处理水分利用效率最高为20.50 kg/（hm^2.mm）.[结论]在宁南干旱半干旱区,施纯磷225 kg/hm^2、纯氮139.2 kg/hm^2、纯钾67.5 kg/hm^2组合,谷平增产效果最大,水分利用效率最高.     

长期施用化肥、猪粪和稻草对红壤性水稻土物理性质的影响

【目的】研究化肥与猪粪和稻草长期配合施用对红壤性水稻土物理性质的影响。【方法】利用1981年建立在湖南省望城县黄金乡第四纪红土发育红壤性水稻土(普通简育水耕人为土)上的肥力定位试验,对化学氮、磷和钾肥料及其与猪粪和稻草长期配合施用的7个处理的土壤物理性质进行了评价。【结果】猪粪、稻草与化肥长期配合施用均可显著降低土壤容重和土粒密度,提高孔隙度,猪粪和稻草的效果基本相似。与化肥处理相比,施用猪粪和稻草显著地促进了5—0.5mm水稳性团聚体的形成和提高土壤团聚体稳定性。猪粪和稻草与化肥配施处理耕层土壤持水量也显著高于化肥处理。【结论】猪粪和稻草与化肥长期配合施用能显著降低土壤容重和土粒密度,提高孔隙度、土壤团聚体稳定性和土壤持水量,是提升土壤物理质量和红壤性水稻土肥力的有效措施。