A conserved family of enzymes that phosphorylate inositol hexakisphosphate

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.     

Positive regulation of Itk PH domain function by soluble IP4

Pleckstrin homology (PH) domain-mediated protein recruitment to cellular membranes is of paramount importance for signal transduction. The recruitment of many PH domains is controlled through production and turnover of their membrane ligand, phosphatidylinositol 3,4,5-trisphosphate (PIP3). We show that phosphorylation of the second messenger inositol 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) establishes another mode of PH domain regulation through a soluble ligand. At physiological concentrations, IP4 promoted PH domain binding to PIP3. In primary mouse CD4+CD8+ thymocytes, this was required for full activation of the protein tyrosine kinase Itk after T cell receptor engagement. Our data suggest that IP4 establishes a feedback loop of phospholipase C-gamma1 activation through Itk that is essential for T cell development.     

Fibroblasts transformed with v-src show enhanced formation of an inositol tetrakisphosphate

The tyrosine kinase pp60v-src, encoded by the v-src oncogene, seems to regulate phosphatidylinositol metabolism. The effect of pp60v-src on control points in inositol phosphate production was examined by measuring the amounts of inositol polyphosphates in Rat-1 cells expressing wild-type or mutant forms of the protein. Expression of v-src-resulted in a five- to sevenfold elevation in the steady-state amount of an isomer of inositol tetrakisphosphate, whereas the concentrations of inositol trisphosphates or other inositol tetrakisphosphates were not affected. The activity of a key enzyme in the formation of inositol tetrakisphosphates, inositol (1,4,5)-trisphosphate 3-kinase, was increased six- to eightfold in cytosolic extracts prepared from the v-src-transformed cells, suggesting that this enzyme may be one target for the pp60v-src kinase and that it may participate in the synthesis of novel, higher order inositol phosphates.     

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.     

PML regulates apoptosis at endoplasmic reticulum by modulating calcium release

The promyelocytic leukemia (PML) tumor suppressor is a pleiotropic modulator of apoptosis. However, the molecular basis for such a diverse proapoptotic role is currently unknown. We show that extranuclear Pml was specifically enriched at the endoplasmic reticulum (ER) and at the mitochondria-associated membranes, signaling domains involved in ER-to-mitochondria calcium ion (Ca(2+)) transport and in induction of apoptosis. We found Pml in complexes of large molecular size with the inositol 1,4,5-trisphosphate receptor (IP(3)R), protein kinase Akt, and protein phosphatase 2a (PP2a). Pml was essential for Akt- and PP2a-dependent modulation of IP(3)R phosphorylation and in turn for IP(3)R-mediated Ca(2+) release from ER. Our findings provide a mechanistic explanation for the pleiotropic role of Pml in apoptosis and identify a pharmacological target for the modulation of Ca(2+) signals.     

Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate

The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.     

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine (PEI) nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMI205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEI and DNA is in the range of 5:1-1:4.Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P＜5 and reached the highest at N/P=5, and began to decrease beyond N/P＞5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.     

血清淀粉样P成分转基因小鼠模型的建立

目的:建立可表达血清淀粉样P成分(SAP)转基因小鼠模型,以研究SAP基因的功能。方法:采用基因重组技术将SAP基因插入到pCMV-Tag5A真核表达载体上。经BciVI限制性内切酶酶切后,琼脂糖凝胶电泳回收2.7 kb目的片断,受精卵原核显微注射法将其注入雄原核,制备转基因小鼠。采用PCR法在基因水平筛选SAP转基因小鼠阳性小鼠;免疫沉淀法结合免疫印迹法在蛋白水平检测SAP基因在PCR阳性转基因小鼠中的表达情况。结果:成功构建了pCMV-Tag5A/mSAP真核表达载体。经显微注射法将2.7 kb线性目的基因片断注射入受精卵雄原核,移植入代孕母鼠,所生127只小鼠,其中PCR鉴定获得10只转基因阳性小鼠。免疫沉淀法和免疫印记法随机检测83#♂founder、7#♀founder与C57 BL/6J小鼠回交F1代PCR阳性转基因小鼠血清中外源SAP基因蛋白水平的表达,发现均有表达。结论:该研究以C57BL/6J小鼠为研究对象,成功地产生能稳定遗传SAP基因的过表达转基因小鼠模型,它将有利于研究SAP基因在炎症、淀粉样沉淀、自身免疫系统中的作用。     

肌醇脂质和cAMP在血小板活化因子诱导血小板聚集中的作用

目的：探讨肌醇脂质和cAMP两个细胞内第二信使系统在血小板活化因子(PAF)诱导血小板聚集过程中所起到的作用和相互关系。方法：采用PKC激动剂PMA和Ca^2 通道A23187,以及PKA激动剂Sp-cAMPS和抑制剂Rp-cAMPS干预的方法,分析观察这两个信使系统在PAF诱导血小板聚集过程中的作用;采用^3H—Inositol和^14C-Adenine双标记液体闪烁技术测定PAF诱导血小板可逆聚集过程中肌醇-1,4,5-三磷酸酯(IP3)和cAMP的水平变化的方法,分析研究这两个信使系统在PAF诱导血小板聚集过程中的相互关系。结果：(1)PMA和A23187能分别增强PAF的血小板聚集效应,而且两者具有协同作用;(2)Rp-cAMP和Sp-cAMPS两者本身都不能引起血小板聚集,但能分别增强和抑制PAF的聚集效应;(3)IP3和cAMP的水平变化分别与血小板的聚集和解聚过程一致。结论：(1)肌醇脂质信使系统是细胞内转导PAF诱导血小板聚集的主要胞内信使系统。(2)降低血小板内cAMP浓度不能诱导聚集,但能增强肌醇脂质信使系统的聚集效应;升高cAMP水平能拮抗肌醇脂质信使系统的作用,这可能是使可逆相聚集的血小板解聚的一个重要机制。     

PEI介导外源基因进入植物细胞的瞬时表达

 【目的】医学方面的大量研究证实,聚乙烯亚胺（polyethylenimine,PEI）作为一种新型阳离子多聚物基因载体可以吸附和浓缩DNA,通过与细胞膜亲和粘附和细胞吞噬作用运载外源基因进入细胞并实现表达。本实验研究旨在探索PEI作为非病毒基因载体介导外源基因进入植物细胞并获得瞬时表达的可能性。【方法】制备PEI/DNA复合物,利用凝胶阻滞分析PEI与DNA的结合情况,采用电子显微镜观察PEI/DNA复合物的形态。以绿色荧光蛋白基因为报告基因研究不同N/P比条件下PEI/DNA复合物对拟南芥原生质体细胞的转化效率,并与PEG转化方法进行比较分析。【结果】PEI与DNA的质量比为5﹕1～1﹕4,PEI可以与DNA稳定结合,形成粒径约100～200 nm的球形复合物。PEI/DNA复合物对拟南芥原生质体细胞的转化效率随其N/P比的增大而提高,N/P＝5时PEI的转化效率达到最高且明显高于PEG介导的转化效率,N/P＞5时转化效率反而下降,容易使细胞破碎和变形。【结论】PEI作为一种新型的外源基因运送载体对拟南芥原生质体细胞具有良好的介导基因转移效果。     

Phosphorylation of proteins by inositol pyrophosphates

The inositol pyrophosphates IP7 and IP8 contain highly energetic pyrophosphate bonds. Although implicated in various biologic functions, their molecular sites of action have not been clarified. Using radiolabeled IP7, we detected phosphorylation of multiple eukaryotic proteins. We also observed phosphorylation of endogenous proteins by endogenous IP7 in yeast. Phosphorylation by IP7 is nonenzymatic and may represent a novel intracellular signaling mechanism.     

Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI

Prefoldins (PFDs) are members of a recently identified, small-molecular weight protein family able to assemble into molecular chaperone complexes. Here we describe an unusually large member of this family, termed URI, that forms complexes with other small-molecular weight PFDs and with RPB5, a shared subunit of all three RNA polymerases. Functional analysis of the yeast and human orthologs of URI revealed that both are targets of nutrient signaling and participate in gene expression controlled by the TOR kinase. Thus, URI is a component of a signaling pathway that coordinates nutrient availability with gene expression.     

Overexpression of IbMIPS1 gene enhances salt tolerance in transgenic sweetpotato

Myo-inositol-1-phosphate synthase(MIPS) is a key rate limiting enzyme in the de novo biosynthesis of myo-inositol in plants.In the present study,the IbMIPS1 gene was introduced into sweetpotato cultivar Xushu 18 and the transgenic plants exhibited significantly enhanced salt tolerance compared with the wild-type(WT).Overexpression of IbMIPSI up-regulated the salt stress responsive genes,including myo-inositol monophosphatase(MIPP),pyrroline-5-carboxylate synthase(P5CS),pyrroline-5-carboxylate reductase(P5CR),psbA,phosphoribulokinase(PRK),and superoxide dismutase(SOD) genes,under salt stress.Inositol and proline content,SOD and photosynthesis activities were significantly increased,whereas malonaldehyde(MDA) and H_2O_2 contents were significantly decreased in the transgenic plants.These findings suggest that the IbMIPS1 gene may enhance salt tolerance of sweetpotato by regulating the expression of salt stress responsive genes,increasing the content of inositol and proline and enhancing the activity of photosynthesis.     

拟南芥AFP4的克隆、原核表达和纯化及其与ABI5的互作

拟南芥ABI5相互作用蛋白AFP4(ABI five binding protein 4)是植物中的一个小分子蛋白,其表达特性与ABI5相似,受ABA诱导。以拟南芥Arabidopsis thaliana L. (Heyn) cv. Columbia为材料,通过PCR扩增了AFP4基因的完整编码序列。扩增片段插入到原核表达载体pET-32a(+)的多克隆位点EcoR Ⅰ和Xho Ⅰ酶切位点之间和酵母双杂交载体pGBKT7的多克隆位点EcoR Ⅰ和Pst Ⅰ酶切位点之间。将ABI5基因的编码序列插入到酵母双杂交载体pGADT7的多克隆位点EcoR Ⅰ和Pst Ⅰ酶切位点之间。重组质粒测序结果表明,所克隆的AFP4和ABI5编码区序列分别与NCBI数据库收录的AFP4基因(GenBank登录号NM_111081.2)和ABI5基因(GenBank登录号NM_129185.3)完全一致。重组原核表达载体含有AFP4片段的重组融合蛋白的理论分子量为53.4 ku。将重组质粒转化至大肠埃希菌表达菌株BL21 Star (DE3)中诱导表达,并经Ni-NTA亲和层析柱分离纯化、SDS-PAGE分析和Western blot鉴定。将含有AFP4和ABI5的酵母双杂交重组质粒共同转化酿酒酵母AH109中进行酵母双杂交。结果表明,重组融合蛋白在大肠埃希菌E. coli BL21 Star (DE3)中表达的适宜条件为:IPTG浓度为0.4 mmol·L^-1、25 ℃下诱导表达6 h。在上述条件下,重组融合蛋白占细胞破碎后上清液总蛋白的41.6%。经Ni-NTA亲和层析柱纯化后,AFP4融合蛋白在SDS-PAGE分析时呈现单一条带。该条带经过抗6xHis标签肽的抗体分析,呈阳性。该条带经酵母双杂交分析结果表明,AFP4和ABI5在酵母细胞中可以相互作用。     

小黑杨碳酸酐酶家族基因表达特性分析

本文克隆了小黑杨CA家族的6个基因成员,分别命名为PsnCA1、PsnCA2、PsnCA3、PsnCA4、PsnCA5和PsnCA6,其中PsnCA4和PsnCA6属于α碳酸酐酶家族,PsnCA1、PsnCA3和PsnCA5属于β碳酸酐酶家族,而PsnCA2属于LbeatH超基因家族。系统进化分析表明,亲缘关系较近的是PsnCA2、PsnCA4和PsnCA6,而PsnCA1、PsnCA3和PsnCA5聚为一大类。通过实时荧光RT-PCR技术研究了小黑杨根、茎、叶中PsnCA基因的表达模式,结果表明:除PsnCA6外,其余5个基因在叶部的表达量显著高于根和茎。在叶部表达量最高的为PsnCA1,在茎部和根部表达最高的为PsnCA3。PsnCA在各部位的表达均受暗处理的影响,其中各基因在根部的表达变化最大,其次为茎和叶。      

IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism

Type 2 and type 3 inositol 1,4,5-trisphosphate receptors (IP3R2 and IP3R3) are intracellular calcium-release channels whose physiological roles are unknown. We show exocrine dysfunction in IP3R2 and IP3R3 double knock-out mice, which caused difficulties in nutrient digestion. Severely impaired calcium signaling in acinar cells of the salivary glands and the pancreas in the double mutants ascribed the secretion deficits to a lack of intracellular calcium release. Despite a normal caloric intake, the double mutants were hypoglycemic and lean. These results reveal IP3R2 and IP3R3 as key molecules in exocrine physiology underlying energy metabolism and animal growth.     

The Role of Extracellular Ca2+Influx, Intracellular Ca2+ Release and Calmodulin in Mouse Egg Fertilization

The effects of various Ca^2 -modifying drugs on moue egg fertilization were studied.Ca^2 chelator,ethylen glycol-bis-(2-aminoethyl)-tetracetic acid(EGTA),and calmodulin(CaM) antagonist,trifluoperzaine (TFP),inhibited fertilization in a dose-dependent manner,whild Ca^2 channel bolcker,verspamil,did not have any effect.When intracellular Ca^2 release was blocked by 8-(N,N-diethylamino) octy 1-3,4,5-trimethoxy-benzonate(TME-8) or the Ca^2 oscillations were inhibited by an inhibitor of endoplasmic reticulum Ca^2 -At-Pase,thapsigargin,the second polar body emission and pronuclear formation were significantly decreased.In contrast,inhibition of intracellular Ca^2 release via bolckage of inositol 1,4,5-triphosphate (IP3) production by neomycin or lithium did not affect fertilization.The results sugest that both extracellular influx,intracellular Ca^2 release and CaM activation are required for mormal fertilization.However,extracellular influx through voltage-gated Ca^2 channel and intracellular release induced by IP3 and not the only pathways for producing Ca^2 transients in moue eggs.