Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells

Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic beta cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (InsP7 or IP7). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP7 were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP7 dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in beta cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP7 in the pancreatic beta cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.     

盐胁迫下植物的分子信号传导

盐胁迫下,植物体内的离子浓度和渗透压发生变化,诱导其产生第二信使(如IP3和活性氧分子等).第二信使调控细胞内Ca2 的水平,引发蛋白磷酸化级联反应,直接诱导或间接由转录因子诱导盐胁迫相关基因的表达,最终植物体表现耐受胁迫、生长受阻或死亡.     

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.     

Protein-tyrosine kinase activity in Saccharomyces cerevisiae

Saccharomyces cerevisiae was examined for tyrosine kinase activity in vitro because this organism offers molecular and genetic approaches for analyzing the role of tyrosine phosphorylation in cellular growth control that are unavailable in higher eukaryotes. Yeast extracts phosphorylated a random copolymer (glutamic acid:tyrosine, 80:20) at tyrosine in a reaction that was linear with respect to time and protein concentration. In the absence of added copolymer, phosphotyrosine was 0.1 percent of the total phosphoamino acids labeled with [gamma-32P]adenosine triphosphate in endogenous yeast proteins. However, specific activities of these reactions were low (approximately 1 percent of those in extracts of chick embryo fibroblasts). Lack of significant incorporation of label from [alpha-32P]adenosine triphosphate into the copolymer or endogenous yeast proteins demonstrated that nucleotide interconversion, adenylylation, and subsequent hydrolysis could not account for the generation of phosphotyrosine observed.     

王浆高产蜜蜂咽下腺磷酸化蛋白质组分析

【目的】通过对王浆高产蜜蜂（Apis mellifera L.）（浙江浆蜂）哺育蜂（6-12 d）咽下腺的磷酸化蛋白质组分析,以期探明蛋白质磷酸化修饰对王浆分泌的生物学意义。【方法】将哺育蜂咽下腺蛋白质液内酶切后,用固相金属离子亲和层析色谱法（IMAC）、强阳离子交换（SCX）及LC-MS/MS和生物信息学分析,对工蜂咽下腺磷酸化蛋白质组进行研究。【结果】在哺育蜂的咽下腺中,共鉴定117个蛋白质,其中6个蛋白质的6条磷酸肽段的8个位点发生磷酸化修饰。这些磷酸化蛋白质是王浆主蛋白1和7的前体、与蛋白翻译合成相关的60S酸性核糖体蛋白P0、P1、P2和60S核糖体蛋白L15。【结论】哺育蜂咽下腺核糖体蛋白发生的磷酸化修饰主要为促进王浆蛋白的高效合成,而磷酸化的王浆蛋白1和7可保证浆蜂在王浆高产的前提下仍保证其王浆具合理的钙磷比,提高营养价值,以满足蜂王产卵和幼虫发育的营养需求。研究结果在蛋白质磷酸化水平上为揭示浆蜂王浆的高产机理奠定了新的理论基础。     

Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates

In budding yeast, phosphate starvation triggers inhibition of the Pho80-Pho85 cyclin-cyclin-dependent kinase (CDK) complex by the CDK inhibitor Pho81, leading to expression of genes involved in nutrient homeostasis. We isolated myo-d-inositol heptakisphosphate (IP7) as a cellular component that stimulates Pho81-dependent inhibition of Pho80-Pho85. IP7 is necessary for Pho81-dependent inhibition of Pho80-Pho85 in vitro. Moreover, intracellular concentrations of IP7 increased upon phosphate starvation, and yeast mutants defective in IP7 production failed to inhibit Pho80-Pho85 in response to phosphate starvation. These observations reveal regulation of a cyclin-CDK complex by a metabolite and suggest that a complex metabolic network mediates signaling of phosphate availability.     

水稻OsV3IP2和OsV3IP3原核表达与鉴定

通过生物信息学分析确定OsV3IP2和OsV3IP3的可溶性肽段,获得可溶性的OsV3IP2和OsV3IP3外源表达蛋白质,将相应的编码区克隆于原核表达载体p ET32a中,进行IPTG诱导表达,利用SDS-PAGE和Western blot检测目的蛋白质。结果表明,利用生物信息学软件对蛋白质的二级结构进行预测,将OsV3IP2、OsV3IP3分别分为2个和4个部分。将其分别克隆到p ET32a后,采用0.2 mol/L IPTG在10、20、30℃诱导表达。经SDS-PAGE和Western Blot分析,证明OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1表达的融合蛋白存在于上清液中,而OsV3IP 3-2、OsV3IP 3-3表达的融合蛋白存在于沉淀中,获得了OsV3IP2-1、OsV3IP 2-2、OsV3IP 3-1可溶性目的蛋白,为进一步体外验证OsVDAC3与OsV3IP2、OsV3IP3的互作奠定了基础。     

Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

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.     

Inhibition of translational initiation by Let-7 MicroRNA in human cells

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNA molecules regulating gene expression in multicellular eukaryotes. In metazoa, miRNAs act by imperfectly base-pairing with the 3' untranslated region of target messenger RNAs (mRNAs) and repressing protein accumulation by an unknown mechanism. We demonstrate that endogenous let-7 microribonucleoproteins (miRNPs) or the tethering of Argonaute (Ago) proteins to reporter mRNAs in human cells inhibit translation initiation. M(7)G-cap-independent translation is not subject to repression, suggesting that miRNPs interfere with recognition of the cap. Repressed mRNAs, Ago proteins, and miRNAs were all found to accumulate in processing bodies. We propose that localization of mRNAs to these structures is a consequence of translational repression.     

草莓果实不同发育时期的蛋白磷酸化水平

【目的】通过分析草莓果实生长发育过程中蛋白磷酸化水平的变化以期揭示蛋白磷酸化在果实生长发育成熟衰老过程中的作用。【方法】以iTraq（isobaric tags for relative and absolute quantification）定量蛋白质组学结合LC-MS/MS（liquid chromatography-mass spectrometry）的方法对草莓果实的5个不同发育时期（小绿果时期、大绿果时期、白果时期、果实成熟期、果实成熟后期）的蛋白磷酸化水平进行综合分析,并对这些鉴定到的磷酸化蛋白进行生物过程分类、亚细胞定位分类和分子功能分类。通过综合分析多个数据库的比对结果,对所鉴定的磷酸化蛋白进行功能的预测。【结果】不同发育时期的磷酸化蛋白有所差异,并且在大绿果时期到果实成熟期磷酸化差异蛋白总数比其他时期显著增加,其中,多数的磷酸化蛋白参与生长发育调控。生物过程分类结果显示,这些磷酸化蛋白多数集中在植物信号转导途径和糖代谢途径中,其中,参与信号转导途径的有17个,参与糖代谢生物过程的有8个。属于MAPK级联途径的有MAPKKK家族的101308592、MAPK家族的470122684和596127083。596127083的磷酸化水平在各个发育时期较为稳定,101308592随发育时期磷酸化水平逐渐升高,而470122684的磷酸化水平从软果期开始急剧下降。亚细胞定位分类结果显示,多数磷酸化蛋白定位在细胞核和细胞质中。分子功能分类结果显示,多数磷酸化蛋白具有转录调节和磷酸化去磷酸化的分子功能,鉴定出与生长发育调节相关的磷酸化蛋白有24个。其中,具有转录调控功能的有4个,参与细胞分裂的有6个,还有一部分磷酸化蛋白与调控生长发育的激素的响应有关,除此之外,还鉴定到3个与果实的成熟软化相关的磷酸化蛋白。另外,一部分蛋白有多种磷酸化修饰方式,其中,SNF1相关蛋白激酶β亚基有3种磷酸化修饰,且其磷酸化水平各不相同。【结论】同种蛋白可能同时存在不止一种磷酸化修饰方式。不同的磷酸化修饰方式的作用不尽相同。不同的时期占主导地位的修饰方式也不尽相同。磷酸化蛋白不仅参与转录调节和细胞分裂分化,还参与果实发育过程中对植物激素的响应和糖的代谢积累,甚至参与果实成熟软化的调节。另外,101308592和470122684可能参与果实生长发育的调控。总之,蛋白磷酸化修饰在草莓果实生长发育过程中起重要的调控作用。     

冰温贮藏对羊肉中蛋白质磷酸化水平的影响

【目的】通过测定冰温贮藏及冷藏过程中肌肉肌浆蛋白与肌原纤维蛋白的磷酸化水平,分析冰温贮藏对蛋白质磷酸化水平的影响,为肉类冰温贮藏品质调控提供理论依据。【方法】选取大尾寒羊与小尾寒羊杂交公羊的背最长肌于冷藏和冰温条件下成熟,分别在0.5 h、12 h、24 h、3 d、5 d、9 d取样测定蛋白激酶活性、肌浆蛋白与肌原纤维蛋白的磷酸化水平。采用蛋白激酶活性测定试剂盒、运用酶联免疫的方法检测各处理组蛋白激酶的活性随贮藏时间的变化;通过SDS-PAGE电泳、荧光染色的方法得到全蛋白染色与磷酸化染色的肌浆蛋白与肌原纤维蛋白条带,运用Quantity One软件分析各处理组各处理时间的肌浆蛋白及肌原纤维蛋白的磷酸化水平。【结果】贮藏初期(0.5—12 h),冰温组蛋白激酶活性显著升高(P0.05),冷藏组蛋白激酶活性则显著降低(P0.05),贮藏12 h—9 d过程中,冰温组蛋白激酶活性均显著高于冷藏组(P0.05)。两处理组蛋白激酶活性均随贮藏时间的延长而逐渐降低,且冰温组蛋白激酶活性均显著高于冷藏组(P0.05)。对肌浆蛋白染色效果图中分布于15—250 k Da的17个蛋白条带逐一进行分析,结果表明贮藏温度极显著影响肌浆蛋白各蛋白条带的磷酸化水平(P0.001)。冷藏组肌浆蛋白整体磷酸化水平先升高后降低,贮藏第3天达到最大值,冰温组肌浆蛋白整体磷酸化水平先降低后升高,最小值出现在贮藏第24天,贮藏第12天至3天时,冷藏组肌浆蛋白整体磷酸化水平高于冰温组(P0.05),贮藏第9天时,冰温组肌浆蛋白整体磷酸化水平高于冷藏组(P0.05)。对肌原纤维蛋白染色效果图中分布于15—250 k Da的20个蛋白条带逐一进行分析,结果表明不同贮藏温度对所有肌原纤维蛋白条带的磷酸化水平均产生极显著影响作用(P0.001)。两处理组肌原纤维蛋白整体磷酸化水平均随贮藏时间的延长呈现先升高后降低的趋势,冷藏组与冰温组最大值分别出现在24 h和12 h,贮藏初期(0.5—12 h),两处理组间肌原纤维蛋白整体磷酸化水平无显著差异(P0.05),贮藏24 h至贮藏期结束,冷藏组肌原纤维蛋白整体磷酸化水平始终高于冰温组(P0.05)。【结论】冰温贮藏通过影响蛋白激酶的活性来调控蛋白质的磷酸化水平,进而通过影响糖酵解途径、肌肉收缩及骨架蛋白降解来间接调控肉品质。     

Myosin rod phosphorylation and the catch state of molluscan muscles

"Catch" is a prolonged state of tension in molluscan smooth muscles shown by mechanical measurements to be associated with the level of protein phosphorylation. Myosin isolated from these muscles is unusual in being phosphorylated in the rod portion by an endogenous kinase, like certain nonmuscle myosins. These findings suggest that the myosin rod is a target for phosphorylation and that this reaction may control the transition from catch to relaxation.     

ATP regulates the phosphorylation and degradation of myofibrillar proteins in ground ovine muscle

Phosphorylation post-translational modification plays an important role in postmortem muscle quality traits. Adenosine triphosphate (ATP) is an energy source and a key substrate of phosphorylation which provides the phosphatase groups to proteins in the presence of protein kinases. However, in postmortem muscle, the effects of ATP content on phosphorylation are poorly studied. The study investigated the effect of ATP on protein phosphorylation and degradation in postmortem ovine muscle. The ground muscle with/without additional ATP were treated/control groups and stored at 25 and 4°C, respectively. The ATP content led to different changes of pH value between the ATP-treated and control groups. The phosphorylation level of myofibrillar proteins was higher (P<0.05) in ATP-treated group compared to the control group at both temperatures, which suggested that ATP played a vital role in postmortem protein phosphorylation. A slower degradation rate of µ-calpain, desmin and troponin T was observed in the ATP-treated group which showed that there was a negative relationship between ATP level and the degradation of proteins. These observations clearly highlighted the role of ATP on the development of meat quality by regulating the phosphorylation and degradation of myofibrillar proteins in postmortem ovine muscle.     

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.     

杨树苗木蛋白质与内源激素的动态变化研究

[目的]研究杨树苗木蛋白质与内源激素的动态变化。[方法]以1年生杨树无性系797杨（P．deltoides×Peuramericana）苗木为材料，研究其叶片和苗木主干皮层中总蛋白质含量的年动态变化。并用酶联免疫法测定同时期叶片和苗木主干皮层中内源激素的含量变化，分析内源激素与蛋白质含量变化的相关性。[结果]1年生杨树苗木叶片和主干皮层中总蛋白质含量变化趋势相反。春季展叶期间叶片总蛋白质含量较高，皮层含量较低；进入秋冬季节，叶片含量降低，皮层开始逐渐积累蛋白质。内源激素季节性变化起伏波动较大，与蛋白质变化密切相关，并间接地调控杨树营养贮藏蛋白质的降解与合成。[结论]探索植物激素在杨树氮素代谢过程中的调控作用，为杨树丰产栽培提供科学依据。     

乳蛋白磷酸化分析方法研究进展

蛋白质的磷酸化是一种可逆性的蛋白质翻译后修饰,在调节蛋白结构和功能方面起重要作用。乳蛋白质的磷酸化与牛奶的稳定性有关。采用蛋白组学方法和技术,在实现乳蛋白质组成鉴定的同时,又能分析蛋白质的磷酸化修饰位点。文章综述了磷酸化蛋白质的检测和富集分离技术及乳蛋白磷酸化修饰的研究进展。     

Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis

Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.     

着色与非着色苹果品种中磷酸化蛋白质的鉴定与分析

【目的】苹果着色是一个重要的质量性状,构建苹果磷酸化蛋白质组并进行鉴定与分析,了解在苹果着色过程中的蛋白质磷酸化,为进一步阐明苹果着色机理提供参考。【方法】以着色‘嘎拉’和非着色‘金冠’两个品种苹果果皮为试材提取总蛋白,用TiO₂富集磷酸化肽段,对富集到的磷酸化肽段进行高效液相色谱分离,利用质谱鉴定磷酸化肽段,采用Mascot2.2和Proteome Discoverer1.4（thermo）软件进行蛋白质查库鉴定,使用的数据库为Uniprot_Maloideae.fasta。对鉴定到的磷酸化肽段和蛋白质进行分析,找出着色与非着色苹果品种中有差异的磷酸化蛋白质及其修饰位点。【结果】在着色苹果品种中鉴定到1 323个蛋白质、3 339个肽段和225个磷酸化肽段,发现磷酸化蛋白质约200-225种;在非着色苹果品种中鉴定到569个蛋白质、1 152个肽段和128个磷酸化肽段,发现磷酸化蛋白质约117-128种。比较分析表明,43个磷酸化肽段仅在非着色苹果品种中发现,139个磷酸化肽段仅在着色苹果品种中发现,在着色苹果品种中有更多的蛋白质发生了磷酸化,其中质膜H⁺-ATPase在两个苏氨酸位点发生磷酸化,过敏原蛋白和两个蛋白激酶在丝氨酸位点发生磷酸化,3个转录因子、1个钾转运蛋白和1个受体激酶都发生磷酸化。在两个苹果品种鉴定出的蛋白质中,发生蛋白质磷酸化的位点主要是丝氨酸,其次是苏氨酸,在酪氨酸位点发生蛋白质磷酸化很少。蛋白质WD40和花青苷合成途径中的相关酶蛋白C4H、4CL、CHS、CHI、F3′H、FLS、LDOX和UFGT在着色过程中未发生磷酸化。在着色和非着色苹果品种中都存在MYBR转录因子,并且都发生了蛋白质磷酸化。【结论】在着色苹果品种中产生了一些特有的磷酸化蛋白质,其中包括已知与着色相关的质膜H⁺-ATPase,苹果着色可能受到蛋白质磷酸化的影响。