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.     

Redox regulation of germline and vulval development in Caenorhabditis elegans

In vitro studies have indicated that reactive oxygen species (ROS) and the oxidation of signaling molecules are important mediators of signal transduction. We have identified two pathways by which the altered redox chemistry of the clk-1 mutants of Caenorhabditis elegans acts in vivo on germline development. One pathway depends on the oxidation of an analog of vertebrate low density lipoprotein (LDL) and acts on the germline through the Ack-related tyrosine kinase (ARK-1) kinase and inositol trisphosphate (IP3) signaling. The other pathway is the oncogenic ras signaling pathway, whose action on germline as well as vulval development appears to be modulated by cytoplasmic ROS.     

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.     

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.     

Autophagy genes are essential for dauer development and life-span extension in C. elegans

Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.     

人参皂苷Rb3对肝糖异生作用机理的研究

为了探讨人参皂苷Rb3在降低血糖方面的分子调控机制,利用HepG2细胞为研究材料,系统分析了人参皂苷Rb3对肝糖异生关键酶PEPCK、G6Pase和转录因子FOXO1、HNF4α的影响。结果表明,人参皂苷Rb3可以显著抑制HepG2细胞肝糖异生途径关键转录因子FOXO1、HNF4α蛋白表达,从而抑制PEPCK和G6Pase酶活性及糖异生作用,该作用能够被AMPK抑制剂Compound C部分阻断,推测人参皂苷Rb3抑制肝糖异生作用是通过激活AMPK信号通路实现。AMPK信号转导通路作为重要的糖脂代谢靶点,在糖尿病及相关代谢类疾病的调控中发挥着重要的作用,为探讨人参皂苷Rb3治疗糖尿病的作用机制提供了新的理论依据。     

Intracellular calcium release mediated by sphingosine derivatives generated in cells

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.     

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.     

TIMP2 promotes intramuscular fat deposition by regulating the extracellular matrix in chicken

The interaction between myocytes and intramuscular adipocytes is a hot scientific topic.  Using a co-culture system, this study aims to investigate the regulation of intramuscular fat deposition in chicken muscle tissue through the interaction between myocyte and adipocyte and identify important intermediary regulatory factors.  Our proteomics data showed that the protein expression of tissue inhibitor of metalloproteinases 2 (TIMP2) increased significantly in the culture medium of the co-culture system, and the content of lipid droplets was more in the co-culture intramuscular adipocytes.  In addition, TIMP2 was significantly upregulated (P<0.01) in muscle tissue of individuals with high intramuscular fat content.  Weighted gene co-expression network analysis revealed that TIMP2 was mainly involved in the extracellular matrix receptor interaction signaling pathway and its expression was significantly correlated with triglyceride, intramuscular fat, C14:0, C14:1, C16:0, C16:1, and C18:1n9C levels.  Additionally, TIMP2 was co-expressed with various representative genes related to lipid metabolism (such as ADIPOQ, SCD, ELOVL5, ELOVL7, and LPL), as well as certain genes involved in extracellular matrix receptor interaction (such as COL1A2, COL4A2, COL5A1, COL6A1, and COL6A3), which are also significantly upregulated (P<0.05 or P<0.01) in muscle tissue of individuals with high intramuscular fat content.  Our findings reveal that TIMP2 promotes intramuscular fat deposition in muscle tissue through the extracellular matrix receptor interaction signaling pathway.     

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.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.     

植物体内肌醇磷脂代谢与渗透胁迫信号转导

磷酸肌醇代谢在生物感受胞外刺激及信号转导中起着重要的作用。在此,介绍了植物体内的肌醇磷脂代谢途径,PI-PLC/IP3途径和磷酸肌醇激酶与渗透胁迫信号转导之间的联系,以及肌醇磷脂代谢参与的渗透胁迫信号转导与ABA之间的关系。     

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.     

烟草WRKY-R1基因的克隆及瞬时表达分析

以烟草根尖组织cDNA为模板,RT-PCR结合电子克隆得到NtWRKY-R1基因的完整ORF,序列分析显示,NtWRKY-R1具有WRKY转录因子家族典型的WRKYGQK保守结构域及C2H2的锌指结构,属于WRKY转录因子家族第Ⅱ类。采用生物信息学方法对NtWRKY-R1蛋白的理化性质、进化关系和磷酸化位点进行预测和分析,结果表明,NtWRKY-R1与茄科植物保守结构域的同源性及系统进化关系最近;磷酸化位点分析显示该蛋白可能通过磷酸化作用修饰,进而对其相应的代谢活动进行调节。通过构建真核表达载体、建立瞬时表达分析体系,结果显示,NtWRKY-R1基因的过表达导致JA信号途径标记基因PDF1.2及烟碱合成关键酶基因PMT1表达量降低,推测NtWRKY-R1基因影响JA信号途径,进而调控烟碱合成。     

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。     

孕酮对绵羊输卵管上皮细胞β-防御素-1 mRNA表达的影响

旨在研究孕酮(P4)对绵羊输卵管上皮细胞内β-防御素-1( SBD-1) mRNA基因表达的影响及可能参与的信号通路,利用10~(-8) mol/L P4培养绵羊输卵管上皮细胞2 h、6 h、12 h、24 h、48 h,利用RT-qPCR技术检测 SBD-1 mRNA的表达变化,选取P4诱导 SBD-1 mRNA表达的最佳时间。分别添加P4核受体阻断剂RU-486、蛋白激酶C (PKC)阻断剂H-7、蛋白激酶A (PKA)阻断剂H-89干预绵羊输卵管上皮细胞1h,再使用P4处理细胞。结果显示,用10~(-8)mol/L P4诱导绵羊输卵管上皮细胞6 h, SBD-1 mRNA表达显著升高;添加RU-486和H-89显著抑制P4诱导的 SBD-1 mRNA的表达;添加H-7后 SBD-1 mRNA的表达量无显著性差异。以上结果表明,P4诱导的绵羊输卵管上皮细胞中 SBD-1 mRNA的表达通过孕酮核受体(PR)和PKA信号通路介导,与PKC信号通路激活无关。     

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway

Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.