A spatial-temporal model of cell activation

A spatial-temporal model of calcium messenger function is proposed to account for sustained cellular responses to sustained stimuli, as well as for the persistent enhancement of cell responsiveness after removal of a stimulus, that is, cellular memory. According to this model, spatial separation of calcium function contributes to temporal separation of distinct phases of the cellular response. At different cellular sites, within successive temporal domains, the calcium messenger is generated by different mechanisms and has distinct molecular targets. In particular, prolonged cell activation is brought about by the interaction of calcium with another spatially confined messenger, diacylglycerol, to cause the association of protein kinase C with the plasma membrane. Activity of the membrane-associated protein kinase C is controlled by the rate of calcium cycling across the plasma membrane. In some instances, a single stimulus induces both protein kinase C activation and calcium cycling and thus causes prolonged activation; but in others, a close temporal association of distinct stimuli brings about cell activation via interaction of these intracellular messengers. Persistent enhancement of cell responsiveness after removal of stimuli is suggested to be due to the continued association, or anchoring, of protein kinase C to the membrane.     

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.     

Stop-transfer regions do not halt translocation of proteins into chloroplasts

Protein targeting in eukaryotic cells is determined by several topogenic signals. Among these are stop-transfer regions, which halt translocation of proteins across the endoplasmic reticulum membrane. Two different stop-transfer regions were incorporated into precursors for a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both chimeric proteins were imported into chloroplasts and did not accumulate in the envelope membranes. Thus, the stop-transfer signals did not function during chloroplast protein import. These observations support the hypothesis that the mechanism for translocation of proteins across the chloroplast envelope is significantly different from that for translocation across the endoplasmic reticulum membrane.     

Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier

Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.     

A G protein directly regulates mammalian cardiac calcium channels

A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.     

Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice

Caveolae are plasma membrane invaginations that may play an important role in numerous cellular processes including transport, signaling, and tumor suppression. By targeted disruption of caveolin-1, the main protein component of caveolae, we generated mice that lacked caveolae. The absence of this organelle impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone. In addition, the lungs of knockout animals displayed thickening of alveolar septa caused by uncontrolled endothelial cell proliferation and fibrosis, resulting in severe physical limitations in caveolin-1-disrupted mice. Thus, caveolin-1 and caveolae play a fundamental role in organizing multiple signaling pathways in the cell.     

The function of a leader peptide in translocating charged amino acyl residues across a membrane

Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous bacteriophage Pf3--which infects Pseudomonas aeruginosa--is 44 amino acids in length and has the same basic structure as the coat protein of bacteriophage M13, which infects Escherichia coli. However, unlike the Pf3 coat protein, the M13 coat protein is synthesized as a precursor (procoat) with a typical leader (signal) sequence, which is cleaved after membrane insertion. Nevertheless, when the gene encoding the Pf3 coat protein is expressed in E. coli, the protein is translocated across the membrane. Hybrid M13 and Pf3 coat proteins were constructed in an attempt to understand how the Pf3 coat protein is translocated without a leader sequence. These studies demonstrated that the extracellular regions of the proteins determined their cellular location. When three charged residues in this region were neutralized, the leader-free M13 coat protein was also inserted into the membrane. Differences in the water shell surrounding these residues may account for efficient membrane insertion of the protein without a leader sequence.     

Regulation of a heart potassium channel by protein kinase A and C

The enzymes adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (protein kinase A) and protein kinase C regulate the activity of a diverse group of cellular proteins including membrane ion channel proteins. When protein kinase A was stimulated in cardiac ventricular myocytes with the membrane-soluble cAMP analog 8-chlorphenylthio cAMP (8-CPT cAMP), the amplitude of the delayed-rectifier potassium current (IK) doubled when recorded at 32 degrees C but was not affected at 22 degrees C. In contrast, modulation of the calcium current (ICa) by 8-CPT cAMP was independent of temperature with similar increases in ICa occurring at both temperatures. Stimulation of protein kinase C by phorbol 12,13-dibutyrate also enhanced IK in a temperature-dependent manner but failed to increase ICa at either temperature. Thus, cardiac delayed-rectifier potassium but not calcium channels are regulated by two distinct protein kinases in a similar temperature-dependent fashion.     

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.     

基于4D label-free技术的水稻成熟种子蛋白质组学研究

【目的】从蛋白水平揭示水稻种子成熟的分子基础，探究调控水稻种子成熟的关键蛋白和代谢通路。【方法】选用授粉后30 d的成熟水稻种子，利用4D label-free定量蛋白质组学进行质谱鉴定，通过生物信息学技术分析蛋白的亚细胞定位、结构域、GO注释和KEGG通路注释。【结果】总共鉴定了3 484个种子成熟期的蛋白，相对分子质量大多在10 000～100 000之间，主要分布于细胞质、细胞核、叶绿体、线粒体和质膜上；结构域主要涉及蛋白质翻译的RNA识别基序和蛋白磷酸化修饰的蛋白激酶结构域；GO分析表明，成熟种子的蛋白主要参与了细胞过程和代谢过程，主要涉及催化活性和结合等功能，大多分布在细胞、细胞组分、细胞器和细胞膜等部位；KEGG分析发现，蛋白主要富集在核糖体、内质网中的蛋白质加工、氧化磷酸化和糖酵解等途径，推测蛋白质的翻译、加工和能量代谢是水稻种子成熟期的主要分子事件；进一步鉴定了脱落酸(Abscisic acid,ABA)信号和吲哚乙酸(Indoleacetic acid,IAA)代谢的相关蛋白，同时也发现了NAC(NAM、ATAF1/2和CUC2)家族的转录因子。【结论】贮藏物质的积累...     

鸡卵泡膜细胞中膜联蛋白A2互作细胞蛋白的筛选及其功能分析

【目的】从鸡卵泡膜细胞蛋白中筛选和鉴定与膜联蛋白A2（ANXA2）相互作用的细胞蛋白并进行功能分析,为深入研究ANXA2调控鸡卵泡发育的作用机制提供理论依据。【方法】制备开产后30周龄贵州黄鸡的卵泡膜细胞,提取卵泡膜总蛋白后利用His Pull-Down联合质谱技术（LC-MS/MS）从卵泡膜细胞中筛选出与鸡ANXA2互作的细胞蛋白,然后通过GO数据库和KEEG数据库分别进行GO功能富集分析及KEEG信号通路注释分析,并利用STRING Version 11.0绘制蛋白互作网络图。【结果】通过His Pull-Down联合LC-MS/MS共鉴定获得41个鸡ANXA2互作细胞蛋白,GO功能富集分析发现这些互作细胞蛋白在分子功能、生物学进程和细胞组成均发挥作用。其中,在分子功能方面主要涉及蛋白结合（占58.06%）、催化活性（占19.35%）、核糖体结构（占16.13%）及细胞骨架结构组成（占6.45%）,在生物学进程方面主要参与细胞骨架（占19.35%）、刺激反应（占19.35%）、翻译（占16.13%）、代谢过程（占12.90%）、细胞迁移（占12.90%）、蛋白折叠（占9.68%）和蛋白运输（占9.68%）,而细胞组分显示以定位于细胞膜的蛋白为主（占32.26%）。鸡ANXA2蛋白互作细胞蛋白参与的KEEG信号通路主要有应激反应、代谢、翻译、信号转导、免疫系统和蛋白定位等。鸡ANXA2互作细胞蛋白互作网络可分为3条,即CNN2-FN1-MYH9-MYH10-ACTN1-CSRP1、ANXA1-ANXA2-ENO1-PRDX4-GPI-ATP5B-PRDX3-HSPA8-TUBB2A和CCT7-CCT4-GNB2L1-ATP5A1-RPS3-RPS3A-RPL23A-RPL22-RPS7;互作细胞蛋白间存在复杂的互作关系,其中又以膜联蛋白A1（ANXA1）与烯醇化酶-1（ENO1）及ANXA2的互作关系最明显。【结论】鸡ANXA2互作细胞蛋白主要参与细胞骨架形成、应对刺激和翻译等生物学过程,涉及应激反应、代谢、翻译、信号转导、免疫及蛋白定位等信号通路。其中,PRDX3、PRDX4、MYH9和TCSC可能通过与ANXA2蛋白相互作用而参与鸡卵巢相关疾病的发生,而ANXA1与ANXA2相互作用可能在鸡卵泡的发育及排卵过程中发挥重要调节作用。     

Transduction of receptor signals by beta-arrestins

The transmission of extracellular signals to the interior of the cell is a function of plasma membrane receptors, of which the seven transmembrane receptor family is by far the largest and most versatile. Classically, these receptors stimulate heterotrimeric G proteins, which control rates of generation of diffusible second messengers and entry of ions at the plasma membrane. Recent evidence, however, indicates another previously unappreciated strategy used by the receptors to regulate intracellular signaling pathways. They direct the recruitment, activation, and scaffolding of cytoplasmic signaling complexes via two multifunctional adaptor and transducer molecules, beta-arrestins 1 and 2. This mechanism regulates aspects of cell motility, chemotaxis, apoptosis, and likely other cellular functions through a rapidly expanding list of signaling pathways.     

Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.     

Uncoupling translocation from translation: implications for transport of proteins across membranes

The segregation of secretory proteins into the cisternae of the endoplasmic reticulum (ER) is normally tightly coupled to their synthesis. This feature distinguishes their biogenesis from that of proteins targeted to many other organelles. In the examples presented, translocation across the ER membrane is dissociated from translation. Transport, which is normally cotranslational, may proceed in the absence of chain elongation. Moreover, translocation across the ER membrane does not proceed spontaneously since, even in the absence of protein synthesis, energy substrates are required for translocation. These conclusions have been extended to the cotranslational integration of newly synthesized transmembrane proteins.     

猪BMSCs成脂分化中细胞膜钙离子通道、钙敏感受体及成脂定向相关基因表达研究

【目的】研究猪骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)定向分化为脂肪细胞过程中细胞膜上钙离子通道、钙敏感受体(Calcium-sensing receptor,Ca SR)基因及成脂定向相关基因的表达。【方法】从5~7日龄仔猪骨髓中分离纯化出猪BMSCs,诱导猪BMSCs成脂分化。油红O法和三酰甘油法检测细胞分化聚酯状况。在成脂分化不同时间(0、1、2、5和10 d)收集细胞,利用荧光定量PCR检测锌指蛋白423(Zinc finger protein423,Zfp423)、脂肪前体细胞因子(Preadipocyte factor 1,Pref-1)、骨形态发生蛋白2(Bone morphogenetic protein 2,BMP2)、骨形态发生蛋白4(Bone morphogenetic protein 4,BMP4)、细胞膜钙离子通道及Ca SR基因的mRNA表达变化。【结果】油红O染色和三酰甘油检测结果表明,成功诱导猪BMSCs成脂分化;定量PCR结果显示,在猪BMSCs成脂分化第5天,成脂定向标志基因Zfp423、脂肪前体细胞标志基因Pref-1及促进成脂分化基因BMP2、BMP4的mRNA相对表达量显著提高(P0.05),说明第5天是猪BMSCs成脂定向形成脂肪前体细胞的关键时期;同时,细胞膜上的电压门控钙离子通道亚基电压依赖型α/δ亚型1(Voltage-dependentalpha-2/delta subunit 1,CACNA2D1)、钙释放激活钙通道调节分子1(Calciumr elease-activated calcium channel modulator 1,Orai1)、瞬时受体电位通道传统型1(Transient receptor potential canonical type 1,TRPC1)、瞬时受体电位通道M型7(Transient receptor potential melastatin 7,TRPM7)、瞬时受体电位通道香草素受体亚型1(Transient receptor potential vanilloid receptor1,TRPV1)基因和Ca SR基因在诱导成脂第5天mRNA相对表达量也显著提高(P0.05),提示细胞膜钙离子通道及Ca SR基因可能参与了猪BMSCs成脂分化过程。【结论】揭示了猪BMSCs成脂分化过程中细胞膜钙离子通道、钙敏感受体及成脂定向相关基因的表达模式。     

基于TMT蛋白组学及生物信息学分析牦牛抗冻差异蛋白

【目的】从蛋白水平阐明牦牛抗寒性能机理,并进一步从营养学角度提高其代谢性能,为牦牛有效抵御外界恶劣气候条件提供科学依据。【方法】应用TMT蛋白组学技术对寒冷季节（1月）和温暖季节（8月）的牦牛抗冻蛋白进行挖掘,并对鉴定到的牦牛抗冻蛋白进行亚细胞定位、结构域、GO功能富集、KEGG信号通路注释、蛋白相互作用等生物信息学分析。【结果】从牦牛耳组织中共鉴定获得21856个肽段（Peptide）,其中特有肽段（Unique peptide）序列为18452个,定量获得4519个蛋白,最终筛选出144个差异蛋白,其中上调蛋白89个、下调蛋白55个。144个牦牛抗冻差异蛋白亚细胞定位到7个条目上,分别是细胞核蛋白56个、细胞质蛋白51个、质膜蛋白24个、细胞外蛋白23个、线粒体蛋白18个、细胞骨架蛋白1个和溶酶体蛋白1个;共鉴定到194个结构域。GO功能富集分析结果显示,生物过程主要富集到细胞过程蛋白79个、代谢过程蛋白70个和生物调控蛋白42个等,分子功能主要富集到结合功能蛋白75个和催化活性蛋白64个等,细胞组分主要富集到细胞部分蛋白89个和细胞蛋白89个等。144个牦牛抗冻差异蛋白在KEGG数据库中注释到205条KEGG信号通路,主要涉及核糖体、氮代谢、胞质DNA感受、动物体内生热作用、氧化磷酸化、白细胞介素-17及钙离子信号等通路。牦牛抗冻差异蛋白相互作用网络分析发现L8IHE5的关联度最高,且冷诱导RNA结合蛋白（CIRP）和HSP70结合蛋白在蛋白相互作用网络中具有更多的相互作用关系。【结论】基于TMT蛋白组学对牦牛抗冻差异蛋白进行挖掘,结果鉴定获得144个抗冻差异蛋白（上调蛋白89个,下调蛋白55个）,其中CIRP和HSP70在冷应激条件下呈上调趋势,能促使牦牛肌体适应低温环境,可作为牦牛抗冻性育种的候选分子标记。     

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.     

Integrins regulate Rac targeting by internalization of membrane domains

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.     

Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast

Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.