多聚磷酸盐在原核和真核生物中的研究进展

多聚磷酸盐(polyphosphate, poly P)广泛存在于自然界无机环境和生命有机体.细菌学研究显示,poly P可增强细胞抵抗外界恶劣环境的能力,促进严瑾反应(strigent response)和孢子形成,促进生物被膜的形成,提高捕食能力,增强细菌毒力等.对真核生物的研究发现,poly P可以促进正常成骨细胞和成纤维细胞分化成熟,参与胞内钙的贮存与释放,刺激一些肿瘤细胞增殖等.本文从现有的信息入手,推测poly P在神经系统中的功能.     

多聚磷酸盐及其在分枝杆菌中的生理功能

结核病仍然是全球性传染病。缩短疗程的新药和新疫苗是控制结核病的关键。研究分枝杆菌的生理功能有助于实现上述目的。多聚磷酸盐在细菌胁迫应答中发挥重要作用。结核分枝杆菌具有两类多聚磷酸盐代谢酶以控制细胞内多聚磷酸盐的动态平衡:多聚磷酸盐激酶和多聚磷酸酸盐水解酶。本文综述多聚磷酸盐在分枝杆菌中的代谢及其生理功能,以期为研究多聚磷酸盐在结核分枝杆菌中的生理功能提供参考。     

多聚磷酸盐在微生物抗环境胁迫中的作用及机制

抗环境胁迫是微生物提高环境适应性和增加生存机会的一个重要策略,探明微生物抗环境胁迫的过程及分子机制对于了解微生物进化和开发微生物资源具有重要意义。多聚磷酸盐（polyphosphate, polyP）在微生物抗环境胁迫中发挥重要作用。在营养限制条件下,polyP可充当微生物的能源来源和信号分子,增强微生物对低营养环境的适应能力。在微生物应对环境胁迫过程中,polyP可作为蛋白质的伴侣,通过蛋白质修饰改变蛋白质结构使其免受失活,从而维持其功能完整性。polyP具有金属螯合能力,可提高微生物对重金属胁迫的抵抗能力。微生物能通过调节polyP的合成来适应环境pH的改变,调节酸碱胁迫过程中的能量消耗。基于polyP抗环境胁迫的特性,通过转基因技术,把polyP合成相关基因转入到农作物中,可以增加农作物体内polyP含量,从而提高农作物抗环境胁迫的能力。利用含有polyP的微生物处理重金属废水,可极大地提高重金属离子的去除效率。同时,微生物中合成的polyP颗粒也能进一步开发为生物活性产品。因此,polyP在微生物抗胁迫中发挥多样化作用,通过各种分子途径提高微生物对环境胁迫的耐受性。加强poly...     

多聚磷酸盐：不仅是异染粒

无机的多聚磷酸盐（inorganic polyphosphate,polyP）是由3个到几百个磷酸根通过高能磷酸酐键聚合而成的聚合物,具有广泛的生物学功能。然而,polyP的所有发现都是在不同的生物体和模式生物中进行研究产生的,因此,这些结果都各自独立,而不能形成一般性结论,即polyP在生物体中到底起什么样的作用。本文综述了polyP在DNA复制、基因转录、翻译和翻译后的修饰、蛋白质的折叠和降解中所发挥的生物学功能。本文尝试将polyP的各种不相关的生物学功能,从中心法则的角度进行了统一认识,希望能为polyP生物学功能的研究带来新的思考。     

多聚磷酸盐及其代谢酶的研究进展

多聚磷酸盐(polyP)是由几个到几百个无机磷酸盐单体通过高能磷酸键聚合而成的线性多聚体,广泛分布于自然界和生物体.本文总结了polyP在生物体中的重要功能,包括基因表达和调控、DNA的摄取、微生物的运动性、对胁迫和饥饿的应答、病原菌的毒性以及对细胞凋亡、血液凝固、细胞钙化、线粒体功能的调节,需要polyP的酶有内切酶、葡萄糖激酶、NAD激酶和AMP磷酸转移酶等.本文对调控polyP的多聚磷酸盐激酶(polyphosphate kinase,ppk)和外切聚磷酸酶(exopolyphosphatase,PPX )的生化性质和结构也进行了总结.同时,结合我们的研究工作,重点分析了结核分枝杆菌中PPX的同源蛋白和可能的生物化学活性.     

多聚磷酸盐激酶双底物通道腔的理性设计及其在无细胞催化生产谷胱甘肽中的应用

三磷酸腺苷(adenosine triphosphate,ATP)是一种重要的辅助因子,参与许多需能的生物催化反应。多聚磷酸盐激酶(polyphosphate kinases,PPK)由于其底物聚磷酸盐廉价易得,可以为消耗ATP的反应提供能量。本研究选择哈氏噬纤维菌(Cytophaga hutchinsonii)来源的ChPPK,进行了底物谱和耐受性分析,通过分子对接和定点突变,理性改造多聚磷酸盐激酶的双底物通道腔来提高PPK酶的催化活性。与野生型相比,筛选得到突变体ChPPK_K81H-K103V的相对酶活提高了326.7%,同时,双突变扩大了ChPPK的底物利用范围与耐受性,提高了该酶的耐热性与耐碱性。基于该ATP再生系统,本研究偶联谷胱甘肽双功能酶GshAB和ChPPK_K81H-K103V,破细胞后采用无细胞催化生产谷胱甘肽,加入5 mmol/L ATP后,该体系6 h可以生产(25.4±1.9) mmol/L的谷胱甘肽,比突变前的催化体系提高了41.9%。优化无细胞催化体系的缓冲液、裂解液菌体量、补料时间后,无细胞体系可产生(45.2±1.8) mmol/L谷胱甘肽,底物l-半胱氨酸的转化率达到90.4%。提高ChPPK生产ATP的能力,可有效增强底物的转化率,降低催化成本,实现了无细胞催化生产谷胱甘肽的高产量、高转化率与高经济价值的统一。本研究提供了一种绿色高效的ATP再生系统,可为消耗ATP的生物催化反应平台提供可持续动力。     

生物细胞中颗粒状多聚磷酸盐细胞器的结构与功能

多聚磷酸盐(polyphosphat,poly P)是一种由数十个或上百个磷酸根聚合而成的生物大分子,以颗粒状、胶体状和溶解状等多种状态存在于各类生物细胞中。生物体中的poly P能够通过分解提供能量;鳌合金属离子来调节细胞内渗透压,维持质膜稳定;与蛋白质或DNA结合稳定其结构,减轻细胞应激损伤。颗粒状多聚磷酸盐细胞器主要指细胞中用于贮存颗粒状poly P、金属阳离子以及蛋白质、氨基酸和少量水等物质的细胞器。在寄生虫细胞中颗粒状聚磷细胞器常称为酸性钙体,而细菌或者其他微生物细胞中则称为异染颗粒,但是随着研究的不断深入,发现酸性钙体和异染颗粒都具有相似的结构特征,遂将其统一定义为颗粒状多聚磷酸盐细胞器。颗粒状聚磷细胞器的发现拓展了生物共同祖先(last universal common ancestor,LUCA)的学说,丰富了原核生物细胞器认知,我们相信该细胞器在生命起源、抗环境胁迫、生物互作和代谢调控等方面具有重要功能,在疾病治疗以及磷生物地球化学循环过程中发挥重要作用。     

细菌基因在哺乳动物细胞中的表达

纯系繁殖的编码黄嘌呤-鸟嘌呤磷酸核糖基转移酶的大肠杆菌(E.coli)基因,同一系列的由SV40DNA衍生的不同载体构成重组体DNA。用这样的重组体DNA转染培养的猴肾细胞,结果产生的转化体能合成大量的易于测定的大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖基转移酶。而且,把这种细菌基因引进到嘌呤核苷酸合成特性缺陷的人莱许-奈恩(Lesch-Nyhan)细胞,这些细胞的此种生理缺陷便得到了纠正。     

哺乳动物滋养层细胞在胚胎植入中的作用

胚胎植入是哺乳动物生殖的关键环节,是一个非常复杂的过程.在胚胎植入过程中,多种着床相关因子、激素在母体-胚胎之间进行多重作用,引发复杂的生理作用,从而完成胚胎着床.在母体-胚胎界面上,胎源性滋养层细胞与母体子宫内膜细胞在信号联系（妊娠识别）和组织紧密连接（胚胎植入）过程中起着决定性作用,尤其是胚源性滋养层细胞,在胚胎植入过程中起主导作用.本文通过对滋养层细胞在胚胎植入中的作用的阐述,为进一步阐明胚胎植入的分子机制提供思路.     

Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography

Polyphosphate is ubiquitous and has a variety of biochemical functions. Among polyphosphate quantification methods, an enzymatic assay using Escherichia coli polyphosphate kinase (PPK), in which polyphosphate is converted to adenosine 5'-triphosphate and quantified by luciferase assay, is the most specific and most sensitive. However, chain-length specificity of the assay has not been analyzed in detail so far. Ion chromatography equipped with an on-line hydroxide eluent generator enabled us to analyze polyphosphate up to 50 inorganic phosphate (P(i)) residues, and we employed this method to investigate the chain-length specificity of PPK in this study. Several fractions of short-chain polyphosphate were prepared by electrophoresis, and the chain-length distribution was analyzed before and after 1-6 h PPK reaction by ion chromatography. Polyphosphates longer than 23 P(i) residues were processed by PPK completely after 1 h incubation, but complete processing of those between 11 and 22 P(i) residues required 6h incubation. Limited processing of polyphosphates of 10 P(i) residues or shorter were observed even after 6h incubation. Metachromasy of Toluidine blue O, an alternative method for polyphosphate quantification, showed broader chain-length specificity although it was not as sensitive as the enzymatic assay. Combination of these two methods would be practically applicable to analysis of polyphosphate dynamics in living organisms.     

Crystallization and characterization of polyphosphate kinase from Escherichia coli

Linear polyphosphate chains have been found to play a key role in bacterial responses to stresses and nutritional depletion, and are necessary for host infection of various pathogens. Polyphosphate kinase (PPK) is a critical enzyme responsible for polyphosphate synthesis in bacteria. PPK knockout mutations in several Gram-negative pathogens identify PPK as an ideal drug target for the development of a new class of antibacterial drugs. To reveal the catalytic mechanism and provide a structural basis for drug discovery, we have purified and crystallized full-length Escherichia coli PPK and its complex with AMP-PNP. The crystals diffract to a resolution of 2.5A and belong to the space group P4(2)2(1)2 with unit-cell parameters a=152.0, b=152.0, and c=150.0 A. Crystal structure of PPK is being determined by the Se-Met MAD experiment.     

The amino acid composition of mammalian and bacterial cells

Summary High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, andEscherichia coli andStaphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions ofEscherichia coli andStaphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.     

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported.     

Different chain length specificity among three polyphosphate quantification methods

Polyphosphate is ubiquitous among living organisms and has a variety of biochemical functions. Arbuscular mycorrhizal fungi have been known to accumulate polyphosphate as a key compound for their function. However, an enzymatic assay using polyphosphate kinase (PPK) reverse reaction, in which polyphosphate is converted to adenosine triphosphate (ATP) and quantified by luciferase assay, failed to detect accumulation of polyphosphate in some mycorrhizal root. When yeast exopolyphosphatase (PPX) was applied to these samples, a much higher polyphosphate level was detected than when the PPK assay was applied. Detailed analysis of substrate chain length specificity of these methods using polyphosphate chain length standards revealed that the PPX method was the most appropriate to detect short-chain polyphosphate. The average chain length of the shortest polyphosphate fraction that could be quantified with more than 50% efficiency was 3 for the PPX method and 38 for the PPK method. It was also suggested that the ratio of the PPK value to the PPX value may be useful as a simple and relative index to compare polyphosphate chain length distribution in different samples.     

A novel mammalian glucokinase exhibiting exclusive inorganic polyphosphate dependence in the cell nucleus

BackgroundHexokinase and glucokinase enzymes are ubiquitously expressed and use ATP and ADP as substrates in mammalian systems and a variety of polyphosphate substrates and/or ATP in some eukaryotic and microbial systems. Polyphosphate synthesising or utilizing enzymes are widely expressed in microbial systems but have not been reported in mammalian systems, despite the presence of polyphosphate in mammalian cells. Only two micro-organisms have previously been shown to express an enzyme that uses polyphosphate exclusively.MethodsA variety of experimental approaches, including NMR and NAD-linked assay systems were used to conduct a biochemical investigation of polyphosphate dependent glucokinase activity in mammalian tissues.ResultsA novel mammalian glucokinase, highly responsive to hexametaphosphate (HMP) but not ATP or ADP as a phosphoryl donor is present in the nuclei of mammalian hepatocytes. The liver enzyme exhibited sigmoidal kinetics with respect to glucose with a S_0.5 of 12 mM, similar to the known kinetics of mammalian ATP-glucokinase. The Km for HMP (0.5 mM) was also similar to that of phosphoryl donors for mammalian ATP-glucokinases. The new enzyme was inhibited by several nucleotide phosphates.ConclusionsWe report the discovery of a polyphosphate-dependent enzyme system in mammalian cells with kinetics similar to established ATP-dependent glucokinase, also known to have a nuclear location. The kinetics suggest possible regulatory or redox protective roles.General significanceThe role of polyphosphate in mammalian systems has remained an enigma for decades, and the present report describes progress on the significance of this compound in intracellular metabolism in mammals.     

Sodium ionic and gating currents in mammalian cells

Ionic and gating currents from voltage-gated sodium channels were recorded in mouse neuroblastoma cells using the path-clamp technique. Displacement currents were measured from whole-cell recordings. The gating charge displaced during step depolarizations increased with the applied membrane potential and reached saturating levels above 20 mV Prolonged large depolarizations produced partial immobilization of the gating charge, and only about one third of the displaced charge was quickly reversed upon return to negative holding potentials. The activation and inactivation properties of macroscopic sodium currents were characterized by voltage-clamp analysis of large outside-out patches and the single-channel conductance was estimated from nonstationary noise analysis. The general properties of the sodium channels in mouse neuroblastoma cells are very similar to those previously reported for various preparations of invertebrate and vertebrate nerve cells. Offprint requests to: O. Moran     

Multigene delivery in mammalian cells: Recent advances and applications

Systems for multigene delivery in mammalian cells, particularly in the context of genome engineering, have gained a lot of attention in biomolecular research and medicine. Initially these methods were based on RNA polymerase II promoters and were used for the production of protein complexes and for applications in cell biology such as reprogramming of somatic cells to stem cells. Emerging technologies such as CRISPR/Cas9-based genome engineering, which enable any alteration at the genomic level of an organism, require additional elements including U6-driven expression cassettes for RNA expression and homology constructs for designed genome modifications. For these applications, systems with high DNA capacity, flexibility and transfer rates are needed. In this article, we briefly give an update on some of recent strategies that facilitate multigene assembly and delivery into mammalian cells. Also, we review applications in various fields of biology that rely on multigene delivery systems.