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。     

植物细胞中蛋白质向叶绿体转运的研究进展

植物细胞中叶绿体的功能主要依赖于叶绿体蛋白, 大部分叶绿体蛋白由核基因组编码, 在细胞质中合成并经过正确的分选后, 通过叶绿体外膜上的Toc复合体和/或内膜上的Tic复合体转运到叶绿体的不同部位。该文主要综述可能参与叶绿体蛋白分选的胞质因子以及Toc和Tic组分如何参与叶绿体蛋白转运的研究进展。     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

多聚磷酸相关蛋白结构及生物学功能

多聚磷酸（polyphosphate,polyP）是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶（polyphosphate kinase,PPK）催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶（exopolyphosphatase,PPX）、内切聚磷酸酶（endopolyphosphatase,PPN）能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD）,使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate,NADP）.多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

多聚磷酸盐：菌体内多功能调控子和环境压力守护者

多聚磷酸盐(polyP)是由几个到上百个磷酸基团组成并由高能磷酸酐键连接的链状无机化合物,广泛地存在于所有生命体中。研究表明,polyP在不适宜的生存条件下会在菌体内聚集,使菌对环境压力有极强的耐受性,它与其相关的酶PPK、PPX、ppp Gpp等一起作出应激反应;在寡营养环境下,polyP作为能量和磷源库,和Rpo S、Lon蛋白及σ因子等一起参与一系列饥饿压力应答,这种存活机制与寡营养菌的生活方式类似。这些反应得益于polyP链状的灵活性和高荷电性,但是其具体的调控机理仍很少为人所知。本文阐述了以4',6-二脒基-2-苯基吲哚(4',6-Diamidino-2-phenylindole,DAPI)为基础的荧光染色方法进行菌体内polyP的定性(荧光或电子显微镜)和定量(荧光分光光度计),重点归纳分析了以polyP为核心的关于细菌适应恶劣生存环境的代替机制。     

高等植物Na+吸收、转运及细胞内Na+稳态平衡研究进展

盐胁迫是影响农业生产的重要环境因素之一。本文对植物Na 吸收的机制和途径、Na 在植物体内的长距离转运以及细胞内Na 稳态平衡的研究进展进行了概述。参与植物Na 吸收与转运的蛋白和通道可能包括HKT、LCT1、AKT和NSCC等。其中,HKT是植物体内普遍存在的一类转运蛋白,能够介导Na 的吸收,其结构中的带电氨基酸残基对于其离子选择性有着非常明显的影响。LCT1是从小麦中发现的一类能够介导低亲和性阳离子吸收的蛋白,然而在典型的土壤Ca2 浓度下LCT1并不能发挥吸收Na 的功能。AKT家族的成员在高盐环境下可能也参与了Na 的吸收。目前虽然还没有克隆到编码NSCC蛋白的基因,但是NSCC作为植物吸收Na 的主要途径的观点已被广泛接受。SOS1和HKT参与了Na 在根部与植株地上部的长距离转运过程,它们在木质部和韧皮部的Na 装载和卸载中发挥重要作用,从而影响植物的抗盐性。另外,由质膜Na /H 逆向转运蛋白SOS1、蛋白激酶SOS2以及Ca2 结合蛋白SOS3组成的SOS复合体对细胞的Na 稳态具有重要的调节作用,单子叶和双子叶植物之间的这种调节机制在结构和功能上具有保守性。SOS复合体与其它位于质膜或液泡膜上的Na /H 逆向转运蛋白以及H 泵一起调节着细胞的Na 稳态。     

Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (P_i), as the preferred P source, have evolved complex systems to survive during P_i starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to P_i. The evidence that the chemotactic response to P_i (P_i taxis) was observed only with cells grown in P_i-limiting medium suggests that P_i taxis plays an important role in scavenging P_i residues under conditions of P_i starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when P_i is added to cells previously subjected to P_i starvation stress. Since polyP can serve as a P source during P_i starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during P_i starvation. In the present review, we summarize our current knowledge on regulation of bacterial P_i taxis and polyP accumulation in response to P_i starvation stress.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

Progress Toward Understanding the Distribution of Accumulibacter Among Full-Scale Enhanced Biological Phosphorus Removal Systems

This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P_i) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P_i and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40–69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi

We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N‐terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two‐electrode voltage clamp showed that TcPho91 is a low‐affinity transporter with a K_m for P_i in the millimolar range, and sodium‐dependency. Epimastigotes overexpressing TcPho91‐green fluorescent protein have significantly higher levels of pyrophosphate (PP_i) and short‐chain polyphosphate (polyP), suggesting accumulation of P_i in these cells. Moreover, when overexpressing parasites were maintained in a medium with low P_i, they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N‐terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PP_i and short‐chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in P_i homeostasis in T. cruzi.     

Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

The biological process for phosphate (P_i) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P_i regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P_i from the medium than the wild-type strain removed.     

Inorganic polyphosphate–an unusual suspect of the mitochondrial permeability transition mystery

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (P_i) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca²⁺-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.     

Inorganic polyphosphate–an unusual suspect of the mitochondrial permeability transition mystery

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (P_i) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca²⁺-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.     

Inorganic polyphosphates in mitochondria

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (∼15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7–10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca²⁺/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P_i from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.