组氨酸磷酸化修饰的鉴定方法及应用

蛋白质磷酸化是广受关注的翻译后修饰类型之一,组氨酸磷酸化作为一种非常见的磷酸化修饰,最早被发现在细菌和低等真核生物信号传导的级联反应中起关键作用。近年来研究显示,其在肿瘤发生发展过程中也可能扮演了重要角色。由于磷酸化组氨酸的化学不稳定性、低丰度、亚化学计量性质、缺乏特异性的富集试剂,导致研究手段缺乏,限制了人们对磷酸化组氨酸修饰底物蛋白质的认识。随着磷酸化组氨酸抗体的设计以及富集和质谱等鉴定方法的发展,更多的磷酸化组氨酸修饰底物被鉴定,从而加速了对磷酸化组氨酸生物学功能的认识。本文介绍了磷酸化组氨酸的化学性质、主要生物学功能,并综述了磷酸化修饰底物富集和鉴定技术等方面的最新进展,同时也要看到组氨酸磷酸化修饰组学研究仍然存在巨大的技术挑战。     

磷酸化组氨酸研究进展

蛋白质磷酸化是生物体内一种广泛存在的蛋白质翻译后修饰形式,这种氨基酸与磷酸基团共价连接的修饰模式对蛋白质结构和功能起到了重要调节作用.目前天然蛋白质中发现的可磷酸化位点主要有9种氨基酸残基,其中包括以磷酰胺连接的磷酸化组氨酸.虽然该磷酸化形式在原核生物与真核生物中都起到了重要的调节作用,但对于其生物学功能的研究长期存在技术困难.由于磷酸化组氨酸本身不同于其他磷酸化氨基酸的化学性质,如存在异构体、化学不稳定等,其在传统的研究方法中容易发生水解去磷酸化.随着现代生物化学与分子生物学技术的不断进步,人们针对含有磷酸化组氨酸的蛋白质构建了新的制备、分离与表征策略,本领域也因此开始迅速发展.本文从磷酸化组氨酸的化学结构入手,分析其两种异构体的主要理化性质与化学反应特性,并概述了基于此发展的新型化学生物学研究手段以及对于磷酸化组氨酸生物功能的研究进展.     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His)蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径.在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统.最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用.     

PTP的结构与功能

蛋白质分子中酪氨酸残基的可逆性磷酸化作为真核生物信号转导的一个重要组成部分,参与了多种细胞功能调节,包括细胞增殖、迁移以及细胞间相互作用等。目前认为这种可逆性的磷酸化调节主要是受控于蛋白酪氨酸激酶（PTK）及蛋白酪氨酸磷酸酯酶（PTP）这两种酶活性的动态平衡。因此,与PTK一样,PTP对于体内各种生命活动起着非常重要的生物学作用。文章综述了近年来PTP在信号转导中的调控作用,特别是其在肿瘤发生、发展过程中的作用、以及其本身的结构与调控的研究进展。     

蛋白组氨酸磷酸酶研究进展

主要概括磷酸酶的种类,原核细胞磷酸组氨酸生物功能及调控,哺乳动物组氨酸残基磷酸化、去磷酸化,以及组氨酸磷酸酶及其底物的最新研究进展. 信号转导在生长发育及细胞功能中起极其重要的作用. 无论在原核还是真核细胞,蛋白质磷酸化是细胞内信号转导的关键机制. 研究最多的可逆的真核蛋白磷酸化,主要发生在含有羟基的丝氨酸、苏氨酸和酪氨酸残基上. 不同的激酶和磷酸酶受不同机制的调节,而调节过程中出现的差异是人类很多疾病的潜在基础. 与大量有关羟基磷酸化氨基酸的报道相比,有关氨基磷酸化氨基酸的报道甚少. 据估计,自然界中存在的磷酸组氨酸比磷酸酪氨酸多10 ~ 100倍,但不如磷酸丝氨酸丰富. 虽然对脊椎动物蛋白质中存在磷酸组氨酸的认识可以追溯到20世纪60年代初, 但由于研究手段的限制,至今对脊椎动物蛋白组氨酸激酶及组氨酸磷酸酶的结构及功能知之甚少. 但是,近几年的研究有突破性的发现,克隆和重组表达哺乳动物组氨酸磷酸酶为研究氨基磷酸化氨基酸的生物功能翻开新的一章.     

真核生物基因组DNA 6mA表观修饰研究进展

众所周知,DNA的6mA修饰在原核生物中,特别是细菌中发挥重要的作用,它控制着许多重要的生物学过程,但这种DNA碱基修饰在真核生物中的存在与生物学功能一直还不清楚。近来的一系列研究表明,6mA在真核生物中不仅存在,并且可能是真核生物的一种新表观遗传学标记,具有重要的表观调控功能。现对最新的真核生物6mA修饰研究进展进行简要综述,回顾并展望这种真核生物新表观修饰的基本特征、潜在生物学功能及未来的研究方向。     

植物的双组分信号系统

双组分系统由感受信号输入的组氨酸(His) 蛋白激酶和调节信号输出的反应调控因子组成,涉及许多原核生物、真菌、黏菌和植物的各种信号转导途径。在植物中,还存在更复杂的包括杂合的His激酶、磷酸传递中间体和反应调控因子的信号系统,称为多步骤双组分系统。最近的研究表明,双组分系统在对环境刺激和生长调节剂(如乙烯、细胞分裂素、光和渗透胁迫)的反应中起重要作用。     

古菌独特的脱氧酮糖酸(ED)葡萄糖酵解途径

葡萄糖通过"中心代谢途径"降解为丙酮酸的过程对于生物体物质及能量的代谢具有重要的作用.古菌的葡萄糖酵解过程具有与真核生物以及细菌葡萄糖代谢显著不同的特征.生化性质分析、基因组学、代谢组学等研究结果表明,古菌糖酵解Embden-Meyerhof(EM)与Entner-Doudoroff(ED)途径具有许多与真核生物及细菌经典的EM与ED途径不同的特异性酶类,其中ED糖酵解代谢又可分为非磷酸化与半磷酸化的糖酵解途径.古菌独特的ED糖酵解途径在代谢路径、酶、调节位点、表达调控、能量转化等方面与真核生物及细菌经典的糖酵解途径均存在明显的差异,反映了其适应极端的生理环境而形成可塑性代谢路径的能力.本文综述了古菌ED葡萄糖降解过程中的各种酶、调控机制以及能量转化特征的最新进展,并对进一步的研究方向做了展望.     

玉米早期花药蛋白质组和磷酸化蛋白质组分析

蛋白质磷酸化修饰是调控其功能的一种重要方式。植物有性生殖过程在农作物产量形成和物种繁衍过程中起着重要作用。作为植物雄性生殖器官的花药,其正常生长发育对于保证形成功能性配子(花粉)以及完成双受精过程至关重要。本研究以重要农作物玉米(B73)为材料,利用Nano UHPLC-MS/MS质谱技术对玉米早期发育的花药在蛋白质组和磷酸化蛋白质组水平进行全面分析,以探究玉米花药发育过程中的蛋白调控网络和磷酸化修饰调控网络。在蛋白质组学分析中,共鉴定到了3 016个多肽,匹配到1 032个蛋白质上。通过Map Man分析,预测到了一些和花药发育相关的蛋白质,例如受体激酶(GRMZM2G082823_P01、GRMZM5G805485_P01等)。另外,在磷酸化蛋白质组学研究中,通过对Ti O2亲和层析富集到的磷酸化多肽进行质谱分析,检测到了257个磷酸化多肽,匹配到210个蛋白质上。我们的数据揭示了玉米花药发育过程中的223个磷酸化位点。与已发现的玉米中的86个磷酸化蛋白质(植物蛋白磷酸化数据库(P3DB):http://www.p3db.org/organism.php)相比,其中203个磷酸化蛋白和218个磷酸化位点为首次揭示。进一步生物信息学分析表明:磷酸化在14-3-3蛋白质、激酶、磷酸酶、转录因子、细胞周期和染色质结构相关的蛋白质介导的玉米早期花药发育过程中起着重要的调控作用。总之,本研究首次在蛋白质组学和磷酸化蛋白质组学水平研究了玉米早期花药发育的蛋白质调控网络,不仅丰富了玉米蛋白质和磷酸化修饰蛋白质数据库,并为利用遗传学和生物化学手段深入研究玉米花药发育的分子调控机理提供了基础。     

转录水平调控中的负调控元件——沉默子

在真核生物、原核生物及病毒的基因组中存在着调控基因表达的顺式元件,沉默子是其中的一种负调控元件,它能够同反式因子协同作用,抑制靶基因的转录活性,在基因表达调控中发挥重要作用。该文主要对沉默子的特性、结构组成及其分类进行简要总结,对沉默子可能作用机制进行简要综述,最后展望沉默子在遗传工程及人类疾病治疗等领域的应用前景。     

Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.     

Role of serine/threonine phosphatase (SP-STP) in Streptococcus pyogenes physiology and virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.     

Two-component signal transduction pathways in Arabidopsis

The two-component system, consisting of a histidine (His) protein kinase that senses a signal input and a response regulator that mediates the output, is an ancient and evolutionarily conserved signaling mechanism in prokaryotes and eukaryotes. The identification of 54 His protein kinases, His-containing phosphotransfer proteins, response regulators, and related proteins in Arabidopsis suggests an important role of two-component phosphorelay in plant signal transduction. Recent studies indicate that two-component elements are involved in plant hormone, stress, and light signaling. In this review, we present a genome analysis of the Arabidopsis two-component elements and summarize the major advances in our understanding of Arabidopsis two-component signaling.     

A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth,cell segregation,and virulence

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.     

双组分系统——细胞识别渗透胁迫信号的感应器

双组分系统是广泛存在于原核和真核细胞中的信号转导系统.主要由组氨酸蛋白激酶(HPK)和响应调节蛋白(RR)两个组分组成. 双组分系统信号通路一般包括信号的输入、HPK自身磷酸化、RR磷酸化、信号输出等环节.对双组分系统信号转导机制及其在渗透胁迫信号识别和传导中的作用进行了综述.     

Mammalian protein histidine kinases

The existence of protein kinases, known as histidine kinases, which phosphorylate their substrates on histidine residues has been well documented in bacteria and also in lower eukaryotes such as yeast and plants. Their biological roles in cellular signalling pathways within these organisms have also been well characterised. The evidence for the existence of such enzymes in mammalian cells is much less well established and little has been determined about their cellular functions. The aim of the current review is to present a summary of what is known about mammalian histidine kinases. In addition, by consideration of the chemistry of phosphohistidine, what is currently known of some mammalian histidine kinases and the way in which they act in bacteria and other eukaryotes, a general role for mammalian histidine kinases is proposed. A histidine kinase phosphorylates a substrate protein, by virtue of the relatively high free energy of hydrolysis of phosphohistidine the phosphate group is easily transferred to either a small molecule or another protein with which the phosphorylated substrate protein specifically interacts. This allows a signalling process to occur, which may be downregulated by the action of phosphatases. Given the known importance of protein phosphorylation to the regulation of almost all aspects of cellular function, the investigation of the largely unexplored area of histidine phosphorylation in mammalian cells is likely to provide a greater understanding of cellular action and possibly provide a new set of therapeutic drug targets.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.     

Comparative proteome bioinformatics: identification of a whole complement of putative protein tyrosine kinases in the model flowering plant Arabidopsis thaliana

Phosphorylation by protein tyrosine kinases is crucial to the control of growth and development of multicellular eukaryotes, including humans, and it also seems to play an important role in multicellular prokaryotes. A plant tyrosine-specific kinase has not been identified yet; hence, plants have been suggested to share with unicellular eukaryote yeast a tyrosine phosphorylation system where a limited number of stress proteins are tyrosyl-phosphorylated only by a few dual-specificity (serine/threonine and tyrosine) kinases. However, preliminary evidence obtained so far suggests that tyrosine phosphorylation in plants depends on the developmental conditions. Since sequencing of the genome of the model flowering plant Arabidopsis thaliana has been recently completed, we have performed a bioinformatic screening of the whole Arabidopsis proteome to identify a model complement of bona fide protein tyrosine kinases. In silico analyses suggest that < 4% of Arabidopsis kinases are tyrosine-specific kinases, whose gene expression has been assessed by a preliminary polymerase chain reaction screening of an Arabidopsis cDNA library. Finally, immunological evidence confirms that the number of Arabidopsis proteins specifically phosphorylated on tyrosine residues is much higher than in yeast.     

The role of protein kinase cascades in stress signal transduction in unicellular eukaryotes

The review summarizes current data on transduction mechanisms of stress signals by protein kinase cascades in unicellular eukaryotes. The role of sensor histidine kinases, tyrosine kinases, PKC, and cyclic nucleotid-dependent kinases are reviewed. Special attention is paid to a comparative analysis of transduction mechanisms of stress signals in vertebrates and unicellular eukaryotes.