N-磷酸化修饰蛋白质的富集和鉴定方法

蛋白质磷酸化修饰在细胞的信号转导、代谢、发育等生命过程中发挥着重要作用。除了研究较为透彻的发生在丝氨酸、苏氨酸和酪氨酸侧链羟基的O-磷酸化修饰之外,近年来,发生在组氨酸、精氨酸和赖氨酸侧链氨基的N-磷酸化修饰受到了越来越广泛的关注。然而,由于N-磷酸化修饰具有独特的P-N键结构,导致其化学稳定性差。尤其是在O-磷酸化肽段富集常用的酸性条件下,N-磷酸化极易丢失。因此,目前对N-磷酸化蛋白质的研究仍处于初始阶段。该文针对蛋白质N-磷酸化修饰的特点、富集和鉴定方法进行了综述,并对其发展前景进行了展望。     

小鼠肝果糖6-磷酸,2-激酶的纯化及动力学研究

经过超离心,聚乙二醇分级沉淀及DEAE-Sephadex,Blue-Sepbarose。磷酸纤维素三种柱层析等一系列过程,纯化了小白鼠肝脏的果糖6-磷酸,2-激酶。经凝胶过滤和SDS聚丙烯酰胺电泳方法测出该酶是由两个相同亚基构成的分子量为110000的蛋白。Mg~(2+)是其活性所必需的,活化方式呈正协同性。酶与底物的结合方式,对果糖6-磷酸表现正协同性,对ATP无协同性,其Km值随果糖6-磷酸的浓度减小而增大,表明酶的作用为顺序机制。活性中心有一个必需氨基酸残基与结合ATP有关,结合后,其侧链pKa由9.5移至9.8。     

利用表面活性剂介导的方法制备AgrC蛋白脂质体

膜蛋白AgrC是金黄色葡萄球菌双组分信号转导系统的感受激酶.其信号转导机制的阐明对于解决细菌耐药问题具有重要意义.目前膜蛋白研究的主要瓶颈是难以获得大量高纯度和功能稳定的蛋白.将目标蛋白表达在大肠杆菌体内,利用表面活性剂将其从细胞膜上溶解,纯化,这一系列步骤容易引起膜蛋白不稳定和功能损失.本文报道了用表面活性剂介导的方法将膜蛋白AgrC镶嵌到脂质体上,即形成蛋白脂质体.脂质体和蛋白脂质体的结构、形貌以及平均粒径分别用透射电镜和动态光散射仪表征.蔗糖密度梯度离心的结果表明蛋白重构效率达80%.硫醇试剂标记实验确定AgrC的细胞质域在脂质体中取向于内侧.体外磷酸化实验表明AgrC蛋白在脂质体中的自我磷酸化活性远远高于表面活性剂中的活性,且其自我磷酸化活性在2周内几乎没有损失.蛋白脂质体的构建不仅解决了膜蛋白的不稳定性问题,也为体外研究AgrC蛋白的结构、功能和信号转导机制提供了新的思路.     

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。     

磷酰化肽链中电子转移的ESR研究

当前生命科学正以前所未有的速度向前发展，从对核酸的深入研究开始过渡到对蛋白质结构与功能的注意，尤其是近年来基因调控研究的重大进展是发现非组蛋白具有高度的磷酸化的特性［1］，研究证明，核内蛋白磷酸化和去磷酸化则是基因表达调控的重要方式之一［2，3］，它是酶活性调节、基因表达调控、细胞转化恶变的关键环节；同时在解决肿瘤的发生和治疗、病毒的感染和诊治、辐射损伤、抗衰老等实际问题上有突出意义，如1992年Krebus和Fisher因为在蛋白的可逆磷酸化方面的突出贡献而获得诺贝尔生理和医学奖．而电子转移是一切化学变化的基础…     

ZnCl2与氧化胰岛素B链结合位点和切割位点的质谱指认

采用电喷雾质谱和串联质谱研究了氧化胰岛素B链与ZnCl₂的键合作用并成功地确定了切割位点。质谱研究显示在pH值2.5及40 ℃条件下，Zn²⁺通过与氧化胰岛素B链的氨基酸侧链His5,His10和Arg22结合，选择性地水解了肽键Asn3-Gln4, His5-Leu6, Gly8-Ser9和Glu21-Arg22。     

胶束溶液中某些氨基酸和二肽的解离常数

用pH电位法测定了在SDS(十二烷基磺酸钠)胶束溶液中甘氨酰丙氨酸和六种氨基酸(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸)的两级酸解离常数 pKa1 和 pKa2,发现与水溶液相比 ,pKa 值均显著增加 ,即SDS胶束形成强烈抑制质子解离.结合1HNMR测定结果 ,讨论了pKa1和 pKa2 随SDS浓度的变化规律以及与各配体自身结构的关系     

混合模式色谱材料Click TE-GSH高效富集多磷酸化肽

多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键.复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤.本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集.我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法.该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽.与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好.本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.     

基于两相预柱的在线二维分离系统用于人肝组织中磷酸肽的规模化鉴定

将C18反相(RP)填充柱和磷酸基强阳离子交换(SCX)整体柱集成于一体的RP - SCX两相预柱,成功用于RPLC - MS/MS系统的自动进样和SCX分级,完成了磷酸肽在线多维分离平台的构建,可以减少样品的损失,极大地提高了系统的集成化水平和检测灵敏度.对人肝组织酶解液富集的磷酸肽进行规模化鉴定,在假阳率小于1％的情况下,共鉴定到3082条非重复的磷酸化肽段、3056个磷酸化位点和1332个磷酸化蛋白.对所鉴定到的磷酸化蛋白进行了功能分类和激酶分析.该实验结果对于了解蛋白质磷酸化在肝脏组织中发挥的生理学作用具有重要意义.     

磷酸化蛋白质组学中的分离富集方法研究进展

蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法.     

Why have serine/threonine/tyrosine kinases been evolutionarily selected in eukaryotic signaling cascades?

The signal transduction systems of eukaryotes are different from those of prokaryotes with respect to their structures and mechanisms. The main signal transduction system of prokaryotes called the two-component system (TCS) is a one-step phosphorelay system composed of a histidine kinase (HK) while the central signal transduction system of eukaryotes called the mitogen-activated protein kinase (MAPK) cascade system (MCS) is a multi-step phosphorelay system composed of serine/threonine/tyrosine kinases (STYKs). The two signal transduction systems are also different in their transphosphorylation mechanisms. HK in the TCS transfers its own phosphate group to the response regulator protein while STYKs in the MCS phosphorylate other proteins using ATP. We were intrigued by the different dynamics resulting from such differences and wondered why STYKs instead of HKs have been evolutionarily selected in eukaryotic signaling cascades. In this paper, we compared the dynamical characteristics of two mathematical models which reflect such differences between the TCS and the MCS, and found that STYKs are more appropriate for cascade structures in eukaryotic signal transduction than HK with respect to the duration and settling time of response signals.     

Activity-based probe for histidine kinase signaling

Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.     

The multi-step phosphorelay mechanism of unorthodox two-component systems in E. coli realizes ultrasensitivity to stimuli while maintaining robustness to noises

E. coli has two-component systems composed of histidine kinase proteins and response regulator proteins. For a given extracellular stimulus, a histidine kinase senses the stimulus, autophosphorylates and then passes the phosphates to the cognate response regulators. The histidine kinase in an orthodox two-component system has only one histidine domain where the autophosphorylation occurs, but a histidine kinase in some unusual two-component systems (unorthodox two-component systems) has two histidine domains and one aspartate domain. So, the unorthodox two-component systems have more complex phosphorelay mechanisms than orthodox two-component systems. In general, the two-component systems are required to promptly respond to external stimuli for survival of E. coli. In this respect, the complex multi-step phosphorelay mechanism seems to be disadvantageous, but there are several unorthodox two-component systems in E. coli. In this paper, we investigate the reason why such unorthodox two-component systems are present in E. coli. For this purpose, we have developed simplified mathematical models of both orthodox and unorthodox two-component systems and analyzed their dynamical characteristics through extensive computer simulations. We have finally revealed that the unorthodox two-component systems realize ultrasensitive responses to external stimuli and also more robust responses to noises than the orthodox two-component systems.     

Phosphorylation‐Responsive Membrane Transport of Peptides

Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation‐responsive membrane transport systems. Dye‐efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell‐penetrating peptides, with EC₅₀ values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label‐free kinase assay.     

Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

Background  

Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress.     

Regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B activity by casein kinase II- and p56lck-mediated phosphorylation

Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56lck in vitro. Phosphoamino acid analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56lck. Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.     

Membrane Sensor Histidine Kinases: Insights from Structural,Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled “Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents” edited by Mary K. Phillips-Jones.     

Histidine kinases and two-component signal transduction systems.

The phosphorylation of histidine is the first step in many signal transduction cascades in bacteria, yeast and higher plants. The transfer of a very reactive phosphoryl group from phosphorylated histidine kinase to an acceptor is an essential step in many cellular signaling responses.     

Label-Free Fluorescent Kinase and Phosphatase Enzyme Assays with Supramolecular Host-Dye Pairs

The combination of the macrocyclic hosts p-sulfonatocalix[4]arene and cucurbit[7]uril with the fluorescent dyes lucigenin and berberine affords two label-free enzyme assays for the detection of kinase and phosphatase activity by fluorescence monitoring. In contrast to established assays, no substrate labeling is required. Since phosphorylation is one of the most important regulatory mechanisms in biological signal transduction, the assays should be useful for identification of inhibitors and activators in high-throughput screening (HTS) format for drug discovery.     

Proteomic identification of p38 MAP kinase substrates using in vitro phosphorylation

Protein phosphorylation is a major mechanism that regulates many basic cellular processes. Identification and characterization of substrates for a given protein kinase can lead to a better understanding of signal transduction pathways. However, it is still difficult to efficiently identify substrates for protein kinases. Here, we propose an integrated proteomic approach consisting of in vitro dephosphorylation and phosphorylation, phosphoprotein enrichment, and 2D‐DIGE. Phosphatase treatment significantly reduced the complexity of the phosphoproteome, which enabled us to efficiently identify the substrates. We employed p38 mitogen‐activated protein kinase (p38 MAP kinase) as a model kinase and identified 23 novel candidate substrates for this kinase. Seven selected candidates were phosphorylated by p38 MAP kinase in vitro and in p38 MAP kinase‐activated cells. This proteomic approach can be applied to any protein kinase, allowing global identification of novel substrates.