植物钙调素结合蛋白研究进展

钙调素(CaM)作为最重要的一类Ca2 传感蛋白可以通过与其下游CaM结合蛋白(CaMBP)作用而调节细胞的生理功能.因此,对CaMBP的研究是揭示CaM作用机制的重要内容,是探明Ca2 -CaM信号转导系统的关键.该文从CaMBP和CaM的结合特性、植物CaMBP的分布以及植物CaMBP的生物学功能等方面综述了植物CaMBP的研究现状和最新进展.     

植物的钙调素亚型

钙调素是一种高度保守的多功能Ca2 结合蛋白,在Ca2 信号转导途径中处于中心环节.近年来的研究表明,CaM亚型在植物中普遍存在,不同的CaM亚型与靶蛋白相互作用,它们的表达特性和生物学功能存在差异.该文介绍了植物 CaM亚型的研究现状和进展.     

钙不依赖性钙调素结合蛋白的研究进展

钙调素是普遍存在于真核生物细胞中、发挥多种生物学调控作用的信号组分.钙调素不仅在有Ca2 情况下通过与钙依赖性钙调素结合蛋白作用而传递信号,也能在相对无Ca2 条件下直接结合钙不依赖性钙调素结合蛋白而传递信号.综述了无钙离子结合钙调素及钙不依赖性钙调素结合蛋白的结构特性、钙不依赖性钙调素结合蛋白的种类及其可能的生物学作用,这将有助于我们深入认识钙调素介导信号途径的特异性、复杂性和多样性.     

植物胞外钙调素与信号转导

植物体需要构建复杂的信号转导体系以调节自身的生长发育过程并适应外界环境的变化,这种功能的实现需要胞内和胞外诸多信号分子的参与,胞外钙调素的发现使人们开始相信植物细胞外多肽信使的存在。胞外钙调素的生物学功能极其广泛,几乎涉及到植物生长发育的各个阶段,其信号转导途径是目前研究得最多也是最为清楚的方面,异三聚体G蛋白、磷脂酶C(PLC)-肌醇三磷酸(IP3)-肌醇三磷酸受体(IP3R)信号通路、活性氧和Ca2 通道之间直接或间接的相互作用是胞外钙调素信号转导的核心。     

钙-钙调素信号系统参与热激信号转导的研究

根据作者实验室的研究工作结合国内外的研究动态讨论热激信号转导的Ca2 -CaM途径。作者实验室的工作表明,钙一钙调素(Ca^2 -CaM)信号系统参与植物热激信号转导。激光共聚焦扫描显微镜的观察结果表明,37℃热激可引起小麦胞内自由Ca。’浓度迅速提高。在Ca^2 存在条件下,热激也引起小麦CaM基因CaM1-2表达及CaM蛋白含量增加。Ca^2 可促进小麦热激基因hsp26和mp70表达和热激蛋白合成,而Ca^2 螯合剂EGTA、Ca^2 通道阻断剂异搏定和LaCl3、CaM抑制剂W7、TFP和CPZ明显降低热激基因hsp26和mp70表达和热激蛋白合成。EGTA、异搏定、TFP或CPZ也阻止小麦耐热性的获得。小麦CaM基因与热激基因的表达动力学研究表明CaM位于热激信号转导的上游,而Ca^2 是启动热激反应的胞内关键因子。凝胶阻滞分析的结果表明,Ca^2 -CaM在热激信号转导中的作用是通过激活热激转录因子的DNA结合活性来实现的。根据大量实验证据,作者提出在植物细胞内存在一条新的热激信号转导途径——钙一钙调素途径。     

钙调素结合蛋白参与调控植物逆境胁迫的研究进展

Ca²⁺是植物体内重要的第二信使,当植物受到各种环境刺激时,细胞内的Ca²⁺浓度瞬间产生变化,并被Ca²⁺信号效应器识别,通过与下游的靶蛋白结合并调节其活性,参与调控植物各种生理活动。钙调素结合蛋白以依赖Ca²⁺或不依赖Ca²⁺的方式结合钙调素。对目前已经鉴定的植物钙调素结合蛋白结构特点进行了综述,并着重介绍了钙调素结合蛋白是如何参与调节植物对生物胁迫和非生物胁迫的反应,为提高作物抗病抗逆能力研究提供理论基础。     

采用GUS标记技术研究钙调素在转基因烟草中的分布

为了监测钙调素 (Ca M)在植物细胞内的分布并探索其生物学功能 ,将水稻 Ca M和 GU S融合基因 (cam.gus)分别置于 35 S启动子和花粉特异启动子 L AT5 2 - 7控制下 ,构建出载体 p MD/ Ca M.GU S和 p BI/ L AT.Ca M.GU S,转化烟草 (N icotiana tobacum cv.SR ) ,GU S染色结果显示 ,Ca M.GU S融合蛋白广泛分布于植物根、茎、叶等组织 ,根尖生长点细胞、根毛细胞、表皮毛细胞、气孔保卫细胞、维管束细胞的细胞质中融合蛋白含量较多。花粉粒中也分布着 Ca M,并在萌发沟处表现出较高的浓度。保卫细胞和表皮毛细胞中 Ca M分布在相邻细胞连接处细胞模内侧 ,呈现出极性分布。研究结果表明 ,细胞内 Ca M的不均匀分布可能与细胞功能相关。     

细胞外钙调素——一种植物中的多肽信使?

钙调素历来被认为是细胞内钙信号的多功能受体蛋白,国内外10多年的研究已证实,它普遍存在于人、动物细胞外与植物质外体.我们的工作证明了钙调素不仅普遍存在于植物细胞外,而且在胞外位点具有促进悬浮培养细胞及其原生质体的增殖、调节花粉萌发与伸长和促进rbc小亚基基因的光不依赖性表达等多种重要生物学功能.在花粉体系中,还证明了胞外钙调素具有跨膜与胞外信号转导机制,其中包括异三聚体G蛋白、PLC/IP3/IP3R和胞内钙信号等组分的参与.因此,认为细胞外钙调素可能是植物中的一种多肽信使,这对传统上认为植物中不存在进行胞间通讯的多肽信使的观点,提出了新的质疑.     

植物免疫应答过程中 Ca2 ＋及 CaM/CML 的功能

钙离子是一个多功能的第二信使,在植物响应各种生理刺激时,Ca2＋参与调节植物的多种生长发育和胁迫适应过程。在这些过程中,Ca2＋信号带有特异性标签,通过Ca2＋结合蛋白及其下游靶蛋白感知不同刺激并翻译成响应的细胞反应。钙调素（CaM）和钙调素类蛋白（CML）是Ca2＋主要感受器,通过调节不同靶蛋白的活性调控多种细胞功能。最近在植物对抗病原菌的防卫反应中有关Ca2＋／CaM信号转导系统的研究取得了一定进展。重点关注植物免疫应答过程中受CaM／CML调控的信号组分的研究,包括参与Ca2＋信号产生和Ca2＋依赖的表达基因组分调控。     

拟南芥钙调素结合蛋白AtIQD26的分离鉴定

钙调素结合蛋白的研究有助于探明钙调素介导的信号转导途径.以拟南芥钙调素亚型2(ACaM2)为钓饵,重组共转化法构建并筛选了酵母双杂交文库.复筛后得到一个阳性克隆.序列测定及分析表明,分离的阳性克隆中包含一个编码钙调素结合蛋白AtIQD26的cDNA片段.凝胶覆盖实验进一步表明,AtIQD26在1 mmol/L Ca2 或1 mmol/L EGTA条件下都能与钙调素结合,说明其存在不依赖于Ca2 的CaM结合特性.GUS染色分析表明,AtIQD26具有普遍的组织表达特性,尤其是在新生的组织中表达量较大;融合荧光蛋白定位显示,AtIQD26在细胞核与质膜附近有分布.AtIQD26与钙调素空间分布的相似性,预示着它们在植物生长发育过程中可能共同发挥作用.     

Calcium and signal transduction in plants

Environmental and hormonal signals control diverse physiological processes in plants. The mechanisms by which plant cells perceive and transduce these signals are poorly understood. Understanding biochemical and molecular events involved in signal transduction pathways has become one of the most active areas of plant research. Research during the last 15 years has established that Ca2+ acts as a messenger in transducing external signals. The evidence in support of Ca2+ as a messenger is unequivocal and fulfills all the requirements of a messenger. The role of Ca2+ becomes even more important because it is the only messenger known so far in plants. Since our last review on the Ca2+ messenger system in 1987, there has been tremendous progress in elucidating various aspects of Ca(2+) -signaling pathways in plants. These include demonstration of signal-induced changes in cytosolic Ca2+, calmodulin and calmodulin-like proteins, identification of different Ca2+ channels, characterization of Ca(2+) -dependent protein kinases (CDPKs) both at the biochemical and molecular levels, evidence for the presence of calmodulin-dependent protein kinases, and increased evidence in support of the role of inositol phospholipids in the Ca(2+) -signaling system. Despite the progress in Ca2+ research in plants, it is still in its infancy and much more needs to be done to understand the precise mechanisms by which Ca2+ regulates a wide variety of physiological processes. The purpose of this review is to summarize some of these recent developments in Ca2+ research as it relates to signal transduction in plants.     

Recruitment of plasma membrane voltage-dependent calcium-permeable channels in carrot cells.

Numerous biological assays and pharmacological studies have led to the suggestion that depolarization-activated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch-clamp techniques to isolated carrot protoplasts has led to direct voltage-clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage-dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole-cell recordings. We show here that large pre-depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage-dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre-depolarization, the greater the recruitment. Pre-depolarizing pulses to +118 mV during 30 s increased the initial calcium currents 5- to 10-fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltage-dependent Ca(2+)-permeable channels are regulated by biological mechanisms which might be induced by large pre-depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage-dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca(2+)-dependent signal transduction.     

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.     

Immunohistochemical evidence for the Na+/Ca2+ exchanger in squid olfactory neurons

The olfactory organs from the squid Lolliguncula brevis are composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour-stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+ exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+ proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+ exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+ during chemosensory responses.     

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.     

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.     

Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.     

Magnesium promotes structural integrity and conformational switching action of a calcium sensor protein

Calcium binding proteins carry out various signal transduction processes upon binding to Ca2+. In general, these proteins perform their functions in a high background of Mg2+. Here, we report the role of Mg2+ on a calcium sensor protein from Entamoeba histolytica (EhCaBP), containing four Ca2+-binding sites. Mg2+-bound EhCaBP exists as a monomer with a conformation different from that of the holo- and apo-EhCaBP. NMR and biophysical data on EhCaBP demonstrate that Mg2+ stabilizes the closed conformation of the apo form. In the presence of Mg2+, the partially collapsed apo-EhCaBP gains stability and structural integrity. Mg2+ binds to only 3 out of 4 calcium binding sites in EhCaBP. The Ca2+ binding affinity and cooperativity of the conformational switching from the "closed" to the "open" state is significantly modulated by the presence of Mg2+. This fine-tuning of the Ca2+ concentration to switch its conformation is essential for CaBPs to carry out the signal transduction process efficiently.     

钙_钙调素信号系统与环境刺激

植物抗逆研究已有很大进展,但传递各种外界刺激的信号通路仍未可知,目前已有一些研究发现很多环境刺激与钙_钙调素系统有关。Ca2+信号系统是很重要的一种信号途径,CaM是目前已知的胞内Ca2+信号受体中最重要的一种,参与了多种生理活动的调节。在热激领域中,研究者已提出Ca2+ CaM系统可能参与了热激反应,在基因调节水平、转录水平、蛋白水平均有Ca2+和CaM参与热激的证据。其它环境刺激也能引起植物体内Ca2+和CaM的一系列变化。这为研究各种环境刺激可能的信号通路提供了基础和依据。