植物中的磷脂酶D信号转导

文章介绍磷脂酶D（PLD）跨膜信号转导作用的研究进展。     

植物中的磷脂酶D

介绍了植物磷脂酶D(PLD)的生化性质、克隆、基因组结构、氨基酸序列结构、活性调控、信号转导和细胞生理功能的研究进展.     

植物信号传导中的磷脂酶

20世纪 80年代早期人们意识到构成细胞膜的磷脂不只是一道将细胞物质与外界隔开的屏障,而且是细胞对外界环境刺激作出应答的物质基础。磷脂酰肌醇（ｐｈｏｓｐｈｏｔｉｄｙｌｉｎｏｓｉｔｏｌ,ＰＩ）不但是构成细胞膜的重要组分（约占细胞膜组分的 10 %）,在细胞内外环境信号的传递方面也起着重要的作用［1］。磷脂酶（ｐｈｏｓｐｈｏｌｉｐａｓｅ）水解磷脂后产生的三磷酸肌醇 （ｉｎｏｓ ｉｔｏｌｔｒｉｓｐｈｏｓｐｈａｔｅ,ＩＰ3 ）／二酰基甘油（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ,ＤＡＧ）、磷脂酸（ｐｈｏｓｐｈａｔｉｄｉｃａｃｉ…     

磷脂酶D的细胞信号转导作用

磷脂酶D(PLD)是一类重要的跨膜信号转导酶类.分别由一个基因家族的不同成员编码.植物PLD的总体域结构相似,只是不同类型之间在某些单元上有重要差异.它们各具独特的生物化学特性.不同的PLD在不同的胁迫类型启动的特定的细胞过程中执行独特的细胞信号转导功能.PLD与其它磷脂酶及Ca2 信使之间有交互作用,形成复杂的信号转导网络.这一网络在不同植物种类、器官、组织和细胞类型中表现出特异性.文章最后讨论了PLD研究中有待揭示的问题并展望了今后的发展方向.     

磷脂酶D和炎症的关系

磷脂酶Ｄ（ＰＬＤ）广泛存在于动物组织细胞中,并受各种胞外信号调节。其主要底物为磷脂酰胆碱（ＰＣ）。ＰＬＣ引起的ＰＣ水解是细胞内重要的信号转导途径。越来越多的证据表明ＰＬＤ和炎症有密切的关系。本文主要介绍ＰＬＤ在呼吸爆发、脱颗粒及花生四烯酸（ＡＡ）释放等方面的研究进展。     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ（PLDδ）基因敲除突变体与野生型植株进行冻害胁迫处理后,比较2种基因型植株的抗冻性。结果发现,经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型,而未经低温驯化的PLD礅除突变体与野生型的抗冻性没有显著差异,表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析,发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节,对脯氨酸和可溶性糖的积累起负调节作用,但是参与低温信号转导物质ABA诱导抗冻性的过程。     

磷脂酶D与病原微生物感染

磷脂酶D(phospholipase D,PLD)普遍存在于细菌,真菌以及哺乳动物中.在病原微生物中,PLD作为毒力决定因子在减数分裂、孢子形成等过程中起作用;在哺乳动物细胞中,PLD主要在胞膜转运、调节有丝分裂和细胞肌动蛋白骨架等一些信号转导中起作用.在病原菌感染宿主细胞的过程中,病原体和宿主细胞的PLD都被激活并发生级联反应,病原菌PLD可调节自身肌动蛋白丝的聚合和重排,并引起宿主细胞局部肌动蛋白丝的集聚,诱导宿主细胞对其吞噬.深入探讨PLD激活对感染发生的调控作用对透彻理解病原菌感染宿主细胞的分子机制具有重要意义.     

磷脂酶D的研究进展

磷脂酶D专一性水解磷脂中的4位酯键,其分布广泛,功能多样,在细胞信号转导,脂质代谢,生物膜形成以及磷脂改性等方面发挥重要作用。文章概述了磷脂酶D的研究现状及最新进展,包括酶源分布、催化特征、底物特一性、酶活调节因子以及其工业应用前景等。     

大孔树脂固定化磷脂酶D

磷脂酶D(PhospholipaseD,EC3.1.4.4,PLD)是催化磷酸酯键水解和碱基交换反应的一类酶的总称.利用PLD的转碱基作用是目前催化合成磷脂酰丝氨酸(PS)的最佳途径.本实验以5种大孔树脂为载体固定化磷脂酶D(PLD)进行了研究.以酶回收率为主要指标,选择了最佳载体和优化了固定化条件.结果表明:非极性阳离子交换树脂H103是最佳固定化载体;其最优固定化条件:加酶液量1.2 mL,固定时间80 min,pH 6.0柠檬酸-柠檬酸钠的缓冲液浓度为10 mmol/L.最佳固定化条件下,固定化之后的PLD比游离PLD酶活提高了三倍.     

Redox Control of Signal Transduction,Gene Expression and Cellular Senescence

Reactive oxygen species (ROS) act as subcellular messengers in such complex cellular processes as mitogenic signal transduction, gene expression, regulation of cell proliferation, replicative senescence, and apoptosis. They serve to maintain cellular homeostasis and their production is under strict control. However, the mechanisms whereby ROS act are still obscure. Here we review recent advances in our understanding of signaling mechanisms and recent data about the involvement of ROS in: (i) the regulation of the mitogenic transduction elements, particularly protein kinases and phosphatases; (ii) the regulation of gene expression; and (iii) the induction of replicative senescence and the role, if any, in aging and age-related disorders.     

Functional Regulation of Phospholipase D Expression in Cancer and Inflammation

Phospholipase D (PLD) regulates downstream effectors by generating phosphatidic acid. Growing links of dysregulation of PLD to human disease have spurred interest in therapeutics that target its function. Aberrant PLD expression has been identified in multiple facets of complex pathological states, including cancer and inflammatory diseases. Thus, it is important to understand how the signaling network of PLD expression is regulated and contributes to progression of these diseases. Interestingly, small molecule PLD inhibitors can suppress PLD expression as well as enzymatic activity of PLD and have been shown to be effective in pathological mice models, suggesting the potential for use of PLD inhibitors as therapeutics against cancer and inflammation. Here, we summarize recent scientific developments regarding the regulation of PLD expression and its role in cancer and inflammatory processes.     

植物缺氧响应相关基因的表达调控机制

对近年来缺氧应答相关基因的分离、克隆及缺氧响应信号转导模式与调控机制作了综述。分析了缺氧应答基因的序列特点,概述了植物感受缺氧胁迫的信号传递模式,阐述了缺氧响应的调控机制并着重讨论了调控因子AtMYB2的调控特点,并对缺氧相关研究进行了展望。     

磷脂酶D(PLD)基因的结构、表达及其表达产物在信号转导中的作用

磷脂酶D(PLDEC 3 .1 .4.4)水解磷脂 (PL) ,磷脂构成生物膜的骨架 ,磷脂酶的激活不仅对细胞的结构和稳定性有很重要的作用 ,而且调控许多重要的细胞生理功能 ,例如PLD在信号转导、小泡运输、有丝分裂、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用。近年来它在跨膜信号转导中的重要作用 ,越来越引起人们的重视 ,成为新的研究热点。介绍了磷脂酶基因的结构特点、亚细胞定位、表达的激活抑制以及其表达产物作为胞内信号分子在植物信号转导中的重要作用。     

Thematic Minireview Series on Phospholipase D and Cancer

Phospholipase D (PLD) signaling plays a critical role in cell growth and proliferation, vesicular trafficking, secretion, and endocytosis. At the cellular level, PLD and its reaction product, phosphatidate, interact with a large number of protein partners that are directly related to the actin cytoskeleton and cell migration. Cancer invasion and metastasis rely heavily on cellular motility, and as such, they have put PLD at center stage in cancer research. This minireview series highlights some of the molecular mechanisms that provide evidence for the emerging tumorigenic potential of PLD, the role of the microenvironment, and putative connections with inflammation. PLD represents a potential target for the rational development of therapeutics against cancer and other diseases.     

The Molecular Basis of Leukocyte Adhesion Involving Phosphatidic Acid and Phospholipase D

Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t_½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t_½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC₅₀ = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t_½ = 3 min at 300 nm (compared with controls of no PA, t_½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation.     

重组人磷脂酶D1/腺病毒表达载体的构建与表达

将磷脂酶D1基因及其功能缺陷点突变基因从真核表达载体pCGNPLD1亚克隆至带有绿色荧光标记蛋白的穿梭质粒pAdTrackCMV中;再与腺病毒骨架载体一起在大肠杆菌BJ5183中进行同源重组;阳性重组子经PacⅠ线性化后,转染入病毒组装细胞系293细胞,成功构建磷脂酰胆碱专一性磷脂酶D1重组腺病毒; 并用该病毒颗粒感染嗜铬细胞瘤细胞PC12细胞,高效表达磷脂酶D1蛋白。证明大蛋白基因,如磷脂酶D1基因的同源重组腺病毒表达构建切实可行,为研究其在细胞内的生理功能提供了有力工具。     

Kinetic Analysis in Mixed Micelles of Partially Purified Rat Brain Phospholipase D Activity and its Activation by Phosphatidylinositol 4,5-Bisphosphate

A partially purified rat brain membrane phospholipase D (PLD) activity was characterized in a mixed micellar system consisting of l-palmitoyl-2-[6-N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)-amino]caproyl-phosphatidylcholine (NBD-PC) and Triton X-100, under conditions where Triton X-100 has a surface dilution effect on PLD activity and the catalytic rate is dependent on the surface concentration (expressed in terms of molar ratio) of NBD-PC. PLD activity was specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂), and the curve of activation versus PIP₂ molar ratio fitted a Michaelis-Menten equation with a K_act value between molar ratios of 0.001–0.002. Maximal activation was observed at a PIP₂ molar ratio of 0.01. Similar values were obtained when activities of partially purified PLD as well as membrane-bound PLD were determined towards pure NBD-PC micelles. In the mixed micellar system PIP₂ was shown to elevate by 6–22 fold the specificity constant of PLD towards NBD-PC (K_A, which is proportional to V_max/K_m). Kinetic analysis of PLD trans-phosphatidylation activity towards ethanol, 1-propanol and 1-butanol revealed a Michaelis-Menten type dependence on alcohol concentration up to 1000, 200 and 80 mM, respectively. While V_max values were similar towards all three alcohols, enzyme affinity increased as the alcohol was longer, and K_m values for ethanol, 1-propanol and 1-butanol were 291, 75 and 16 mM (respectively). PLD specificity constants (K_A) towards ethanol, 1-propanol and 1-butanol were shown to be respectively 260, 940 and 5,920 times higher than to water, the competing substrate. 1-Propanol and 1-butanol inhibited PLD activity above 400 and 100 mM, respectively. The present results indicate that partially purified PLD obeys surface dilution kinetics with regard to its phospholipid substrate PC and its cofactor PIP₂, and that in the presence of alcohols, its transphosphatidylation activity may be analyzed as a competitive reaction to the hydrolysis reaction.     

PLD 的生化特性及在信号传导中的作用

磷脂酶 D(PLD)是一种分解磷脂的多功能酶,磷脂酶可激活调控许多重要的细胞生理功能,在信号转导、小泡运输、有丝分裂、激素作用的发挥、细胞骨架组装、防御反应以及种子萌发和衰老过程中都起重要作用．主要介绍了磷脂酶基因的生化特性及在植物信号转导中的作用．