植物蛋白质S-亚硝基化修饰的检测与分析

S-亚硝基化是一种重要的蛋白质翻译后修饰方式, 是指一氧化氮(NO)基团共价连接至靶蛋白特定半胱氨酸残基的自由巯基, 从而形成S-亚硝基硫醇(SNO)的过程。S-亚硝基化修饰广泛存在于各有机体中, 通过改变蛋白质生化活性、稳定性、亚细胞定位以及蛋白质-蛋白质相互作用等机制而调控不同的生物学过程或信号通路。在蛋白质S-亚硝基化检测分析方法中, 最为广泛使用的是生物素转化法(biotin switch assay), 其基本原理是首先封闭未被修饰的自由巯基, 进而将被修饰的SNO基团特异地还原为自由巯基并使用生物素将其特异标记。被生物素标记的半胱氨酸残基(即被修饰位点)可进一步通过蛋白质免疫印迹和/或质谱等方法进行检测分析。该文详细描述了植物蛋白质样品的体内和体外生物素转化法的实验流程, 并对实验过程中的注意事项进行了讨论。     

蛋白质巯基亚硝基化———一种典型氧化还原依赖的蛋白质翻译后修饰

综述了蛋白质巯基亚硝基化修饰的特点、检测方法、功能研究、相关疾病和发展态势.蛋白质巯基亚硝基化(S-nitrosation)是指一氧化氮(nitricoxide,NO)及其衍生物修饰蛋白质半胱氨酸(cysteine,Cys)巯基—SH生成—SNO,其是一种典型的氧化还原依赖的蛋白质翻译后修饰,也是一氧化氮发挥其广泛信号转导作用的新的重要途径.     

蛋白质巯基亚硝基化与帕金森病

巯基亚硝基化(S-nitrosylation,SNO),即蛋白质中半胱氨酸的巯基与亚硝基基团(NO基团)形成共价键,是一氧化氮(NO)在体内发挥细胞信号转导作用的机制之一。NO通过使某些蛋白质发生SNO,进而可能参与神经退行性疾病如帕金森病(PD)发生的病理机制。深入认识帕金森病发病机制,对人们探索此类神经退行性疾病的新疗法具有重要意义。     

一氧化氮的功能及其作用机制(Ⅱ)——蛋白质巯基亚硝基化修饰

一氧化氮的功能多样,其作用机制也是复杂而相互关联的,是多靶点、多机制同时作用的调控网络。除了经典的cGMP依赖的信号通路外,一氧化氮还能通过对蛋白质的半胱氨酸巯基进行蛋白质翻译后修饰而起作用。蛋白质巯基亚硝基化修饰(protein S-nitrosation)是活性氮对蛋白质半胱氨酸巯基的一种蛋白质翻译后修饰,在一氧化氮的作用机制中占有重要位置。本综述简要总结蛋白质巯基亚硝基化修饰的功能及作用机制。     

人类S-亚硝基化蛋白组微阵列芯片检测研究进展

<正>NO通过对蛋白质的S-亚硝基化修饰广泛参与细胞生理活动的调节,然而S-亚硝基化底物尚不完全清楚。来自约翰霍普金斯医学院细胞工程院的研究团队近期利用自发研制的人类高密度蛋白微阵列芯片对16,368种人类蛋白进行了亚硝基化检测,并确定了834种潜在可被亚硝基化修饰的蛋白质,其中95种蛋白质中131条肽链上的138个半胱氨酸残基被证实为亚硝基化位点。传统对亚硝基化位点的研究存在物种偏差以及检测靶点数量的局限,而本研究应用的人类蛋白组芯片在很大程度上克     

一氧化氮相关的巯基亚硝基化修饰及其对血管功能的调控

一氧化氮(nitric oxide,NO)作为重要的血管舒张活性因子已成共识。近年来,NO的非c GMP依赖调控机制——巯基亚硝基化修饰受到广泛关注。巯基亚硝基化属于蛋白质翻译后修饰,广泛参与调控生物体内各种生理病理过程。本综述将从NO相关的蛋白质巯基亚硝基化的发生和调控等方面简要介绍近年来相关工作的研究进展,并着重阐述巯基亚硝基化修饰在血管生理及相关疾病中发挥的调节作用。     

蛋白质巯基亚硝基化参与调控一氧化氮介导的心肌缺血预适应

一氧化氮（nitricoxide,NO）作为一种重要的信使分子参与缺血预适应（ischemic preconditioning,IPC）心肌保护。目前普遍认为NO通过经典的NO／cGMP依赖的信号转导途径调节线粒体ATP敏感性钾（ATP-sensitive potassium,KATP通道来发挥其保护作用,然而越来越多的数据表明NO还可能通过蛋白质巯基亚硝基化（S-nitrosylation）来发挥生理功能。蛋白质巯基亚硝基化,即蛋白质半胱氨酸巯基与NO基团形成共价键,是一种氧化还原依赖的蛋白质翻译后可逆修饰。蛋白质巯基亚硝基化不仅可以改变蛋白质的结构和功能,而且还可以阻抑目标半胱氨酸的进一步氧化修饰。IPC增加S-亚硝基硫醇（S-nitrosothi01）含量,引起蛋白质巯基亚硝基化。S-亚硝基硫醇还能发挥药理性预适应作用,抵抗心肌缺血,再灌注损伤。因此,蛋白质巯基亚硝基化是IPC心肌保护的一种重要途径,参与抵抗细胞内氧化应激和亚硝化应激（nitrosative stress）。     

硫化氢介导的S-巯基化修饰及其化学检测技术

硫化氢(hydrogen sulfide, H₂S)是继一氧化氮和一氧化碳之后的第3种内源性气体信号分子,通过影响细胞信号通路调节机体各个系统,具有广泛的生理和病理作用。近年的研究发现,H₂S的作用主要通过对靶蛋白进行S-巯基化修饰,改变其结构,影响其活性、稳定性以及蛋白质之间互相作用,进而调控细胞内信号通路及相关生物学过程。本文基于国内外对S-巯基化修饰的研究,主要综述了：S-巯基化修饰的研究现状,详细总结了目前已揭示S-巯基化修饰及其具体半胱氨酸(cysteine, Cys)位点的研究,并进一步总结了S-巯基化修饰与S-亚硝基修饰之间的关系;S-巯基化修饰的反应类型,3种化学检测(马来酰亚胺法、改良生物素转换法和标记转换法)方法的检测原理和特点,通过S-巯基化修饰检测的条件和应用范围,将这3种方法进行对比,并对各方法在检测过程中应注意的事项进行了讨论。根据作者在S-巯基化修饰研究方面积累的经验,重点阐述马来酰亚胺法检测过程中,细胞和组织样品的制备及其较详细的实验步骤。此外,本文还讨论了当前S-巯基化修饰检测方法存在的不足及该领域未来亟待解...     

氧化还原信号转导的分子机制

氧化还原调控参与多种生物学过程,包括细胞增殖、分化和凋亡等的细胞信号转导和基因表达调控,因而在细胞生命活动中扮演着非常重要的角色。细胞内各种氧化还原介质,如活性氧(reactive oxygen species,ROS)和活性氮(reactive nitrogen species,RNS)等,能对多种蛋白质在半胱氨酸残基上进行可逆性修饰。ROS或RNS对靶蛋白的氧化还原修饰方式主要有巯基／二硫键转换反应、S-亚硝基化及谷胱甘肽化等,这些修饰方式构成了胞内氧化还原信号转导的主要机制。     

硫氧还蛋白的共价修饰

硫氧还蛋白是细胞中普遍存在的低分子量蛋白质,为生物体所必需。硫氧还蛋白、硫氧还蛋白还原酶和烟酰胺腺嘌呤二核苷磷酸组成硫氧还蛋白系统,调节细胞的氧化还原状态。硫氧还蛋白不仅维持细胞的氧化还原平衡,还具有抗凋亡及促进细胞增殖等功能。原核细胞的硫氧还蛋白仅含有两个半胱氨酸残基,真核细胞的硫氧还蛋白除了活性中心的两个半胱氨酸残基外,通常还有另外的半胱氨酸残基。这些半胱氨酸残基的共价修饰使硫氧还蛋白具有了更丰富的功能。硫氧还蛋白的共价修饰包括谷胱甘肽化、巯基氧化、亚硝基化和烷基化。     

Inhibition of the catalytic activity of rhodanese by S-nitrosylation using nitric oxide donors

Rhodanese (EC 2.8.1.1.) from bovine liver contains four reduced cysteine groups. The –SH group of cysteine 247, located in a rhodanese active centre, transfers sulfane sulfur in a form of hydrosulfide (–S–SH) from appropriate donors to nucleophilic acceptors. We aimed to discover whether S-nitrosylation of critical cysteine groups in rhodanese can inhibit activity of the enzyme by covalent modification of –SH groups.

The inhibition of rhodanese activity was studied with the use of a number of nitric oxide (NO) donors. We have successfully confirmed using several methods that the inhibition of rhodanese activity is a result of the formation of stable S-nitrosorhodanese.

Low molecular weight NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO), inactivate rhodanese and are much more effective in this regard (100% inhibition at 2.5 mM) than such known inhibitors of this enzyme, as N-ethylmaleimide (NEM) (25 mM < 50%) or sulfates(IV) (90% inhibition at 5 mM). On the other hand, sodium nitroprusside (SNP) and nitrites inhibit rhodanese activity only in the presence of thiols, which suggests that S-nitrosothiols (RSNO) also have to participate in this reaction in this case.

A demonstration that rhodanese activity can be inhibited as a result of S-nitrosylation suggests the possible mechanism by which nitric oxide may regulate sulfane sulfur transport to different acceptors.     

一氧化氮介导蛋白质亚硝基化与甲基化协调植物非生物胁迫的分子机制

一氧化氮(NO)作为一种具有活性的小分子物质参与众多动植物生理活动。在蛋白转录后修饰方面,NO主要以S-亚硝基化(S-nitrosylation)的形式参与。而甲基化作为另一种蛋白翻译后修饰,在DNA损伤及m RNA翻译方面具有重要作用。虽然近年来有关这2种蛋白翻译后修饰方面的研究成果较多,但是2种途径之间是否存在相互作用却报道较少。近期,我国科学家发现NO可以通过S-亚硝基化修饰PRMT5的第125位半胱氨酸,正向调节该精氨酸甲基转移酶活性。prmt5-1突变体表现出严重的发育障碍且对非生物胁迫敏感。通过互补第125位半胱氨酸点突变PRMT5基因,使之转化为不可被S-亚硝基化修饰的氨基酸后,拟南芥(Arabidopsis thaliana)植株可恢复突变体的发育障碍,但无法恢复其非生物胁迫敏感表型。实验同时证明,PRMT5蛋白第125位半胱氨酸的S-亚硝基化修饰参与调节NaCl诱导的精氨酸对二甲基化。该研究引领了蛋白S-亚硝基化和蛋白甲基化修饰新方向,开辟了新的研究领域,同时为相关研究树立了新的榜样。     

不同基质栽培的药用鲍姆桑黄孔菌体外抗肿瘤活性的比较研究

比较3种不同栽培技术的鲍姆桑黄孔菌Sanghuangporus baumii——3年生栎树段木鲍姆桑黄孔菌、桑枝代料鲍姆桑黄孔菌和发酵鲍姆桑黄孔菌中多糖、总酚、黄酮及总三萜含量和体外抗肿瘤活性。分别采用硫酸-蒽酮、福林-酚、亚硝酸-硝酸铝和香草醛-冰醋酸法对4种主要成分进行含量测定。选用人大细胞肺癌细胞（H460）、人前列腺癌细胞（PC3）、人乳腺癌细胞（MDA-MB-231）、人肝癌细胞（SMMC-7721和BEL-7402）和人神经母细胞瘤细胞（SH-SY5Y）为待检细胞株,采用CCK-8检测细胞活力。结果显示,发酵鲍姆桑黄孔菌多糖含量最高（29.64%）,代料鲍姆桑黄孔菌和3年生段木鲍姆桑黄孔菌多糖含量均低于2.57%;2种代料鲍姆桑黄孔菌的总酚和黄酮含量较高,而发酵鲍姆桑黄孔菌最低（0.24%和0.68%）,尤其是代料鲍姆桑黄孔菌1号的总酚和黄酮含量高达4.02%和32.13%;三萜含量在4种栽培法的鲍姆桑黄孔菌中差异不明显。体外对6种肿瘤细胞增殖抑制能力最强的均为代料鲍姆桑黄孔菌,其IC₅₀值最低,其次是3年生段木鲍姆桑黄孔菌,发酵鲍姆桑黄孔菌在本研究设置的浓度范围内无明显的细胞毒性。结果表明：不同栽培方式鲍姆桑黄孔菌产生体外抗肿瘤活性不同,代料鲍姆桑黄孔菌最强,其次是3年生段木鲍姆桑黄孔菌,发酵鲍姆桑黄孔菌活性最差,这种活性差异与其有效成分含量高低直接相关;鲍姆桑黄孔菌发挥抗肿瘤活性的物质基础主要是总酚和黄酮。     

Cellular Thiol Production and Oxidation of Low-Density Lipoprotein

Compelling evidence suggests that low-density lipoprotein (LDL) is oxidized by cells within the arterial intima and that, once oxidized, it is profoundly atherogenic. The precise mechanism(s) by which cells promote the oxidation of LDL in vivo are not known; in vitro, however, oxidation of LDL can be enhanced by a number of differing mechanisms, including reaction with free and protein-bound metal ions, thiols, reactive oxygen species, lipoxygenase, myeloperoxidase and peroxynitrite. This review is concerned with the mechanisms by which cells enhance the oxidation of LDL in the presence of transition metals; in particular, the regulation, pro- and anti-oxidant consequences, and mechanism of action of cellular thiol production are examined, and contrasted with thiol-independent oxidation of LDL in the presence of transition metals.     

Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA

-erythro-5,6,7,8-Tetrahydrobiopterin (BH₄), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH₄ exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH₄. Thus, TH activity was observed only in the presence of exogenous BH₄. We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH₄ in the human TH-transfected cells. The effect of exogenous BH₄ was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 μM endogenous (6R)-BH₄. The rate of uptake of both BH₄ isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 μM but was nearly saturated at 1 mM BH₄. TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH₄ dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH₄ was approximately three times as active as (6S)-BH₄. In contrast, in PC12h cells which contained endogenous (6R)-BH₄ (approximately 100 μM), exogenous (6R)-BH₄ activated DOPA formation maximally at 500 μM about 10-fold, while (6S)-BH₄ activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.     

Oxidative damage to surfactant protein D in pulmonary diseases

Surfactant protein D is an important innate host defence molecule that has been shown to interact with a variety of pathogens and to play a role in surfactant homeostasis. The aim of this study was to examine the influence of oxidation on surfactant protein D in different lung diseases. Bronchoalveolar lavage fluids (BALFs) from patients with different grade of protein oxidation were examined for changes in the primary chain and the quaternary structure of surfactant protein D. Significant changes of quaternary surfactant protein-D (SP-D) structure were detected under oxidative conditions in vitro and in vivo. The functional capacity of surfactant protein D to agglutinate bacteria was impaired by oxidation. We conclude that surfactant protein D is an important target of free radicals generated in the lungs. Host defence may be impaired due to the oxidation of surfactant protein D and may contribute to the suppurative lung diseases like cystic fibrosis (CF).     

植物中验证蛋白相互作用的Pull-down和Co-IP技术

蛋白互作在细胞生命活动中发挥关键作用,在不同时空层面上参与多种细胞学过程,因此研究蛋白互作对理解分子调控网络至关重要。通常情况下,利用酵母双杂交系统筛选植物蛋白互作必须通过体外和体内系统进行验证。Pull-down和Co-IP是验证植物蛋白互作的常用技术。Pull-down被广泛用于体外验证蛋白间的直接互作;而在植物活体内,利用本氏烟草(Nicotiana benthamiana)叶片瞬时表达蛋白,继而通过Co-IP进行鉴定是目前验证蛋白互作最简单且最有效的方法之一。该文对GST Pull-down和烟草瞬时表达系统中Co-IP技术原理及实验方案进行详细描述,以期为验证植物蛋白互作提供参考。     

鲍姆桑黄五个品种栽培特性及功能成分的比较分析

测定比较了鲍姆桑黄5个品种的菌丝生长速度、原基分化时间、农艺性状和产量,子实体多糖、总黄酮、总三萜和总酚含量,以及其对ABTS+自由基、DPPH自由基和羟自由基的清除能力.结果 表明:相同培养条件下,鲍姆桑黄不同品种菌丝生长速度、原基分化时间、农艺性状、产量、活性成分及抗氧化能力均存在差异;鲍姆桑黄HN01菌丝生长速度...     

Fractal structure and conformational entropy of protein chain

The structures of 25 proteins arbitrarily chosen are investigated by fractal geometry, and their fractal dimensions (D_f) and conformational entropies S(N₀) are calculated by Havlin—Ben Avraham and Monte Carlo method, respectively. Comparison of the D_f and S(N₀) gives the relation: D_f = 1.532 - 3.00 × 10⁻⁴ S(N₀). The entropy data obtained by Monte Carlo method for the chain of random self-avoiding walks confirm the prediction of renormalization group: S(N₀) = 1.544N₀ + 0.1667 In N₀ + 0.1570 where N₀ is the number of residues in a protein chain. Both the D_f and S(N₀) reflect the conformational properties of a protein molecular chain. The idea resulting from the present communication suggests that the thermodynamic behaviours of proteins may be related to multifractals.