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）。     

蛋白质精氨酸甲基化参与基因转录后调控的研究进展

蛋白质精氨酸甲基化是真核生物中一种广泛存在并在进化上保守的蛋白质翻译后修饰,由蛋白质精氨酸甲基转移酶(PRMT)催化完成。动物中的研究表明,PRMT通过催化多种RNA结合蛋白的精氨酸甲基化而参与调控细胞多种重要的生命过程,如RNA代谢、细胞增殖以及信号转导等。概述真核生物中精氨酸甲基化对不同的RNA结合蛋白的功能调控,并重点阐述该翻译后修饰在转录后加工过程中的重要作用;介绍高等植物拟南芥中蛋白质精氨酸甲基转移酶参与转录后调控的最新研究进展,并对精氨酸甲基化修饰参与调控植物RNA结合蛋白的功能及今后可能的研究方向进行讨论。     

蛋白亚硝基化研究进展及其在植物抗病中的作用

蛋白亚硝基化（S-nitrosylation）是一种在一氧化氮作用下与蛋白半胱氨酸巯基共价结合,使巯基-SH转化为-SNO的反应。作为一种氧化还原依赖的翻译后调控形式,蛋白亚硝基化对多种蛋白的功能具有调节作用,越来越多的证据表明蛋白亚硝基化在植物抗病中发挥重要的作用。简要介绍了蛋白巯基亚硝基化的特点、检测方法、功能研究以及在植物抗病调节方面的最新进展。     

植物一氧化氮合成代谢与信号转导研究进展与展望

一氧化氮(nitric oxide, NO)是有机体内一种重要的气体信号小分子,通过介导S-亚硝基化修饰、酪氨酸硝基化修饰等翻译后修饰,影响蛋白的稳定性和活性.在植物中, NO调控生长发育和胁迫响应等多个生物学过程,并与植物激素、活性氧等信号分子之间形成复杂的交互调控网络,精细调控植物生长发育的各阶段,以维持植物的正常生命活动.本文概述了NO的合成与代谢、作用机制,以及NO在植物生长发育、胁迫响应中的重要生物学功能.     

植物亚硝基谷胱甘肽还原酶在胁迫反应中的作用研究

信号分子一氧化氮(Nitric oxide,NO)参与植物的许多生理反应过程,例如:萌发、气孔的关闭、侧根的发育以及生物与非生物的胁迫反应过程等,主要的调控形式是与半胱氨酸上的硫基发生可逆的S-亚硝基化作用。NO的半衰期很短,这限制了它在细胞中的生理功能,与胞内含硫基的分子形成的S-亚硝基硫醇(S-nitrosothiols,SNOs)的化学性质稳定,在植物的生长发育及抗逆过程中SNOs参与NO的运输、扩散、储存以及蛋白的翻译后修饰过程。谷胱甘肽(Glutathione,GSH)与NO发生S-亚硝基化作用形成S-亚硝基谷胱甘肽(S-nitrosoglutathione,GSNO),GSNO作为NO的储存与转运形式,可以把NO转到靶蛋白上,使靶蛋白发生亚硝基化。亚硝基谷胱甘肽还原酶(S-Nitrosoglutathione reductase,GSNOR)是生物体中的一类保守蛋白,通过还原亚硝基谷胱甘肽从而调节细胞内NO及亚硝基硫醇(S-nitrosothiols,SNOs)水平,保护机体免受亚硝化的胁迫,间接的调控的细胞的氧化状态。GSNO是一个天然的NO储存库,GSNOR是调节机体亚硝基化水平的关键基因。主要对GSNOR参与的植物生长发育、生物与非生物胁迫等过程进行了概述,探讨GSNOR在植物生长发育及胁迫反应中的作用机制,将有助于我们对NO生理功能的了解,旨在为将来GSNOR的研究提供理论参考和思路。     

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     

组蛋白精氨酸甲基化修饰对基因转录的调控

总结了组蛋白精氨酸甲基化修饰体系的最新研究进展．组蛋白精氨酸甲基化修饰在基因转录调控中发挥着十分重要的作用,这类修饰由蛋白精氨酸甲基转移酶（PRMTs）介导,其中PRMT1和PRMT4的甲基化修饰与基因的转录激活作用相关,PRMT5和PRMT6的甲基化修饰则与基因的转录抑制作用相关．组蛋白精氨酸的甲基化是一个动态的可逆过程,催化组蛋白精氨酸的去甲基化是由“精氨酸去甲基化酶”介导的．     

蛋白质精氨酸甲基化修饰在糖代谢调节中的作用

蛋白质精氨酸甲基化是重要的细胞翻译后修饰方式,参与众多生命过程. 精氨酸的甲基化修饰与糖代谢相关疾病如糖尿病、糖耐量异常密切相关. 蛋白质精氨酸甲基化转移酶(protein arginine methyltransferases, PRMTs)活性下降及表达异常是糖代谢疾病的重要发病基础. 目前研究表明,PRMT1、PRMT4、PRMT5在糖代谢调节中均扮演重要角色,与糖代谢关键酶如磷酸烯醇式丙酮酸羧基激酶、葡萄糖6磷酸酶,胰岛素受体 胰岛素受体配体1 磷脂酰肌醇3激酶通道及其它通路密切相关. 给予甲基化抑制剂MTA及siRNA干扰甲基化则可引发糖代谢紊乱,进而诱发糖代谢疾病. 糖尿病药物罗格列酮、氨基胍与蛋白质精氨酸甲基化也有一定联系. 深入研究蛋白质精氨酸甲基化与糖代谢调节之间的联系及机制,可为防治糖代谢疾病及相关并发症提供更多的理论依据.     

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.     

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。     

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.     

The RNA N6-methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis

The mRNA modification N⁶-methyladenosine(m⁶A)plays vital roles in plant development and biotic and abiotic stress responses.The RNA m⁶A demethylase ALKBH9 B can remove m⁶A in alfalfa mosaic virus RNA and plays roles in alfalfa mosaic virus infection in Arabidopsis.However,it is unknown whether ALKBH9 B also exhibits demethylation activity and has a biological role in endogenous plant mRNA.We demonstrated here that mRNA m⁶A modification is in...     

Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis

Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.     

Evidence for Ca-dependent protein phosphorylation in vitro in guard cells from Vicia faba L.

Protein phosphorylation in vitro was investigated in guard cells from Vicia faba. A number of proteins with apparent molecular masses of 72, 67, 57, 52, 49, 44, 37, and 26 kDa were phosphorylated when guard-cell extract was incubated with [γ-³²P]ATP under Ca²⁺-free conditions. In the presence of Ca²⁺ at 1 μM, several proteins with apparent molecular masses of 125, 83, 41, 31, and 25 kDa were newly phosphorylated. These Ca²⁺-dependent protein phosphorylations were suppressed by (8R^*,9S^*,11S^*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a- triazadibenzo[a,g]cycloocta[cde]trinden-1-one (K-252a), a wide-range inhibitor of protein kinases, suggesting that the protein phosphorylations were mediated by protein kinases. Several proteins were phosphorylated in vitro in mesophyll extract from Vicia. In contrast to guard cells, there was no detectable Ca²⁺-dependent protein phosphorylation in mesophyll cells. 1-(5-Indonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), an inhibitor of myosin light chain kinase (MLCK), and an antagonist of calmodulin (CaM), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins in guard cells. Fractionation experiments revealed that the Ca²⁺-dependent phosphorylated proteins with molecular masses of 41 and 25 kDa were present in the mitochondria, and the 125- and 31-kDa proteins in the cytosol. These results suggest that Ca²⁺-dependent protein phosphorylation occurs markedly in guard cells, and that Ca²⁺-dependent phosphorylation of 41- and 25-kDa proteins may be catalyzed by MLCK or MLCK-like protein kinase in guard cells.     

Sm protein methylation is dispensable for snRNP assembly in Drosophila melanogaster

Sm proteins form stable ribonucleoprotein (RNP) complexes with small nuclear (sn)RNAs and are core components of the eukaryotic spliceosome. In vivo, the assembly of Sm proteins onto snRNAs requires the survival motor neurons (SMN) complex. Several reports have shown that SMN protein binds with high affinity to symmetric dimethylarginine (sDMA) residues present on the C-terminal tails of SmB, SmD1, and SmD3. This post-translational modification is thought to play a crucial role in snRNP assembly. In human cells, two distinct protein arginine methyltransferases (PRMT5 and PRMT7) are required for snRNP biogenesis. However, in Drosophila, loss of Dart5 (the fruit fly PRMT5 ortholog) has little effect on snRNP assembly, and homozygous mutants are completely viable. To resolve these apparent differences, we examined this topic in detail and found that Drosophila Sm proteins are also methylated by two methyltransferases, Dart5/PRMT5 and Dart7/PRMT7. Unlike dart5, we found that dart7 is an essential gene. However, the lethality associated with loss of Dart7 protein is apparently unrelated to defects in snRNP assembly. To conclusively test the requirement for sDMA modification of Sm proteins in Drosophila snRNP assembly, we constructed a fly strain that exclusively expresses an isoform of SmD1 that cannot be sDMA modified. Interestingly, these flies were viable, and snRNP assays revealed no defects in comparison to wild type. In contrast, dart5 mutants displayed a strong synthetic lethal phenotype in the presence of a hypomorphic Smn mutation. We therefore conclude that dart5 is required for viability when SMN is limiting.     

Dirhodium(II) tetrakis[N,N-dimethyl-2-pyrrolidone-5(S)-carboxamide]. Structural effects on enantioselection in metal carbene transformations

The preparation and structural characterization of dirhodium(II) tetrakis[N,N-dimethyl-2-pyrrolidone-5(S)-carb- oxamide], Rh₂(5S-DMAP)₄, a new sterically-demanding catalyst for enantioselective metal carbene transformations, is described. The pyrrolidone ligands are arrayed around the dirhodium(II) core with two oxygen and two nitrogen donor atoms, each oriented cis, bound to each octahedral rhodium. The crystal structure of this compound has been determined to be that of Rh₂(5S-DMAP)₄(CH₃CN)₂·CH₃CN·6H₂O: space group P2₁2₁2₁ with cell constants a= 12.685(4), b=15.050(3), c=24.035(4) Å; V=4588.5(1.9) ų, Z=4, R=0.0316, Rh---Rh DISTANCE =2 4538(5) Å. Decreased activity for diazodecomposition catalyzed by Rh₂(5S-DMAP)₄ is observed, and enantiocontrol for cyclopropanation and carbon-hydrogen insertion is lower than expected by analogy to the corresponding di- rhodium(II) tetrakis[methyl 2-pyrrolidone-5(S)-carboxylate], Rh₂(5S-MEPY)₄ Electronic stabilization of the in- termediate metal carbene is absent in reactions catalyzed by Rh₂(5S-DMAP)₄.     

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation

Although the Trithorax histone methyltransferases ATX1–5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1–5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylationmainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.