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。     

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。     

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）。     

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。     

硫巯基化：一种新的蛋白质翻译后修饰

随着对硫化氢（hydrogen sulfide,H2S）生理效应的研究,蛋白质硫巯基化（S-sulfhydration）修饰已进入人们的视野。已知依赖于H2S的蛋白质硫巯基化是继磷酸化（phosphorylation）、泛素化（ubiquitylation）、乙酰化（acetylation）和S-亚硝基化（S-nitrosylation）等之后的一种新的蛋白质翻译后修饰方式。对动物的研究表明,蛋白质硫巯基化修饰通过影响蛋白质活性和功能,从而在细胞内信号通路中发挥重要的调控作用。最近的研究结果提示,硫巯基化修饰还参与调节植物新陈代谢和形态建成。本文阐述了依赖于H2S的蛋白质硫巯基化的作用机制、检测方法和生理功能,并提出硫巯基化修饰也可能参与植物细胞信号转导的观点。     

转录因子的巯基亚硝基化修饰及其生理学意义

巯基亚硝基化(S-nitrosylation)修饰是一种一氧化氮(nitric oxide, NO)介导的氧化还原依赖的、可逆性蛋白质翻译后修饰。生理条件下,S-nitrosylation通过调控蛋白质的稳定性、蛋白质活性、亚细胞定位及蛋白质-蛋白质相互作用,在维持细胞稳态中发挥重要作用。而在多种病理条件下,蛋白质S-nitrosylation及其产物表现出异常的升高或降低。转录因子又称反式作用因子,通过识别并结合调控元件而影响基因转录。本文简要综述转录因子的S-nitrosylation修饰的研究进展及其生理学意义。     

阿兹海默症中S-亚硝基化蛋白质组确定及功能分析

<正>蛋白质的S-亚硝基化修饰在阿兹海默症(AD)发病机制中的影响已有初步研究成果,实验证明阿兹海默症中错误折叠的蛋白质形成与聚集均与蛋白质亚硝基化修饰有关联。近期,来自巴基斯坦卡拉奇大学和德国哥廷根大学医学中心的研究团队对阿兹海默症中发生S-亚硝基化修饰的蛋白质进行了筛查,并确定出45个蛋白靶点,其中超氧岐化酶(SOD2)[Mn]、果糖     

蛋白质酪氨酸硝化和细胞信号转导

蛋白质硝基酪氨酸作为一氧化氮(NO)衍生的蛋白质翻译后修饰产物,被认为是许多生理和病理条件下的生物标志物。本文综述了蛋白质酪氨酸硝化可以作为信号调节元件与已知的信号途径相关,包括NO、蛋白质酪氨酸激酶、丝裂原激活蛋白激酶、T-淋巴细胞、转录因子NF-κB、Ca2 等。同时也论证了蛋白质酪氨酸硝化作为信号转导元件的可能性。     

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.     

Nonlinear laser photomodification of nucleic acids induced by intercalating dyes

Results of a cycle of investigations of two-quantum affinity modification of nucleic acids (NA) are presented. The modification is induced by laser excitation of chromophoric molecules which are in intercalative complexes with NA. The following subjects are considered: theoretical basis of the two-quantum affinity modification: experimental investigation of nonlinear scission of DNA; theory and experiment on the light--induced diffusion of DNA. The latter is an effect which accompanies the scission and allows one to obtain information on it. The modification specificity and universality in a dye type are established experimentally. Influence of free radicals, oxygen, heating and hydrodynamical phenomena in bulk are excluded. The modification has been shown to be dependent on NA structure (secondary and tertiary) and to provide information on it. Total aggregate of the data obtained is in agreement with the suggested modification mechanism which is based on the radiationless transfer of two-quantum excitation energy from the chromophore to NA.     

Superiority of single covalent modification in specificity: From deterministic to stochastic viewpoint

Covalent modification(s) are required in many signaling pathways. It has been discussed from a deterministic viewpoint that dual covalent modification is more favorable than single covalent modification for signaling specificity. However, whether this conclusion is feasible in stochastic situation has not yet been studied. To study the role of covalent modification in the specificity of a stochastic signaling pathway, we here simulate the dynamics of a transiently stimulated signaling pathway, considering the influence of the stochasticity arising from the low molecule number of reactants. It turns out that the specificity of dual covalent modification would be worse than that of single covalent modification when the number of molecules is in some biologically plausible range. We further discuss some factors that have potential influence on specificity, such as the rates of the upstream reaction cycle of the covalent modification(s), the duration and the magnitude of the transient stimulus. Our numerical results indicate that whether dual or single covalent modification(s) is better in specificity also depends on these factors. Superiority of single covalent modification in specificity would arise if the stimulus is weak and transient, or if it is embedded downstream of a reaction whose activation rate is slow while deactivation rate is fast. The relevance of these conclusions to signal transduction is briefly discussed.     

Adaptation in bacterial chemotaxis: CheB-dependent modification permits additional methylations of sensory transducer proteins

Sensory transduction in E. coli consists of two phases, excitation and adaptation, both of which involve the methyl-accepting chemotaxis proteins (MCPs). These molecules relay transmembrane signals and are reversibly methylated during adaptation of E. coli to environmental stimuli. Each MCP contains multiple sites of methylation, and we identified six of these sites in MCPI. Recently, a second covalent modification of MCPs has been identified, which is not methylation. This modification, designated CheB-dependent modification, is stimulated by repellents and causes a net increase in the negative charge of MCPI and MCPII by one or two charges. We demonstrate that one CheB modification occurs on the methyl-accepting methionine-and lysine-containing tryptic peptide in MCPI and MCPII, and the second CheB modification is on an arginine-containing tryptic peptide. The CheB modification allows three additional methyl groups to be incorporated into the methyl-accepting methionine-lysine peptide, while not actually creating all of these methylation sites. The two CheB modifications occur sequentially. A possible mechanism by which CheB modification permits additional methylations and the role of CheB modification in bacterial chemotaxis are discussed.     

The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1

Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IkappaBalpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity-ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.     

An improved method for the sensitive monitoring of low density lipoprotein modification by myeloperoxidase

The aim of this investigation was to compare an improved fluorometric method with an UV absorbance assay for their ability to monitor low density lipoprotein (LDL) modification by myeloperoxidase (MPO) and to evaluate determining factors influencing the modification of LDL. Using absorbance at 234 nm to study the kinetics of LDL aggregation, and a native fluorescence assay for protein oxidation, we found that all components of the MPO/H2O2/Cl- system may have rate determining effects on LDL modification. While the lipoprotein modification rate correlated positively with enzyme concentration, variation of the concentration of H2O2 had a biphasic effect on the maximal rate of LDL modification with both methods. Furthermore, a positive association was found between the maximal rate of LDL modification and the acidity of the medium, with a pathophysiologically relevant optimal rate at a slightly acidic pH of 5-6, but hardly any modification above pH 6.8. In summary, both methods provide simple and useful tools for the continuous monitoring of LDL modification by the MPO/H2O2/Cl- system, but the more sensitive fluorometric method is preferable, since it allows the application of experimental conditions which are much closer to the situation in vivo.     

超氧化物歧化酶翻译后修饰的研究进展

超氧化物歧化酶(superoxide dismutase,SOD)是生物体内存在的一种抗氧化金属酶,它能够催化超氧阴离子自由基歧化生成氧(O2)和过氧化氢(H2O2),在机体氧化与抗氧化平衡中起到至关重要的作用,且与很多疾病的发生、发展密不可分.对SOD的活性调节一直是研究热点,大多数研究都集中在转录水平(基因表达)和...     

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg39 and Arg85, which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg10, while Arg33 appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg39 and Arg85 are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.     

Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.