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)和翻译水平(酶蛋白合成)两个方面。随着研究的深入,发现蛋白质翻译后修饰(PTM)对SOD的酶活性有重要影响。近年来,研究蛋白质翻译后修饰对SOD的酶活性的影响越来越受到重视。总结了硝基化、磷酸化、S-谷胱甘肽化、糖基化、乙酰化、次磺酸化、亚磺酸化、SUMO化等几种SOD翻译后的修饰方式,讨论了修饰后对SOD酶活性的影响和生理意义,并对SOD翻译后修饰的发展及面临的挑战进行了展望,为相关疾病的研究、治疗及靶向药物的研制提供了理论基础。     

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.     

Effect of chemical modification on the cryoprecipitation of monoclonal human cryoglobulin M

The effect of the chemical modification of lysine, histidine, arginine, tyrosine, tryptophan residues and carboxylic groups on the cryoproperties of monoclonal human cryoglobulin M has been studied. The modification of 35-40 lysine residues and that of 42-45 arginine residues in the molecule of cryo-IgM has been shown to result in practically complete inhibition of the cryoprecipitation. The same effect is observed on the modification of 60 histidine residues per molecule and on modification of 50 or 51 carboxylic groups. At the same time the modification of practically all the reagent-exposed tryptophan (10 residues per molecule) and tyrosine residues (55 residues per molecule) does not lead to any noticeable decrease in the cryoprecipitation. The conformations of the modified and native proteins are identical according to the circular dichroism data.     

The mechanisms for the effects of dietary lipid modification on autoimmune diseases: the role of membrane lipid composition in antigen presenting cells.

Several mechanisms have been proposed to explain the effects of dietary lipid modification on autoimmune diseases. One of these being the modification of cell membranes which affects immune functions. In order to further define this mechanism, the author proposes that dietary lipid modification could affect antigen presentation, an immune function responsible for initiating cell-mediated immune responses in body defense or autoimmune diseases, through membrane lipid composition modification.     

Structural and functional features of the 5-methylcytosine distribution in the eukaryotic genome

DNA methylation is an integral part of the mechanism of a remodeling and modification of the chromatin structure. The global complex net of chromatin modification and remodeling reactions is still to be determined, and studies of the mechanisms controlling the epigenetic processes of histone modification and DNA methylation are in their infancy. Cytosine methylation occurs predominantly in CpG sequences of the eukaryotic genome, and it also takes place at symmetric CpHpG and nonsymmetric CpHpH sites (where H is A, T, or C). The modification efficiency of the three types of DNA methylation sites depends on their genomic localization. Different regions of the eukaryotic genome are remarkable for their methylation features: CpG-islands, CpG-island shores, differentially methylated regions of imprinted genes, and regions of nonalternative site-specific modification. The three canonical sites (CpG, CpHpG, and CpHpH) differ in DNA methylation efficiency depending on their nucleotide context. An epigenetic code of DNA methylation can be assumed with context differences playing a specific functional role. The review summarizes the main up-to-date data on the structural and functional features of site-specific cytosine methylation in eukaryotic genomes. Pathogenesis-related alterations in the methylation pattern of the eukaryotic genome are considered.     

蓖麻碱的提取、纯化、改性及其杀虫活性研究

蓖麻饼中含有生物碱等毒性物质,主要杀虫活性物质为蓖麻毒蛋白和蓖麻碱,蓖麻碱是蓖麻中的主要毒素之一,具有一定的生物活性。本文研究了蓖麻碱的提取、纯化,以及将所得蓖麻碱再进一步进行改性,探讨改性方法。采用红外光谱方法对蓖麻碱改性前后变化进行对比;并对提取、纯化以及改性过程中的各个环节的物质进行杀虫实验,对实验结果进行观察。结果表明,蓖麻碱的主要杀虫活性基团为氰基。     

Modulation of the hydrophobicity of glutamine synthetase by mixed-function oxidation

Oxidative modification of Escherichia coli glutamine synthetase renders the enzyme susceptible to proteolytic degradation by a specific protease purified from the bacterium; native enzyme is not a substrate for the protease. A model oxidizing system consisting of ascorbate, iron, and oxygen was used to generate a series of glutamine synthetases of increasing oxidative modification. We assessed the effect of oxidative modification on the surface hydrophobicity of the glutamine synthetases, utilizing hydrophobic chromatography on a phenyl matrix. Initial exposure to the oxidizing system caused inactivation of the enzyme and generated a protein that was more hydrophilic than the native form; it was not a substrate for the protease. Continued exposure to the oxidizing system yielded a protein with additional oxidative modification. This form was distinctly more hydrophobic than the native form and it was very susceptible to proteolytic attack by the purified protease. Thus, oxidative modification modulates the surface hydrophobicity of glutamine synthetase, and this modulation can control susceptibility to proteolysis.     

Changes in the post-translational modification of lysosomal enzymes during development of Dictyostelium

The form of post-translational modification present on two lysosomal enzymes--acid phosphatase and alpha-mannosidase--changes as part of the developmental program of Dictyostelium discoideum. Prior to 8 h of development, all enzyme molecules are of a single modification type (early form enzyme). Starting at 8 h of development, enzyme molecules with a second type of modification (late-form enzymes) begin to appear in the cell. We separated the early and late forms of these enzymes from each other by chromatography on DEAE-cellulose. We found that the change in protein modification affects the enzymes' in vitro properties. The early and late forms of both of these enzymes differ in thermostability and susceptibility to proteolytic inactivation. We also found that the late form of alpha-mannosidase is preferentially secreted. We suggest that by synthesizing molecules with a second form of modification, the cell confers new characteristics to its lysosomal enzymes.     

Nature of tryptophan photooxidation products in lysozyme in the presence of methylene blue]

One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue.     

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.     

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.     

Biophysical studies of the modification of poly(rG) . poly(rC) by cisplatin. Relations to the biological activity of the complex

The integrity of the double-stranded complex polyriboguanylic.polyribocytidylic acid [poly(rG).poly(rC)] modified by antitumour cis-diamminedichloroplatinum(II)(cis-DDP) was studied with the aid of differential pulse polarography and terbium fluorescence measurement. The modification was made to level corresponding to rb = 0.05 (rb is defined as the number of platinum atoms covalently bound per one nucleotide residue). Two modes of the modification of the polynucleotide complex were employed: The action of cis-DDP on poly(G) before formation of the complex with poly(C) and on the complex already formed from non-modified polynucleotides. It was shown that in the latter case modification disordered the integrity of the complex only negligibly. while in the former case the modification resulted in a noticeably more extensive disturbance of the double-stranded polynucleotide complex. Moreover, the modification of the complex (after its formation) at rb = 0.02 led to improved interferon-inducing and antiviral activity of poly(rG).poly(rC) tested on mice infected by influenza virus. It was suggested that the combined effects of interferon-inducing and antiviral activities of poly(rG).poly(rC) and antiviral activity of cis-DDP may result in an increased effect over and above what may be expected from the actions of the two modalities separately.     

Quantitative estimation of the modification of mutagenic effect in experiments on mammals using a regression modification index

A regressive modification index (RMI = beta M - beta/beta.100) is suggested to quantitatively evaluate modification of the mutagenic effect in experiments on mammals. It is based on the correlation of regressive equation "dose mutagen--effects" coefficients beta M and beta in variants with and without modifier, respectively. Regressive modification indices of various doses of phenobarbital are calculated in experiments on rats with cyclophosphamide and fotrin.     

维生素C抑制低密度脂蛋白的氧化修饰

研究了不同浓度维生素 C 对 Cu²⁺诱导的低密度脂蛋白(LDL)氧化修饰的抑制作用,通过测定硫代巴比妥酸反应物质(TBARS),荧光物质(lipofusion)扫描及琼脂糖电泳,显示一定浓度的维生素 C 在24h 内对 LDL 的氧化修饰具有抑制作用,并呈现量效效应.提示维生素 C 作为体内存在的一种抗氧化物,可抑制 LDL的氧化修饰,从而在防治动脉粥样硬化的发生具有一定意义.     

Influence of Shifting Positions of Ser, Thr, and Cys Residues in Prenisin on the Efficiency of Modification Reactions and on the Antimicrobial Activities of the Modified Prepeptides

Since the recent discovery that the nisin modification and transport machinery can be used to produce and modify peptides unrelated to nisin, specific questions arose concerning the specificity of the modification enzymes involved and the limits of their promiscuity with respect to the dehydration and cyclization processes. The nisin leader peptide has been postulated to fulfill a recognition and binding function required for these modifications. Here, we investigated whether the relative positions of the modifiable residues in the nisin prepeptide, with respect to the leader peptide, could influence the efficiency of their modification. We conducted a systematic study on the insertion of one to four alanines in front of either ring A or ring D to change the “reading frame” of modifiable residues, resulting in altered distance and topology of the modifiable residues relative to the leader. The insertion of N-terminal and hinge-located Ala residues had only a modest influence on the modification efficiency, demonstrating that the “phasing” of these residues relative to the leader peptide is not a critical factor in determining modification. However, in all cases, but especially with the N-terminal insertions, the antimicrobial activities of the fully modified nisin species were decreased.     

N-type inactivation and the S4-S5 region of the Shaker K+ channel

The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.     

Seasonal dynamics of activity of oxidative modification of proteins and oxidation-inhibiting enzymes in the blood of goby Neogobius melanostomus inhabiting the Black Sea and the Sea of Azov

Seasonal dynamics of activity of oxidative modification of proteins and oxidation-inhibiting enzymes was studied in the blood of round goby Neogobius melanostomus that inhabits the coastal areas of the Black Sea and the Sea of Azov. The activity of oxidative modification of proteins and oxidation-inhibiting processes depend on the physiological state of a peculiar specimen, on the hydrochemical parameters of the environment, and on the anthropogenic load in the studied areas during different seasons.     

Cranial vault modification as a cultural artifact: a comparison of the Eurasian steppes and the Andes.

This paper details the practice of intentional cranial vault modification in the Eurasian steppes as well as in the pre-Columbian Andes focusing on the similarities and differences in how the practice was used to respond to changes in society. The appearance of vault modification in the steppes and the forms seen in the cemeteries of the Syr Darya and Amu Darya River deltas are discussed. Temporal changes in the pattern of modification are also investigated, especially the dramatic homogenization of the custom resulting from the conquests of the Huns. This is contrasted with incidences of cranial modification in the south-central Andes, including the appearance of deliberate head shaping as well as shifts in the practice during the expansion of the Bolivian Altiplano state of Tiwanaku. Similarities in the use of cranial vault modification between these unrelated areas and in the alterations of the practice resulting from foreign contact are considered in light of vault modification's role as a malleable cultural artifact.     

Genetic Control of the Secondary Modification of Deoxyribonucleic Acid in Escherichia coli

The wild-type restriction and modification alleles of Escherichia coli K-12 and B were found to have no measurable effect on the patterns of methylated bases in the deoxyribonucleic acid (DNA) of these strains. The genetic region controlling the methylation of cytosine in E. coli K-12 was mapped close to his, and the presence or absence of this gene in E. coli B or E. coli K had no effect on the restriction and modification properties of these strains. Thus, only a few of the methylated bases in the DNA of these strains are involved in host modification, and the biological role of the remainder remains obscure.