蛋氨酸亚砜/蛋氨酸亚砜还原酶荧光检测研究进展

蛋白质蛋氨酸亚砜化是一种重要的氧化还原依赖的蛋白质翻译后修饰,不仅是氧化应激的重要标志物之一,也是一种蛋白质功能调控开关可影响活性氧信号转导,与一系列疾病尤其是神经退行性疾病的发生发展密切相关。 在许多生物体中,蛋氨酸亚砜还原酶是目前已经发现的唯一能将蛋白质蛋氨酸亚砜还原为蛋氨酸的物质,可以修复氧化损伤蛋白,恢复蛋白质功能,调控细胞氧还平衡,对相关疾病的治疗具有非常重要的意义。 本文重点介绍蛋氨酸亚砜和蛋氨酸亚砜还原酶的结构和催化机理,综述蛋氨酸亚砜和蛋氨酸亚砜还原酶荧光探针的部分研究进展,对该领域的研究前景进行展望。     

Type I Photosensitized Oxidation of Methionine†

Methionine (Met) is an essential sulfur‐containing amino acid, sensitive to oxidation. The oxidation of Met can occur by numerous pathways, including enzymatic modifications and oxidative stress, being able to cause relevant alterations in protein functionality. Under UV radiation, Met may be oxidized by direct absorption (below 250 nm) or by photosensitized reactions. Herein, kinetics of the reaction and identification of products during photosensitized oxidation were analyzed to elucidate the mechanism for the degradation of Met under UV‐A irradiation using pterins, pterin (Ptr) and 6‐methylpterin (Mep), as sensitizers. The process begins with an electron transfer from Met to the triplet‐excited state of the photosensitizer (Ptr or Mep), to yield the corresponding pair of radicals, Met radical cation (Met^?+) and the radical anion of the sensitizer (Sens^??). In air‐equilibrated solutions, Met^?+ incorporates one or two atoms of oxygen to yield methionine sulfoxide (MetO) and methionine sulfone (MetO₂), whereas Sens^?? reacts with O₂ to recover the photosensitizer and generate superoxide anion (O₂^??). In anaerobic conditions, further free‐radical reactions lead to the formation of the corresponding dihydropterin derivatives (H₂Ptr or H₂Mep).     

Determination of lipid hydroperoxides in low density lipoprotein from human plasma using high performance liquid chromatography with chemiluminescence detection

A high performance liquid chromatographic system with chemiluminescence detection (HPLC-CL) was used for determining phospholipid hydroperoxides in human plasma low density lipoprotein (LDL). This system involved separation of phospholipids from LDL-total lipids with normal phase silica gel HPLC and post-column detection of hydroperoxide-dependent chemiluminescence produced by luminol oxidation during the reaction of hydroperoxide with cytochrome c-haeme. By using HPLC-CL, we could detect phosphatidylcholine hydroperoxide (PCOOH) in human plasma LDL, and the LDL-PCOOH concentration was significantly higher in patients with atherosclerosis and hyperlipidemia than that of healthy volunteers. The LDL-PCOOH level was proportional to the plasma total cholesterol concentration.     

A Facile Method for the Separation of Methionine Sulfoxide Diastereomers,Structural Assignment,and DFT Analysis

Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO₂ chromatography and allows obtaining both diastereomers in purities exceeding 99 %. ¹H NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2 kcal mol⁻¹, highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.     

蛋氨酸亚砜还原酶及其在白内障发生发展中的作用

蛋氨酸(Met)是生物体内很容易被氧化的氨基酸之一,氧化应激条件下,生成S型和R型蛋氨酸亚砜(MetO), 晶状体蛋白中MetO的增加与晶状体老化和白内障形成相关。生物体内存在着两种不同的蛋氨酸亚砜还原酶(Msr),即MsrA和B,分别能特异性地作用于自由或结合在蛋白质中的S-MetO和R-MetO,将MetO修复为Met,从而避免了蛋白质结构和功能的改变。在哺乳动物中,MsrA以单基因形式存在,而MsrB有3种异构体,分别为MsrB1,MsrB2和MsrB3,其中MsrB1是一个硒蛋白,又被称为硒蛋白R(SelR)。本文介绍了Msrs的基因表达、分布和亚细胞定位,比较了MsrA和MsrBs蛋白结构和催化机制的异同,讨论了晶状体蛋白Met残基的氧化与白内障形成和发展的关系。现有的这些研究结果表明Msrs作为一类特异性的抗氧化还原酶,通过对MetO的修复,在抑制晶状体的损伤方面发挥重要作用。此外,MsrB1作为一个硒蛋白受机体硒水平的调节,因此,通过补硒保持晶状体适当的硒浓度以维持MsrB1的活性,对白内障的形成和发展可能具有一定的预防作用。     

Interactions between sphingomyelin and cholesterol in low density lipoproteins and model membranes

This work examines three related, but previously unexplored, aspects of membrane biophysics and colloid science in the context of atherosclerosis. First, we show that sphingomyelinase (SMase)-induced aggregation of low density lipoproteins (LDLs), coupled with LDL exposure to cholesterol esterase (CEase), results in nucleation of cholesterol crystals, long considered the hallmark of atherosclerosis. In particular, this study reveals that the order of enzyme addition does not effect the propensity of LDL to nucleate cholesterol crystals, raising the possibility that nucleation can proceed from either the intra- or extracellular space. Second, we demonstrate that ceramide-rich aggregates of LDL release cholesterol to neighboring vesicles far more rapidly, and to a greater extent, than does native LDL. A likely explanation for this observation is displacement of cholesterol from SM–Chol rafts by “raft-loving” ceramide. Third, we demonstrate that a time-independent Förster resonance energy transfer (FRET) assay, based on dehydroergosterol and dansylated lecithin and used previously to study cholesterol nanodomains, can be used to measure raft sizes (on the order of 10 nm) in model membrane systems. Taken together, these observations point to the possibility of an extracellular nucleation mechanism and underscore the important role that biological colloids play in human disease.     

Determination of protein-bound methionine oxidation in the hippocampus of adult and old rats by LC-ESI-ITMS method after microwave-assisted proteolysis

Protein-bound methionine (Met) oxidation has been associated with normal aging and a variety of age-related diseases, including Alzheimer’s disease and Parkinson’s disease. Monitoring the changes of protein-bound methionine content in the brain in response to normal aging and oxidative stress is of great interest and could be used as an indicator of oxidative stress of rats in pathological conditions. We have developed a rapid analytical method for the determination of oxidized products of protein-bound methionine in rat brain. The assay involved rapid acid proteolysis with microwave irradiation and solid-phase extraction of the free amino acids followed by LC-ESI-ITMS analysis. Detection was achieved in positive ionization with an ion trap mass spectrometer operating in multiple-reaction monitoring mode. The calibration curves of the analytes were linear (r ² > 0.99) in the range between 0.098 and 1.560 μg/mL. Intra- and inter-day relative standard deviation percentages were <9% and <8%, respectively. The assay performance was sufficient to support a rapid analytical tool for monitoring brain protein-bound methionine oxidation levels. The content of protein-bound Met and methionine sulfoxide (MetO) in the hippocampus of adult and old rats with or without H₂O₂ treatment was determined by employing the new method. The content of protein-bound MetO was significantly increased in old rats after exposure to H₂O₂. This result indicates increased sensitivity to Met oxidation in the hippocampus of old rats.     

Homeostasis as regulated by activated macrophage. VII. Suppression of serum cholesterol level by LPSw (a lipopolysaccharide from wheat flour) in WHHL (Watanabe heritable hyperlipidemic) rabbit.

The effect of LPSw (a lipopolysaccharide from wheat flour) on cholesterol catabolism was examined using WHHL (Watanabe heritable hyperlipidemic) rabbit, which is an experimental model of familial hyperlipidemia. The serum cholesterol level of the animal decreased by the addition of LPSw to drinking water. Following cessation of the addition of LPSw to the drinking water, the cholesterol level was decreased for 30 to 40d and then gradually elevated. The serum level of apolipoprotein B, which is a constituent of apolipoprotein of low density lipoprotein (LDL), also decreased in accord with serum cholesterol at a nearly coincident rate. Conversely, the level of apolipoprotein A-I, which is a constituent of apolipoprotein of high density lipoprotein (HDL), did not change, nor did HDL-cholesterol. Furthermore, the atherosclerosis risk factor, expressed as the ratio of apolipoprotein B to apolipoprotein A-I, was decreased by LPSw administration.     

Electron Transfer Reactions of Photochemically Generated Ruthenium(III)–Polypyridyl Complexes with Methionines

The oxidation of methionine (Met) plays an important role during biological conditions of oxidative stress as well as for protein stability. Ruthenium(III)–polypyridyl complexes, [Ru(NN)₃]³⁺, generated from the photochemical oxidation of the corresponding Ru(II) complexes with molecular oxygen, undergo a facile electron transfer reaction with Met to form methionine sulfoxide (MetO) as the final product. Interaction of [Ru(NN)₃]³⁺ with methionine leads to the formation of >S^+● and (>S∴S<)⁺ species as intermediates during the course of the reaction. The interesting spectral, kinetic, and mechanistic study of the electron transfer reaction of four substituted methionines with six [Ru(NN)₃]³⁺ ions carried out in aqueous CH₃CN (1:1, v/v) by a spectrophotometric technique shows that the reaction rate is susceptible to the nature of the ligand in [Ru(NN)₃]³⁺ and the structure of methionine. The rate constants calculated by the application of Marcus semiclassical theory to these redox reactions are in close agreement with the experimental values.     

Anion-exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl-ligated gel with a perchlorate-containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion‐exchange high‐performance liquid chromatography using a column filled with nonporous, diethylaminoethyl‐ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high‐density lipoprotein, low‐density lipoprotein, intermediate‐density lipoprotein, very‐low‐density lipoprotein and chylomicrons) were eluted with step‐wise increases in a concentration of NaClO₄. The post‐column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein‐cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within‐day and between‐day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high‐fat diet increased lipoprotein‐cholesterol concentrations by 6‐ to 77‐fold. The method described here was nevertheless able to quantitate levels of lipoprotein‐cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models. Copyright © 2011 John Wiley & Sons, Ltd.     

Modulation of Substrate Specificity upon Modification of Cholesterol Oxidase from Brevibacterium Sterolicum by Hydrogen Peroxide: Evidence for a Peripheral Site

Evidence for a peripheral site that recognizes micelles where cholesterol is inserted, is presented for cholesterol oxidase from Brevibacterium sterolicum (CHOD). The Ω loop part of this site contains methionine residues. Their oxidation is reflected in a different selectivity to the micelles. When CHOD is oxidized with hydrogen peroxide, preferential introduction of two oxygen atoms by hydrogen peroxide to the enzyme is observed by the electrospray mass spectrometry. The modified enzyme does not oxidize cholesterol inserted in H‐Triton X‐100/sodium cholate mixed micelles. However, cholesterol solubilized in microemulsions, in biphasic media or inserted in hydroxypropyl‐β‐cyclodextrin is oxidized by the modified enzyme. This modified enzyme oxidizes pregn‐5‐en‐3β‐ol solubilized in mixed micelles, and soluble steroids, such as 3β‐hydroxyandrost‐5‐en‐17β‐carboxylic acid or 3β‐hydroxyandrost‐5‐en‐17‐one. So the modification does not occur at the active site, but at the peripheral site (methionine 81), abolishing the recognition of the micelles and thus inactivating the enzyme specifically towards cholesterol inserted in the micelles.     

Tuneable Transient Thermogels Mediated by a pH‐ and Redox‐Regulated Supramolecular Polymerization

A multistimuli‐responsive transient supramolecular polymerization of β‐sheet‐encoded dendritic peptide monomers in water is presented. The amphiphiles, which contain glutamic acid and methionine, undergo a glucose oxidase catalyzed, glucose‐fueled transient hydrogelation in response to an interplay of pH and oxidation stimuli, promoted by the production of reactive oxygen species (ROS). Adjusting the enzyme and glucose concentration allows tuning of the assembly and the disassembly rates of the supramolecular polymers, which dictate the stiffness and transient stability of the hydrogels. The incorporation of triethylene glycol chains introduces thermoresponsive properties to the materials. We further show that repair enzymes are able to reverse the oxidative damage in the methionine‐based thioether side chains. Since ROS play an important role in signal transduction cascades, our strategy offers great potential for applications of these dynamic biomaterials in redox microenvironments.     

Mutations affecting the binding, internalization, and lysosomal hydrolysis of low density lipoprotein in cultured human fibroblasts, lymphocytes, and aortic smooth muscle cells.

Studies comparing the metabolism of low density lipoprotein (LDL) in normal cells and in cells cultured from patients with homozygous familial hypercholesterolemia have disclosed the existence of a receptor for plasma LDL. This receptor has been identified on the surface of human fibroblasts, lymphocytes, and aortic smooth muscle cells. An extension of these studies to cell strains derived from patients with other single gene defects in cholesterol metabolism has provided additional insight into the normal mechanisms by which cells regulate their cholesterol content and how alterations in these genetic control mechanisms may predispose to atherosclerosis in man.     

Peptide Amphiphile Supramolecular Nanostructures as a Targeted Therapy for Atherosclerosis

The rising prevalence of cardiovascular disease worldwide necessitates novel therapeutic approaches to manage atherosclerosis. Intravenously administered nanostructures are a promising noninvasive approach to deliver therapeutics that reduce plaque burden. The drug liver X receptor agonist GW3965 (LXR) can reduce atherosclerosis by promoting cholesterol efflux from plaque but causes liver toxicity when administered systemically at effective doses, thus preventing its clinical use. The ability of peptide amphiphile nanofibers containing apolipoprotein A1–derived targeting peptide 4F to serve as nanocarriers for LXR delivery (ApoA1‐LXR PA) in vivo is investigated here. These nanostructures are found to successfully target atherosclerotic lesions in a mouse model within 24 h of injection. After 8 weeks of intravenous administration, the nanostructures significantly reduce plaque burden in both male and female mice to a similar extent as LXR alone in comparison to saline‐treated controls. Furthermore, they do not cause increased liver toxicity in comparison to LXR treatments, which may be related to more controlled release by the nanostructure. These findings demonstrate the potential of supramolecular nanostructures as safe, effective drug nanocarriers to manage atherosclerosis.     

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.     

Controlling the Host–Guest Interaction Mode through a Redox Stimulus

A proof‐of‐concept related to the redox‐control of the binding/releasing process in a host–guest system is achieved by designing a neutral and robust Pt‐based redox‐active metallacage involving two extended‐tetrathiafulvalene (exTTF) ligands. When neutral, the cage is able to bind a planar polyaromatic guest (coronene). Remarkably, the chemical or electrochemical oxidation of the host–guest complex leads to the reversible expulsion of the guest outside the cavity, which is assigned to a drastic change of the host–guest interaction mode, illustrating the key role of counteranions along the exchange process. The reversible process is supported by various experimental data (¹H NMR spectroscopy, ESI‐FTICR, and spectroelectrochemistry) as well as by in‐depth theoretical calculations performed at the density functional theory (DFT) level.     

A heparin modified polypropylene non‐woven fabric membrane adsorbent for selective removal of low density lipoprotein from plasma

In the present work, a new porous low density lipoprotein (LDL) adsorbent membrane is prepared by ⁶⁰Co γ‐ray irradiation‐induced grafting copolymerization of polypropylene (PP) non‐woven fabric with acrylic acid, followed by immobilizing heparin covalently. The amount of carboxyl group and heparin on the resultant PP non‐woven fabric is determined by titration and colorimetry, respectively. The new adsorbent membrane is characterized by attenuated total reflection Fourier transform infrared spectroscopy, scanning electron microscope, and contact angle microscopy. Static adsorption and hemoperfusion tests indicate this new adsorbent can efficiently and selectively remove LDL from human plasma. Meanwhile, good adsorption of triglyceride (TG) is also obtained. The best result is achieved by the adsorbent membrane P_0.45‐A₁₅‐H, where 33.3 ± 2.9 µg of LDL‐C, 14.7 ± 1.9 µg of high density lipoprotein cholesterol (HDL‐C), 64.9 ± 4.3 µg of total cholesterol (TC), and 202.4 ± 5.7 µg of TG are removed from human plasma per square centimeter. Hemocompability and toxicity tests show this new adsorbent membrane has good blood compatibility and low toxicity. Considering the adsorbent performance, safety, low cost, and simple preparation, this new adsorbent membrane has potential clinical application for removal of LDL. Copyright © 2013 John Wiley & Sons, Ltd.     

Metabolomics analysis of the therapeutic mechanism of Semen Descurainiae Oil on hyperlipidemia rats using 1H‐NMR and LC–MS

Semen descurainiae oil (SDO) is an important traditional Chinese medicine that was recently discovered to have the function of reducing blood lipids. Metabolomics analyses of plasma, liver and kidney in rats were performed using ¹H‐NMR and LC–MS to illuminate the lower blood lipid concentration effect of SDO, and niacin was considered as the active control. The measure of total cholesterol (TC) and low‐density‐lipoprotein cholesterol (LDL‐C) in plasma showed that SDO treatment decreased significantly the content of TC and LDL‐C. An orthogonal partial least squares–discriminant analysis approach was applied to identify the different metabolic profiles of plasma, liver and kidney in rats and to detect related potential biomarkers. The results suggested that the metabolic profiles of the control group and hyperlipidemia group showed significant difference and the SDO and niacin group had effective anti‐hyperlipidemia function. The biomarkers primarily concern lipid metabolism, amino acid metabolism and glycometabolism, and the change in biomarkers indicated that hyperlipidemia could cause the unbalance of these metabolic pathways in vivo. SDO reduced blood lipids by repairing amino acid and lipid metabolism.     

A pilot proteomic study of protein markers in autism spectrum disorder

Autism spectrum disorder (ASD) diagnosis is increasing, with 1/88 children believed to be affected by the disorder, with a most recent survey suggesting numbers as high as 1/50. Treatment and understanding of ASD causes is a pressing health concern. ASD protein biomarkers may provide clues about ASD cause. Protein biomarkers for ASDs could be used for ASD diagnosis, subtyping, treatment monitoring, and identifying therapeutic targets. Here, we analyzed the sera from seven children with ASD and seven matched controls using Tricine gel electrophoresis (Tricine‐PAGE) and LC‐MS/MS. Overall, we found increased levels of apolipoproteins ApoA1 and ApoA4, involved in cholesterol metabolism and of serum paraoxanase/arylesterase 1, involved in preventing oxidative damage, in the sera of children with ASD, compared with their matched controls. All three proteins are predicted to interact with each other and are parts of high‐density lipoproteins. Further studies are needed to validate these findings in larger subject numbers.