The oxidative environment and protein damage

Proteins are a major target for oxidants as a result of their abundance in biological systems, and their high rate constants for reaction. Kinetic data for a number of radicals and non-radical oxidants (e.g. singlet oxygen and hypochlorous acid) are consistent with proteins consuming the majority of these species generated within cells. Oxidation can occur at both the protein backbone and on the amino acid side-chains, with the ratio of attack dependent on a number of factors. With some oxidants, damage is limited and specific to certain residues, whereas other species, such as the hydroxyl radical, give rise to widespread, relatively non-specific damage. Some of the major oxidation pathways, and products formed, are reviewed. The latter include reactive species, such as peroxides, which can induce further oxidation and chain reactions (within proteins, and via damage transfer to other molecules) and stable products. Particular emphasis is given to the oxidation of methionine residues, as this species is readily oxidised by a wide range of oxidants. Some side-chain oxidation products, including methionine sulfoxide, can be employed as sensitive, specific, markers of oxidative damage. The product profile can, in some cases, provide valuable information on the species involved; selected examples of this approach are discussed. Most protein damage is non-repairable, and has deleterious consequences on protein structure and function; methionine sulfoxide formation can however be reversed in some circumstances. The major fate of oxidised proteins is catabolism by proteosomal and lysosomal pathways, but some materials appear to be poorly degraded and accumulate within cells. The accumulation of such damaged material may contribute to a range of human pathologies.     

Characterisation of photo-oxidation products within photoyellowed wool proteins: tryptophan and tyrosine derived chromophores.

Understanding the photodegradation of complex protein systems represents a significant goal in protein science. The photo-oxidation and resultant photoyellowing of wool in sunlight is a severe impediment to its marketability. However, although some photomodifications have been found in irradiated model amino acid systems, direct identification of the chromophoric photoproducts responsible for photoyellowing in irradiated wool itself has proved elusive. We here describe the direct characterisation and location of yellow chromophores and related photomodifications within the proteins of photoyellowed wool fabric, utilising a quasi-proteomic approach. In total, eight distinct photoproducts were characterised. Of these, five were derived from tryptophan; namely hydroxytryptophan, N-formylkynurenine, kynurenine, residues consistent with the dehydration of kynurenine, and hydroxykynurenine, while three were derived from tyrosine; namely dihydroxyphenylalanine, dityrosine, and a cross-linked residue consistent with a hydroxylated dityrosine residue. Fourteen modified peptide sequences were identified and the positions of modification for thirteen of these were located within the primary structure of known wool proteins. The nature of the photoproducts characterised offer valuable insight into the reaction pathways followed in the UV-induced photoyellowing of wool proteins.     

Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.

Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.     

Comparison of Communities of Stored Product Mites in Grain Mass and Grain Residues in the Czech Republic

In storage facilities one can find grain either in stored grain mass or in grain residues in the store corners or machinery. Although it is claimed that grain residues are serious pest reservoirs since they harbor numbers of stored product arthropods and are connected via continuous emigration with grain mass, the documentation for this is not convincing. Therefore in 78 selected grain stores, we simultaneously sampled the grain mass and residues in order to compare concurrent mite communities in these two different habitats. We found 30 species in about 614 000 individuals in residues and 23 species in about 20 000 individuals in grain mass. Canonical correspondence analysis (CCA) of transformed abundance data showed differences in the communities of mites in grain mass and residues: (i) species associated to grain residues (e.g. Tyrophagus longior, Tydeus interruptus, Acarus farris and Cheyletus eruditus) and (ii) species associated to both grain mass and grain residues (e.g. Tarsonemus granarius, Acarus siro, Tyrophagus putrescentiae, Lepidoglyphus destructor and Cheyletus malaccensis). Although the residue samples had more mites and higher species diversity than the stored grain mass, no correlation in mite abundance and species numbers between samples from grain residues and grain mass was found, thereby indicating low connectivity of these two habitats.     

Gas chromatography-mass spectrometry identification of photoproducts of hexahydroquinoline derivatives: potential calcium channel antagonists

Photodegradation products of hexahydroquinoline derivatives (HHQ) have been analysed with gas chromatography-mass spectrometry (GC-MS). The photodegradation was carried out under the conditions recommended in the first version of the document issued by the International Conference on Harmonization (ICH), currently in force in the studies of photochemical stability of drugs and therapeutic substances. The study was performed on the compounds having two chlorine atoms at different positions of the phenyl ring. Photodegradation of dichlorophenyl derivatives of HHQ resulted in formation of one or three photoproducts. The main product of their decomposition was aromatic compound formed as a result of dehydrogenation of the dihydropyridine ring. The most often observed fragmentation pathway of the photoproducts formed was elimination of methyl and methoxy radicals from the ester groups. The fragmentation of the photoproducts containing one chlorine atom at the ortho-position of the phenyl ring occurred through elimination of chlorine radical.     

Role of the phenylalanine 260 residue in defining product profile and alcoholytic activity of the α-amylase AmyA from Thermotoga maritima

Some α-amylases besides catalyzing the hydrolysis of α-1,4 glycosidic bonds in starch are also capable of carrying out some transglycosylation activity. The importance of aromatic residues near the catalytic site in determining the ratio of these two competing activities has been remarked in the past. In the present work we investigated the role of residue 260 in the product profile of the α-amylase AmyA from Thermotoga maritima. This phenylalanine residue, two positions after the glutamic acid/base catalyst was substituted by both tryptophan and glycine residues, showing opposite behaviors. The tryptophan mutant displayed a very similar product profile pattern to that of the wild-type enzyme; while the mutant Phe260Gly showed a higher transglycosylation/hydrolysis ratio. When the Phe260Trp mutation was constructed in the context of His222Gln, a mutant we have already reported with an increased transglycosylation/hydrolysis ratio and a higher alcoholysis activity, the resultant enzyme showed an apparent higher hydrolysis/transglycosylation ratio and a change to shorter products pattern than the single mutant enzyme, still maintaining the increased alcoholytic activity provided by the His222Gln mutation. The mutant Phe260Gly, on the other hand showed by itself a higher alcoholytic activity, similar to that of the His222Gln mutant.     

A geometrical constraint approach for reproducing the native backbone conformation of a protein

It is known that the backbone conformation of a protein can be reproduced with precision once a correct contact map (two-dimensional representation showing residue pairs in contact) is given as geometrical constraints. There is, however, no way to infer the correct contact map for a protein of unknown structure. We started with one-dimensional constraints using the quantity N14 (the number of neighboring residues within the radius of 14 Å). Since the plot of N14 along a chain shows a good correlation with the corresponding amino acid sequence, the N14 profile obtained from the X-ray structure is predictable from the sequence. Construction of backbone conformations under a given N14 profile was carried out in the following two steps: (1) a contact map from the N14 profile was produced by taking the product of N14 values of every two residues; (2) backbone conformations were generated by applying the distance geometry technique to distance constraints given by the contact map. If present, disulfide bonds in a protein, as well as the secondary structure, were treated as additional constraints, and both cases with or without the additional information were examined. The method was tested for 11 proteins of known structure, and the results indicated that the reproduced conformation was fairly good, using an X-ray structure for comparison, for small proteins of less than 80 residues long. The basic assumption and effectiveness of the present method were compared with those of previous studies employing the geometrical constraint approach. It has become clear that the specific, one-dimensional information (e.g., N14 profile) is more effective than nonspecific, two-dimensional constraints, such as average interresidue distances between particular types of amino acids. © 1993 Wiley-Liss, Inc.     

Effects of bio‐based residue amendments on greenhouse gas emission from agricultural soil are stronger than effects of soil type with different microbial community composition

With the projected rise in the global human population, agriculture intensification and land‐use conversion to arable fields is anticipated to meet the food and bio‐energy demand to sustain a growing population. Moving towards a circular economy, agricultural intensification results in the increased re‐investment of bio‐based residues in agricultural soils, with consequences for microbially mediated greenhouse gas (GHG) emission, as well as other aspects of soil functioning. To date, systematic studies to address the impact of bio‐based residue amendment on the GHG balance, including the soil microorganisms, and nutrient transformation in agricultural soils are scarce. Here, we assess the global warming potential (GWP) of in situ GHG (i.e., CO₂, CH₄, and N₂O) fluxes after application of six bio‐based residues with broad C : N ratios (5–521) in two agricultural soils (sandy loam and clay; representative of vast production areas in north‐western Europe). We relate the GHG emission to the decomposability of the residues in a litter bag assay and determined the effects of residue input on crop (common wheat) growth after incubation. The shift in the bacterial community composition and abundance was monitored using IonTorrent^TM sequencing and qPCR, respectively, by targeting the 16S rRNA gene. The decomposability of the residues, independent of C : N ratio, was proportional to the GWP derived from the GHG emitted. The soils harbored distinct bacterial communities, but responded similarly to the residue amendments, because both soils exhibited the highest mean GWP after addition of the same residues (sewage sludge, aquatic plant material, and compressed beet leaves). Our results question the extent of using the C : N ratio alone to predict residue‐induced response in GHG emission. Taken together, we show that although soil properties strongly affect the bacterial community composition, microbially mediated GHG emission is residue dependent.     

A gene for a Class II DNA photolyase from<Emphasis Type="Italic"> Oryza sativa</Emphasis>: cloning of the cDNA by dilution-amplification

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.     

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

Background  

The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium.     

Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method

An Fc fusion protein expressed in Escherichia coli contains Met1 and Asp2 residues at the N terminus and an active peptide attached to the C terminus of the Fc region. Due to the unique amino acid sequence of Fc, many commonly used proteolysis methods have severe drawbacks for characterizing degradations of Met1 and Asp2 residues. A novel method has been developed to effectively characterize the degradations by employing a limited endoproteinase Glu-C digestion. The limited digestion generates a dimeric peptide of (Met1-Glu14)(2) due to specific cleavage at the residue Glu14 of the N terminus. This peptide together with its degraded products, including Met1 oxidation and Asp2 isomerization, can be identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimization of digestion procedure and linearity of quantification are also described. This approach was successfully used in a photostability study to assess the product stability of an Fc fusion peptibody.     

Exploring metallodrug–protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound

Electrospray ionisation mass spectrometry was used to analyse the reactions of metal compounds with mixtures of selected proteins. Three representative medicinally relevant compounds, cisplatin, transplatin and the organometallic ruthenium compound RAPTA-C, were reacted with a pool of three proteins, ubiquitin, cytochrome c and superoxide dismutase, and the reaction products were analysed using high-resolution mass spectrometry. Highly informative electrospray ionisation mass spectra were acquired following careful optimisation of the experimental conditions. The formation of metal–protein adducts was clearly observed for the three proteins. In addition, valuable information was obtained on the nature of the protein-bound metallofragments, on their distribution among the three different proteins and on the binding kinetics. The platinum compounds were less reactive and considerably less selective in protein binding than RAPTA-C, which showed a high affinity towards ubiquitin and cytochrome c, but not superoxide dismutase. In addition, competition studies between cisplatin and RAPTA-C showed that the two metallodrugs have affinities for the same amino acid residues on protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Human tissue transglutaminase is inhibited by pharmacologic and chemical acetylation

Human tissue transglutaminase (TGM2) is implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's, Parkinson's and expanded polyglutamine (polyQ) diseases. TGM2 promotes formation of soluble and insoluble high molecular weight aggregates by catalyzing a covalent linkage between peptide‐bound Q residues in polyQ proteins and a peptide‐bound Lys residue. Therapeutic approaches to modulate the activity of TGM2 are needed to proceed with studies to test the efficacy of TGM2 inhibition in disease processes. We investigated whether acetylation of Lys‐residues by sulfosuccinimidyl acetate (SNA) or aspirin (ASA) would alter the crosslinking activity of TGM2. Acetylation by either SNA and/or ASA resulted in a loss of >90% of crosslinking activity. The Lys residues that were critical for inhibition were identified by mass spectrometry as Lys⁴⁴⁴, Lys⁴⁶⁸, and Lys⁶⁶³. Hence, acetylation of Lys‐residues may modulate the enzymatic function of TGM2 in vivo and offer a novel approach to treatment of TGM2 mediated disorders.     

Sequence based residue depth prediction using evolutionary information and predicted secondary structure

Background  

Residue depth allows determining how deeply a given residue is buried, in contrast to the solvent accessibility that differentiates between buried and solvent-exposed residues. When compared with the solvent accessibility, the depth allows studying deep-level structures and functional sites, and formation of the protein folding nucleus. Accurate prediction of residue depth would provide valuable information for fold recognition, prediction of functional sites, and protein design.     

A fast method for the determination of fractional contributions to solvation in proteins

A fast method for the calculation of residue contributions to protein solvation is presented. The approach uses the exposed polar and apolar surface of protein residues and has been parametrized from the fractional contributions to solvation determined from linear response theory coupled to molecular dynamics simulations. Application of the method to a large subset of proteins taken from the Protein Data Bank allowed us to compute the expected fractional solvation of residues. This information is used to discuss when a residue or a group of residues presents an uncommon solvation profile.     

Identification of 2-deoxyribonolactone at the site of neocarzinostatin-induced cytosine release in the sequence d(AGC)

Neocarzinostatin- (NCS) induced release of cytosine from the deoxycytidylate residues of d(AGC) sequences of duplex oligonucleotides leaves a damaged sugar residue with intact phosphodiester linkages [Kappen, L.S., Chen, C., & Goldberg, I.H. (1988) Biochemistry 27, 4331-4340]. In order to isolate and characterize the sugar damage product, drug-treated duplex d(AGCGAGC*G) (the single target C* residue has 3H in its 5- and 5'-positions) was enzymatically digested to mononucleosides. High-pressure liquid chromatographic analysis of the digest revealed drug-induced products which could be cleanly separated by thin-layer chromatography (TLC) into two components: product a (Rf0.47) and product 1 (Rf0.87). The more polar product a was further purified by adsorption onto DEAE-Sephadex A-25. After elution with HCl and lyophilization, this material behaved like product 1 on TLC. Readjustment to alkaline pH caused its quantitative conversion back to product a. On electrophoresis product 1 behaved like a neutral compound, and the negatively charged product a migrated just behind formate. On the basis of the various chemical and biochemical characteristics of the lesion and apparent 3H abstraction by NCS from the C-1' position, it appears that the two interconvertible products a and 1 are respectively the acid (carboxylate) and lactone forms of 2-deoxyribonic acid. The structure of the sugar damage product was confirmed by gas chromatography/mass spectrometry. The amount of 2-deoxyribonolactone recovered is about 60% of the cytosine released on a molar basis, showing that it is the major, if not the only, product associated with cytosine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatographic determination and identification of acyl migration and photodegradation products of furosemide 1-O-acyl glucuronide

Stability of furosemide glucuronide, the major metabolite of furosemide, was studied in order to accurately assess the glucuronidation of furosemide. Furosemide glucuronide was purified by high-performance liquid chromatography, and the mass spectrum of furosemide glucuronide showed the molecular ion peaks [M−H]⁻ at 505 and 507 (m/z). Furosemide glucuronide was photodegraded to the compound, which was shown more hydrophilic than furosemide glucuronide by high-performance liquid chromatography assay. The photodegradation product of furosemide glucuronide was hydrolyzed to one of the photodegradation products of furosemide by β-glucuronidase, indicating that the photodegradation product of furosemide glucuronide possessed a glucuronic acid moiety. Furthermore, the mass spectrum of the photodegradation product of furosemide glucuronide exhibited molecular ion peaks [M−H]⁻ at 487 and [M−2H+2Na]⁻ at 509, indicating the chlorine displacement of furosemide glucuronide by a hydroxyl group. Furosemide glucuronide was unstable in an aqueous solution (pH=7.4), and presumed acyl migration isomers of furosemide glucuronide (furosemide glucuronide-isomers) were detected by high-performance liquid chromatography equipped with photodiode array UV detector. The UV spectra of seven furosemide glucuronide-isomers were closely similar to that of furosemide glucuronide but not furosemide. Exposing a mixture of furosemide glucuronide and furosemide glucuronide-isomers to light resulted in the production of new compounds. UV spectra of photodegradation products of furosemide glucuronide-isomers were closely similar to those of photodegradation product of furosemide glucuronide. These results suggested that furosemide glucuronide-isomers were also photodegraded, resulting in the displacement of chlorine by a hydroxyl group as in furosemide glucuronide.     

蛹虫草饲料添加剂的研究现状及展望

蛹虫草饲料添加剂包括蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、蛹虫草菌固液发酵产物、微生物发酵蛹虫草残基等产品.蛹虫草饲料添加剂含有粗蛋白、粗脂肪、氨基酸等营养成分,以及虫草素、腺苷、多糖等活性成分,在畜禽、反刍动物、水产品等动物养殖中的应用均获得较好的效果.对蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养...     

Isolation and structure elucidation of the major photodegradation products of loteprednol etabonate

Photodegradation of loteprednol etabonate (5), a steroid anti-inflammatory drug, in the solid state, in aqueous suspension, and in aqueous acetonitrile solution has been investigated. Analysis by HPLC showed that the profile of photodegradation products in the solid state was qualitatively similar to that in the aqueous suspension, although the profile in the aqueous acetonitrile solution was considerably different. The major photodegradation products were isolated from the aqueous suspension and the aqueous acetonitrile solution by using preparative reversed-phase HPLC and their structures were elucidated on the basis of spectroscopic data. Photolysis in the solid state and in aqueous suspension yielded three rearrangement products, chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate (8), chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate (9), and chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate (10). In aqueous acetonitrile solution, 10 was the major product, however, 8 and 9 were not obtained. Pathways for the formation of these compounds from loteprednol etabonate (5) are proposed.     

Use of mass spectrometry to probe the nucleophilicity of cysteinyl residues of prolyl hydroxylase domain 2

Prolyl hydroxylase domain 2 (PHD2) plays an important role in hypoxic sensing in humans. Here we report studies on the reactivity of cysteinyl residues of the catalytic domain of PHD2 using an approach in which nondenaturing electrospray ionization–mass spectrometry (ESI–MS) analyses were combined with the use of a thiol library and residue substitution. Among the seven cysteinyl residues of the PHD2 catalytic domain, Cys201 was found to be predominantly modified by thiols or N-ethylmaleimide. Selective modification of Cys201 was further demonstrated with methanethiosulfonate, a spin-labeled probe. The modified PHD2 will be useful in electron paramagnetic resonance studies on PHD2. The results demonstrate the use of a combined library/residue substitution/ESI–MS approach for analyzing residue reactivity.