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Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO(4) buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10-100mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations. 相似文献
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Marcin Popielarski Halszka Ponamarczuk Marta Stasiak Lidia Michalec Radoslaw Bednarek Maciej Studzian Lukasz Pulaski Maria Swiatkowska 《Biochemical and biophysical research communications》2018,495(2):1635-1641
Integrins belong to a family of transmembrane receptors that mediate cell migration and adhesion to ECM. Extracellular domains of integrin heterodimers contain cysteine-rich regions, which are potential sites of thiol-disulfide exchanges. Rearrangements of extracellular disulfide bonds regulate activation of integrin receptors by promoting transition from an inactive state into a ligand-binding competent state. Modifications of integrin disulfide bonds dependent on oxidation-reduction can be mediated by Protein Disulfide Isomerse (PDI). This paper provides evidences that binding to integrin ligands initiate changes in free thiol pattern on cell surface and that thiol-disulfide exchange mediated by PDI leads to activation of integrin subunit α11. By employing co-immunoprecipitation and confocal microscopy analysis we showed that α11β1 and PDI create complexes bounded by disulfide bonds. Using surface plasmon resonance we provide biochemical evidence that PDI can interact directly with integrin subunit α11. 相似文献
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Protein thiol oxidation in health and disease: techniques for measuring disulfides and related modifications in complex protein mixtures 总被引:6,自引:0,他引:6
Eaton P 《Free radical biology & medicine》2006,40(11):1889-1899
Oxidant species are known to contribute to disease and dysfunction in biological systems. However, evidence has been progressively accumulating that demonstrates a more fundamental role for many oxidant species in the regulation of everyday function of healthy cells. Redox dependent signaling events involving the post-translational, oxidative modification of proteins has now been accepted as an important regulatory process, although the full extent of such mechanisms is yet to be determined. Some protein cysteinyl thiols are known to be susceptible to a number of redox-dependent modifications, including an interchange between the reduced thiol and several different oxidized disulfide states. Here, the role of oxidants as regulatory entities is reviewed, as are the many different ways protein disulfide formation can be analysed in complex protein mixtures. This includes an overview of many of the Proteomic strategies that can be used to identify proteins that form disulfides when pro-oxidizing conditions arise in cells, as well as related methods for studying intermediates that may precede disulfide formation. 相似文献
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P B Moore 《Journal of molecular biology》1973,79(4):615-632
Ribosome preparations respond heterogeneously to thiol blocking agents such as N-ethylmaleimide. About 55% of the particles rapidly lose the ability to catalyze peptide bond formation. The remaining 45% are immune. The only function related to peptide bond formation that appears abnormal in inactivated ribosomes is messenger RNA-directed transfer RNA binding. The change, however, is small relative to the alteration in activity observed. Thus it appears that thiol blocking, which selectively damages 30 S subunits, renders about half the 30 S subunits unable to co-ordinate properly with 50 S subunits in peptide bond formation. The implications of these results with regard to ribosome heterogeneity are discussed. 相似文献
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Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications 总被引:3,自引:0,他引:3
Holtgrefe S Gohlke J Starmann J Druce S Klocke S Altmann B Wojtera J Lindermayr C Scheibe R 《Physiologia plantarum》2008,133(2):211-228
Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues. 相似文献
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Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures
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We have demonstrated earlier that protein microenvironments were conserved around disulfide‐bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non‐enzymatic classes. Modifications studied were—disulfide formation, thio‐ether formation, metal‐binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high‐resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups—buried‐hydrophobic, intermediate and exposed‐hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non‐enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha‐helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non‐enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non‐enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine‐related diseases. 相似文献
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Unimod is a database of protein modifications for use in mass spectrometry applications, especially protein identification and de novo sequencing. It contains accurate and verifiable values, derived from elemental compositions, for the mass differences introduced by both natural and artificial modifications. 相似文献
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Elliott Shaw 《The protein journal》1984,3(1):109-120
Peptidyl diazomethyl ketone derivatives inactivate thiol proteases by affinity labeling. The peptide sequence permits the reagents to be targeted to individual proteases according to their specificity. Alkylation of the active center thiol group takes place. The reagents are not reactive to simple thiols such as mercaptoethanol and appear to inactivate thiol proteases by acting as suicide substrates. Other classes of proteases are not affected. Initial results indicate that peptidyl diazomethyl ketones can also be used to inactivate thiol proteases in vivo. 相似文献
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Obese visceral adipose tissue remodeling and dysfunction, based on chronic inflammation and local immunological changes, play
major roles in the metabolic syndrome. Therefore, an in vivo visualization technique has been developed to assess the dynamic
interplay between multiple cell types in obese adipose. In vivo imaging revealed close spatial and temporal interrelationships
between angiogenesis and adipogenesis, which were augmented in obese adipose tissue. In addition, increased leukocyte–platelet–endothelial
cell interactions were observed in the microcirculation, a hallmark of inflammation. Upregulated expression of adhesion molecules
contribute to the local activation of inflammatory processes. We also found that large numbers of CD8+ effector T cells infiltrated
into the obese adipose tissue, playing major roles in inflammatory macrophage infiltration into obese adipose tissue, the
induction and maintenance of inflammation, and systemic insulin resistance. Our results demonstrate the power of our imaging
technique to analyze multi-cellular interactions in inflammation in vivo and to evaluate new therapeutic interventions. 相似文献
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Antiviral heat treatment is routinely used in the bioprocessing of therapeutic proteins as a means of reducing viral load. However, in protein formulations containing sucrose this form of bioprocessing can lead to protein modifications. Using a model protein, hen egg white lysozyme, we investigated the effects of antiviral heat treatments in the presence of sucrose on protein integrity during subsequent long-term protein storage. Although heat treatment alone resulted in protein modification, subsequent medium- to long-term storage of both lyophilized and liquid samples at room temperature or above led to further protein modifications. The majority of these modifications were due to the formation of glycation and advanced glycation end products via the reaction of reducing sugars and their autoxidation products (derived from hydrolyzed sucrose) with function groups on the protein surface. These findings have implications for the improvement of therapeutic protein bioprocessing to ensure protein product quality. 相似文献
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Angeliki Pournara Maria Kippler Teresa Holmlund Rebecca Ceder Roland Grafström Marie Vahter Karin Broberg Annika E. Wallberg 《Cell biology and toxicology》2016,32(4):275-284
Arsenic, an established carcinogen and toxicant, occurs in drinking water and food and affects millions of people worldwide. Arsenic appears to interfere with gene expression through epigenetic processes, such as DNA methylation and post-translational histone modifications. We investigated the effects of arsenic on histone residues in vivo as well as in vitro. Analysis of H3K9Ac and H3K9me3 in CD4+ and CD8+ sorted blood cells from individuals exposed to arsenic through drinking water in the Argentinean Andes showed a significant decrease in global H3K9me3 in CD4+ cells, but not CD8+ cells, with increasing arsenic exposure. In vitro studies of inorganic arsenic-treated T lymphocytes (Jurkat and CCRF-CEM, 0.1, 1, and 100 μg/L) showed arsenic-related modifications of H3K9Ac and changes in the levels of the histone deacetylating enzyme HDAC2 at very low arsenic concentrations. Further, in vitro exposure of kidney HEK293 cells to arsenic (1 and 5 μM) altered the protein levels of PCNA and DNMT1, parts of a gene expression repressor complex, as well as MAML1. MAML1 co-localized and interacted with components of this complex in HEK293 cells, and in silico studies indicated that MAML1 expression correlate with HDAC2 and DNMT1 expression in kidney cells. In conclusion, our data suggest that arsenic exposure may lead to changes in the global levels of H3K9me3 and H3K9Ac in lymphocytes. Also, we show that arsenic exposure affects the expression of PCNA and DNMT1—proteins that are part of a gene expression silencing complex. 相似文献