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Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by ?oxidant-mediated thiol-disulfide reactions? via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

Cross-links formed within and between proteins are a major cause of protein dysfunction, and are postulated to drive the accumulation of protein aggregates in some human pathologies. Cross-links can be formed from multiple residues and can be reversible (usually sulfur-sulfur bonds) or irreversible (typically carbon-carbon or carbon-heteroatom bonds). Disulfides formed from oxidation of two Cys residues are widespread, with these formed both deliberately, via enzymatic reactions, or as a result of unintended oxidation reactions. We have recently demonstrated that new protein-glutathione mixed disulfides can be formed through oxidation of a protein disulfide to a thiosulfinate, and subsequent reaction of this species with glutathione. Here we investigate whether similar reactions occur between an oxidized protein disulfide, and a Cys residues on a second protein, to give novel protein cross-links. Singlet oxygen (1O2)-mediated oxidation of multiple proteins (?-lactalbumin, lysozyme, beta-2-microglobulin, C-reactive protein), and subsequent incubation with the Cys-containing protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH), generates inter-protein cross-links as detected by SDSPAGE, immunoblotting and mass spectrometry (MS). The cross-link yield is dependent on the 1O2 concentration, the presence of the original protein disulfide bond, and the free Cys on GAPDH. MS with 18O-labeling has allowed identification of the residues involved in some cases (e.g. Cys25 from the Cys25-Cys80 disulfide in beta2-microglobulin, with Cys149 or Cys244 of GAPDH). The formation of these cross-links results in a loss of GAPDH enzymatic activity. These data provide ?proof-of-concept? for a novel mechanism of protein cross-link formation which may help rationalize the accumulation of cross-linked proteins in multiple human pathologies.

The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO?). We hypothesized that exposure to ROO? would generate specific modifications and inter- and intraprotein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2?-azobis(2-methylpropionamidine) dihydrochloride) as a ROO? source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 ?C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO?). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLCMS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.

Photosensitized protein oxidation is a promising tool for medical procedures such as photochemical tissue bonding (PTB). We have recently reported that the binding of rose Bengal, a sensitizer employed in PTB, to lysozyme modulates the photooxidation and crosslinking of this protein. In this work we examined the photo - oxidation and crosslinking of lysozyme mediated by riboflavin (RF) an endogenous sensitizer also employed in PTB. We hypothesized that since RF does not bind strongly to proteins, the mechanism(s) and extent of enzy- matic inactivation, amino acid modification and protein crosslinking would be dependent on the presence of O2, and differ to that induced by rose Bengal. This hypothesis was tested using UV -visible spectrophotometry, isothermal titration calorimetry (ITC), SDS-PAGE gels, quantification of amino acid consumption, and LC -MS analysis of sites of modification and crosslinks. Under N2, limited damage was detected arising from type 1 (radical) chemistry with formation of specific intra- (Tyr20-Tyr23) and inter- (Tyr23-Trp108) molecular cross - links. In contrast, the presence of O2 triggered extensive protein damage through mixed type 1 and type 2 ( 1 O2) mechanisms leading to Trp, Met, Tyr and His oxidation, loss of enzymatic activity and protein dimerization. LC - MS analysis provided evidence for crosslinking via radical -radical recombination reactions (Trp28-Tyr53), and secondary reactions involving nucleophilic attack of the side -chain amine of Lys116 on carbonyl groups. Overall, this behavior is in marked contrast to that detected with rose Bengal indicating that the mechanisms and sites of photo -oxidative damage, and consequences for protein function, can be modulated by the choice of sensitizing dye.

Quinones can modify biological molecules through both redox-cycling reactions that yield radicals (semiquinone, superoxide and hydroxyl) and via covalent adduction to nucleophiles (e.g. thiols and amines). Kinetic data indicate that Cys residues in GSH and proteins are major targets. In the studies reported here, the interactions of a prototypic quinone compound, p-benzoquinone (BQ), with the key redox protein, thioredoxin-1 (Trxl) were examined. BQ binds covalently with isolated Trx1 forming quinoprotein adducts, resulting in a concentration-dependent loss of enzyme activity and crosslink formation. Mass spectrometry peptide mass mapping data indicate that BQ forms adducts with all of the Trxl Cys residues. Glutathione (GSH) reacts competitively with BQ, and thereby modulates the loss of activity and crosslink formation. Exposure of macrophage-like (J774A.1) cells to BQ results in a dose-dependent loss of Trx and thioredoxin reductase (TrxR) activities, quinoprotein formation, and a decrease in GSH levels without a concomitant increase in oxidized glutathione. GSH depletion aggravates the loss of Trx and TrxR activity. These data are consistent with adduction of GSH to BQ being a primary protective pathway. Reaction of BQ with Trx in cells resulted in the activation of apoptosis signal-regulating kinase 1 (ASK1), and p38 mitogen-activated protein kinase (MAPK) leading to apoptotic cell death. These data suggest that BQ reacts covalently with Cys residues in Trx, including at the active site, leading to enzyme inactivation and protein cross-linking. Modification of the Cys residues in Trx also results in activation of the ASK1/p38-MAPK signalling pathway and promotion of apoptotic cell death.

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring O-1(2) formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H-2 O-18 and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a K-d of 4.8 mu M, while data from ITC studies, yielded a K-d of 0.68 mu M as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (O-1(2)) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

Objective: Currently, there are no disease-modifying osteoarthritis drugs (DMOADs) approved for osteoarthritis. It is hypothesized that a subtype of OA may be driven by inflammation and may benefit from treatment with anti-inflammatory small molecule inhibitors adopted from treatments of rheumatoid arthritis. This study aimed to investigate how small molecule inhibitors of intracellular signaling modulate cartilage degradation and formation as a pre-clinical model for structural effects. Design: Bovine cartilage explants were cultured with oncostatin M (OSM) and tumour necrosis factor alpha (TNF-alpha) either alone or combined with the small molecule inhibitors: SB203580 (p38 inhibitor), R406 (Spleen tyrosine kinase (Syk) inhibitor), TPCA-1 (Inhibitor of kappa B kinase (Ikk) inhibitor), or Tofacitinib (Tofa) (Janus kinases (Jak) inhibitor). Cartilage turnover was assessed with the biomarkers of degradation (AGNx1 and C2M), and type II collagen formation (PRO-C2) using ELISA. Explant proteoglycan content was assessed by Safranin O/Fast Green staining. Results: R406, TPCA-1 and Tofa reduced the cytokine-induced proteoglycan loss and decreased AGNx1 release 3.7-, 43- and 32-fold, respectively. SB203580 showed no effect. All inhibitors suppressed C2M at a concentration of 3 mu M. TPCA-1 and Tofa increased the cytokine reduced PRO-C2 3.5 and 3.7-fold, respectively. Conclusion: Using a pre-clinical model we found that the inhibitors TPCA-1 and Tofa inhibited cartilage degradation and rescue formation of type II collagen under inflammatory conditions, while R406 and SB203580 only inhibited cartilage degradation, and SB203580 only partially. These pre-clinical data suggest that TPCA-1 and Tofa preserve and help maintain cartilage ECM under inflammatory conditions and could be investigated further as DMOADs for inflammation-driven osteoarthritis.

Various redox compounds are known to influence the structure of the gluten network in bread dough, and hence its strength. The cereal thioredoxin system (NTS), composed of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) and thioredoxin (Trx), is a major reducing enzymatic system that is involved in seed formation and germination. NTS is a particularly interesting tool for food processing due to its heat stability and its broad range of protein substrates. We show here that barley NTS is capable of remodeling the gluten network and weakening bread dough. Furthermore, functional wheat Trx that is present in the dough can be recruited by the addition of recombinant barley NTR, resulting in dough weakening. These results confirm the potential of NTS, especially NTR, as a useful tool in baking for weakening strong doughs, or in flat product baking.

Introduction: Protein cross-links are common in biological systems and are generated both deliberately as a part of normal metabolism and also accidently as a result of exposure to external factors such as oxidation and glycation stresses. These cross-links can be both positive and negative for biological function, with high levels of inappropriate cross-links being associated with multiple human pathologies, as well as loss of protein structure and function in the food, agricultural, and pharmaceutical fields.Areas covered: This review covers recent advances in the detection and identification of protein cross-links arising from oxidation reactions, mediated by both radicals and two-electron oxidants. Information on both enzymatic and nonenzymatic cross-linking is reviewed, both where this is intentional, as part of normal metabolism, and accidental and a potential cause of disease.Expert commentary: The advantages and drawbacks of various methods available for the detection, identification, and quantification of these species are discussed, as well as some of the mechanisms and processes known to give rise to these species.

Indications of dementia disease include deterioration of memory, thinking, behaviour, and the ability to perform everyday activities. Any of these symptoms can lead to stress and difficulties organizing everyday life. As a way to view factors that support human health and well-being despite stressful situations, Antonovsky introduced a salutogenic model. This model proposes that sense of coherence primarily determines physical and mental health i.e. psychological well-being. Having a sense of coherence in everyday life can reduce the impact of stress on the individual in everyday life. The study’s aim of this study was to explore how participants in existing support groups scored on the Sense of Coherence Scale (SOC), and what they perceived as contributory factors to a meaningful, manageable, and comprehensible everyday life in the presence of dementia using a mixed method. Persons with dementia had the highest scores on the SOC scale and their partners the lowest. Persons with dementia expressed that being with others who understood them made their everyday life comprehensible and manageable. Their partners expressed that learning about dementia was helpful in managing and comprehending everyday situations. The adult children expressed that it was meaningful to care for their parents and they scored slightly higher than the partners on the SOC scale. Long-term ongoing support supplemented with information and social support can contribute to the sense of coherence in persons with dementia and their next of kin.