Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the ß-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation.

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO-) in vitro. We found that ONOO- exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO- induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.

The cysteine residue in beta-lactoglobulin reacts with oxidized tyrosine residues in beta-casein to give casein-lactoglobulin dimers.

Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and ß-casein. Ubiquitin and ß-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or ß-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized ß-casein reacts efficiently with the thermally unfolded ß-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.

Cross-linking and modification of fibronectin by peroxynitrous acid: Mapping and quantification of damage provides a new model for domain interactions.

Fibronectin (FN) is an abundant glycoprotein found in plasma and the extracellular matrix (ECM). It is present at high concentrations at sites of tissue damage, where it is exposed to oxidants generated by activated leukocytes, including peroxynitrous acid (ONOOH) formed from nitric oxide (from inducible nitric oxide synthase) and superoxide radicals (from NADPH oxidases and other sources). ONOOH reacts rapidly with the abundant tyrosine and tryptophan residues in ECM proteins, resulting in the formation of 3-nitrotyrosine, di-tyrosine, and 6-nitrotryptophan. We have shown previously that human plasma FN is readily modified by ONOOH, but the extent and location of modifications, and the role of FN structure (compact versus extended) in determining these factors is poorly understood. Here, we provide a detailed LC-MS analysis of ONOOH-induced FN modifications, including the extent of their formation and the sites of intramolecular and intermolecular cross-links, including Tyr-Tyr, Trp-Trp, and Tyr-Trp linkages. The localization of these cross-links to specific domains provides novel data on the interactions between different modules in the compact conformation of plasma FN and allows us to propose a model of its unknown quaternary structure. Interestingly, the pattern of modifications is significantly different to that generated by another inflammatory oxidant, HOCl, in both extent and sites. The characterization and quantification of these modifications offers the possibility of the use of these materials as specific biomarkers of ECM modification and turnover in the many pathologies associated with inflammation-associated fibrosis.

Exceptionally rich keratinolytic enzyme profile found in the rare actinomycetes Amycolatopsis keratiniphila D2T.

The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC-MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120 h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates. KEY POINTS: • A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases. • This actinomycete efficiently converts bristle meal to a protein hydrolysate. • Proteome analysis identified 57 proteases in its secretome.

Oxidative Crosslinking of Peptides and Proteins: Mechanisms of Formation, Detection, Characterization and Quantification.

Covalent crosslinks within or between proteins play a key role in determining the structure and function of proteins. Some of these are formed intentionally by either enzymatic or molecular reactions and are critical to normal physiological function. Others are generated as a consequence of exposure to oxidants (radicals, excited states or two-electron species) and other endogenous or external stimuli, or as a result of the actions of a number of enzymes (e.g., oxidases and peroxidases). Increasing evidence indicates that the accumulation of unwanted crosslinks, as is seen in ageing and multiple pathologies, has adverse effects on biological function. In this article, we review the spectrum of crosslinks, both reducible and non-reducible, currently known to be formed on proteins; the mechanisms of their formation; and experimental approaches to the detection, identification and characterization of these species.

Two novel S1 peptidases from Amycolatopsis keratinophila subsp. keratinophila D2T degrading keratinous slaughterhouse by-products.

Two proteases, named C- and T-like proteases, respectively, were purified from the culture supernatant of Amycolatopsis keratinophila subsp. keratinophila D2T grown on a keratinous slaughterhouse by-product of pig bristles and nails as sole nitrogen and carbon source. The two proteases belong to peptidase family S1 as identified by mass spectrometric peptide mapping, have low mutual sequence identity (25.8%) and differ in substrate specificity. T-like protease showed maximum activity at 40 °C and pH 8-9, and C-like protease at 60 °C and pH 8-10. Peptides released from the keratinous by-product were identified by mass spectrometry and indicated P1 specificity for arginine and lysine of T-like and alanine, valine and isoleucine of C-like protease as also supported by the activity of the two proteases towards synthetic peptide and amino acid substrates. The specific activities of the C- and T-like proteases and proteinase K on keratin azure and azokeratin were comparable. However, C- and T-like proteases showed 5-10-fold higher keratin/casein (K/C) activity ratios than that of another S1 and two keratin-degrading S8 peptidases used for comparison. The findings support that the range of peptidase families considered to contain keratinases should be expanded to include S1 peptidases. Furthermore, the results indicated the quite thermostable C-like protease to be a promising candidate for use in industrial degradation of keratinous slaughterhouse by-products.

Mass-Spectrometry-Based Identification of Cross-Links in Proteins Exposed to Photo-Oxidation and Peroxyl Radicals Using 18O Labeling and Optimized Tandem Mass Spectrometry Fragmentation.

Protein cross-links are formed in regulated biochemical processes in many biological systems, but they are also generated inadvertently via the reactions of exogenous or endogenous oxidants. Site-specific identification and characterization of such cross-links is challenging, and the goal was, therefore, to develop mass-spectrometry-based approaches tailored for proteins subjected to oxidative challenges that also are applicable for the analysis of complex samples. Using trypsin-mediated 18O isotopic labeling, different types of data acquisition workflows, and designated database software tools, we successfully identified tyrosine-tyrosine, tyrosine-tryptophan, tyrosine-lysine, and histidine-lysine cross-links in proteins subjected to sensitizer-mediated photo-oxidation with rose bengal or chemical oxidation with peroxyl radicals generated from the water-soluble compound 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Subsequently, AAPH was also applied to a protein extract from the Gram-positive bacterium Lactococcus lactis, demonstrating the feasibility to identify tyrosine-tyrosine, tyrosine-tryptophan, and tryptophan-tryptophan cross-linked peptides in a complex system. Different fragmentation techniques were evaluated, and it was observed that higher-energy collisional dissociation (HCD) resulted in a higher number of identified cross-link peptides, while electron-transfer dissociation supplemented with HCD (EThcD) generally provides higher fragment ion coverage of the cross-linked peptides.

Identification and characterization of protein cross-links induced by oxidative reactions.

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.

Unrestricted Mass Spectrometric Data Analysis for Identification, Localization, and Quantification of Oxidative Protein Modifications.

Oxidation generates multiple diverse post-translational modifications resulting in changes in protein structure and function associated with a wide range of diseases. Of these modifications, carbonylations have often been used as hallmarks of oxidative damage. However, accumulating evidence supports the hypothesis that other oxidation products may be quantitatively more important under physiological conditions. To address this issue, we have developed a holistic mass spectrometry-based approach for the simultaneous identification, localization, and quantification of a broad range of oxidative modifications based on so-called "dependent peptides". The strategy involves unrestricted database searches with rigorous filtering focusing on oxidative modifications. The approach was applied to bovine serum albumin and human serum proteins subjected to metal ion-catalyzed oxidation, resulting in the identification of a wide range of different oxidative modifications. The most common modification in the oxidized samples is hydroxylation, but carbonylation, decarboxylation, and dihydroxylation are also abundant, while carbonylation showed the largest increase in abundance relative to nonoxidized samples. Site-specific localization of modified residues reveals several "oxidation hotspots" showing high levels of modification occupancy, including specific histidine, tryptophan, methionine, glutamate, and aspartate residues. The majority of the modifications, however, occur at low occupancy levels on a diversity of side chains.

Seed thioredoxin h.

Thioredoxins are nearly ubiquitous disulfide reductases involved in a wide range of biochemical pathways in various biological systems, and also implicated in numerous biotechnological applications. Plants uniquely synthesize an array of thioredoxins targeted to different cell compartments, for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses on occurrence, reaction mechanisms, specificity, target protein identification, three-dimensional structure and various applications. The aim is to provide a general background as well as an update covering the most recent findings. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Lactococcus lactis thioredoxin reductase is sensitive to light inactivation.

Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar kcat values (∼25 s(-1)) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax=460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.

Two Lactococcus lactis thioredoxin paralogues play different roles in responses to arsenate and oxidative stress.

Thioredoxin (Trx) maintains intracellular thiol groups in a reduced state and is involved in a wide range of cellular processes, including ribonucleotide reduction, sulphur assimilation, oxidative stress responses and arsenate detoxification. The industrially important lactic acid bacterium Lactococcus lactis contains two Trxs. TrxA is similar to the well-characterized Trx homologue from Escherichia coli and contains the common WCGPC active site motif, while TrxD is atypical and contains an aspartate residue in the active site (WCGDC). To elucidate the physiological roles of the two Trx paralogues, deletion mutants ΔtrxA, ΔtrxD and ΔtrxAΔtrxD were constructed. In general, the ΔtrxAΔtrxD strain was significantly more sensitive than either of the ΔtrxA and ΔtrxD mutants. Upon exposure to oxidative stress, growth of the ΔtrxA strain was diminished while that of the ΔtrxD mutant was similar to the wild-type. The lack of TrxA also appears to impair methionine sulphoxide reduction. Both ΔtrxA and ΔtrxD strains displayed growth inhibition after treatment with sodium arsenate and tellurite as compared with the wild-type, suggesting partially overlapping functions of TrxA and TrxD. Overall the phenotype of the ΔtrxA mutant matches established functions of WCGPC-type Trx while TrxD appears to play a more restricted role in stress resistance of Lac. lactis.

Gibberellic acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.

The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.

Serological assessment of neutrophil elastase activity on elastin during lung ECM remodeling.

BACKGROUND: During the pathological destruction of lung tissue, neutrophil elastase (NE) degrades elastin, one of the major constituents of lung parenchyma. However there are no non-invasive methods to quantify NE degradation of elastin. We selected specific elastin fragments generated by NE for antibody generation and developed an ELISA assay (EL-NE) for the quantification of NE-degraded elastin. METHODS: Monoclonal antibodies were developed against 10 NE-specific cleavage sites on elastin. One EL-NE assay was tested for analyte stability, linearity and intra- and inter-assay variation. The NE specificity was demonstrated using elastin cleaved in vitro with matrix metalloproteinases (MMPs), cathepsin G (CatG), NE and intact elastin. Clinical relevance was assessed by measuring levels of NE-generated elastin fragments in serum of patients diagnosed with idiopathic pulmonary fibrosis (IPF, n = 10) or lung cancer (n = 40). RESULTS: Analyte recovery of EL-NE for human serum was between 85% and 104%, the analyte was stable for four freeze/thaw cycles and after 24 h storage at 4°C. EL-NE was specific for NE-degraded elastin. Levels of NE-generated elastin fragments for elastin incubated in the presence of NE were 900% to 4700% higher than those seen with CatG or MMP incubation or in intact elastin. Serum levels of NE-generated elastin fragments were significantly increased in patients with IPF (137%, p = 0.002) and in patients with lung cancer (510%, p < 0.001) compared with age- and sex-matched controls. CONCLUSIONS: The EL-NE assay was specific for NE-degraded elastin. The EL-NE assay was able to specifically quantify NE-degraded elastin in serum. Serum levels of NE-degraded elastin might be used to detect excessive lung tissue degradation in lung cancer and IPF.

Glycopeptide enrichment using a combination of ZIC-HILIC and cotton wool for exploring the glycoproteome of wheat flour albumins.

Hydrophilic liquid chromatography (HILIC) is used extensively as a sample preparation step for glycopeptide enrichment in proteome research. Here, we have applied cotton wool and a zwitterionic HILIC (ZIC-HILIC) resin in solid-phase extraction microcolumns to provide a higher loading capacity and broader specificity for glycopeptide enrichment. This strategy was applied to tryptic digests of wheat flour albumin extracts followed by simulataneous site-specific (18)O labeling and deglycosylation using peptide-N-glycosidase A (PNGase A) in H(2)(18)O. Subsequent LC-MS/MS analysis allowed for assignment of 78 N-glycosylation sites in 67 albumin proteins. Bioinformatic analysis revealed that several of the identified glycoproteins show sequence similarity to known food allergens. In addition, the potential impact of some of the identified glycoproteins on wheat beer quality is discussed.

A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase.

The ubiquitous disulfide reductase thioredoxin (Trx) regulates several important biological processes such as seed germination in plants. Oxidized cytosolic Trx is regenerated by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (NTR) in a multistep transfer of reducing equivalents from NADPH to Trx via a tightly NTR-bound flavin. Here, interactions between NTR and Trx are predicted by molecular modelling of the barley NTR:Trx complex (HvNTR2:HvTrxh2) and probed by site directed mutagenesis. Enzyme kinetics analysis reveals mutants in a loop of the flavin adenine dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent on these two residues, suggesting a distinct mode for NTR:Trx recognition. Comparison between the HvNTR2:HvTrxh2 model and the crystal structure of the Escherichia coli NTR:Trx complex reveals major differences in interactions involving the FAD- and NADPH-binding domains as supported by our experiments. Overall, the findings suggest that NTR:Trx interactions in different biological systems are fine-tuned by multiple intermolecular contacts.

Lactococcus lactis TrxD represents a subgroup of thioredoxins prevalent in Gram-positive bacteria containing WCXDC active site motifs.

Three protein disulfide reductases of the thioredoxin superfamily from the industrially important Gram-positive Lactococcus lactis (LlTrxA, LlTrxD and LlNrdH) are compared to the "classical" thioredoxin from Escherichia coli (EcTrx1). LlTrxA resembles EcTrx1 with a WCGPC active site motif and other key residues conserved. By contrast, LlTrxD is more distantly related and contains a WCGDC motif. Bioinformatics analysis suggests that LlTrxD represents a subgroup of thioredoxins from Gram-positive bacteria. LlNrdH is a glutaredoxin-like electron donor for ribonucleotide reductase class Ib. Based on protein-protein equilibria LlTrxA (E°'=-259mV) and LlNrdH (E°'=-238mV) show approximately 10mV higher standard state redox potentials than the corresponding E. coli homologues, while E°' of LlTrxD is -243mV, more similar to glutaredoxin than "classical" thioredoxin. EcTrx1 and LlTrxA have high capacity to reduce insulin disulfides and their exposed active site thiol is alkylated at a similar rate at pH 7.0. LlTrxD on the other hand, is alkylated by iodoacetamide at almost 100 fold higher rate and shows no activity towards insulin disulfides. LlTrxA, LlTrxD and LlNrdH are all efficiently reduced by NADPH dependent thioredoxin reductase (TrxR) from L. lactis and good cross-reactivity towards E. coli TrxR was observed with LlTrxD as the notable exception.

The role of extracellular matrix quality in pulmonary fibrosis.

This review discusses the role of extracellular matrix (ECM) quality in the pathogenesis of pulmonary fibrosis (PF). In PF, the highly ordered structure of collagens and elastin within the ECM of the lung is severely disrupted and lacks its original tissue quality. Discussions about the ECM have focused on the role of protein quantity in relation to the progression of PF, while the importance of lung ECM quality, defined by the levels of ECM protein modifications and by the protein distribution in lung tissue, has not been properly addressed. The quality and function of proteins may be altered by different post-translational modifications (PTMs), such as cross-linking, proteolytic cleavage, citrullination, misfolding and glycosylation. This paper is the first to review key data from the literature related to the lung ECM at the molecular level, relate these to changes observed at a macroscopic level and evaluate which PTMs most likely contribute to PF. This paper also reviews the role of novel neo-epitope-specific biomarkers in the early diagnosis and prognosis of fibrotic disorders. We discuss and argue that the altered quality of the individual ECM proteins contributes to the progression of PF and may also lead to the increased quantity of lung proteins. Thus, both quantity and quality appear to be of utmost importance.

Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination.

At inflammatory sites, immune cells generate oxidants including H2O2. Myeloperoxidase (MPO), released by activated leukocytes employs H2O2 and halide/pseudohalides to form hypohalous acids that mediate pathogen killing. Hypochlorous acid (HOCl) is a major species formed. Excessive or misplaced HOCl formation damages host tissues with this linked to multiple inflammatory diseases. Previously (Redox Biology, 2020, 28, 101331) we reported that iodide (I⁻) modulates MPO-mediated protein damage by decreasing HOCl generation with concomitant hypoiodous acid (HOI) formation. HOI may however impact on protein structure, so in this study we examined whether and how HOI, from peroxidase/H2O2/I⁻ systems ± Cl⁻, modifies proteins. Experiments employed MPO and lactoperoxidase (LPO) and multiple proteins (serum albumins, anastellin), with both chemical (intact protein and peptide mass mapping, LC-MS) and structural (SDS-PAGE) changes assessed. LC-MS analyses revealed dose-dependent iodination of anastellin and albumins by LPO/H2O2 with increasing I⁻. Incubation of BSA with MPO/H2O2/Cl⁻ revealed modest chlorination (Tyr286, Tyr475, ∼4 %) and Met modification. Lower levels of these species, and extensive iodination at specific Tyr and His residues (>20 % modification with ≥10 µM I⁻) were detected with increasing I⁻. Anastellin dimerization was inhibited by increasing I⁻, but less marked changes were observed with albumins. These data confirm that I⁻ competes with Cl⁻ for MPO and is an efficient HOCl scavenger. These processes decrease protein chlorination and oxidation, but result in extensive iodination. This is consistent with published data on the presence of iodinated Tyr on neutrophil proteins. The biological implications of protein iodination relative to chlorination require further clarification.