Correction: Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc.

[This corrects the article DOI: 10.1371/journal.ppat.1000977.].

Linear epitopes of bony fish ß-parvalbumins.

Introduction: Fish ß-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised. Methods: High-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish ß-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens. Results: Interindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects. Conclusions: Altogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.

Failure of prion protein oxidative folding guides the formation of toxic transmembrane forms.

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

Metamorphism in TDP-43 prion-like domain determines chaperone recognition.

The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and co-chaperones primarily recognize the structured elements in TDP-43´s prion-like domain. Significantly, while HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in disease.

Oxidation of Helix-3 methionines precedes the formation of PK resistant PrP.

While elucidating the peculiar epitope of the alpha-PrP mAb IPC2, we found that PrPSc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the alpha-fold, suggesting oxidation may facilitate the conversion of PrPC into PrPSc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrPC, but could not recognize most PrPSc bands unless they were vigorously reduced. Next, we showed that the alpha-Met pAbs did not recognize newly formed PrPSc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrPC to PrPSc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

Major shrimp allergen peptidomics signatures and potential biomarkers of heat processing.

It is important to develop tools that can be used to understand the effects of processing on allergenic foods in order to achieve personalized food labeling. To evaluate the effect of heating on the allergy-relevant structural properties of tropomyosin (TM), arginine kinase (AK), myosin light chain (MLC) and sarcoplasmic calcium-binding protein (SCP) shrimp allergens, trypsin digests of raw, fried and baked shrimp extracts were analyzed by peptidomics and epitope correlations. Processing altered the number of peptides released from the distinct allergens, and each treatment generated a specific epitope-matched peptide allergen fingerprint. Among the four allergens, TM led to a number of released peptides and epitope changes being detected, and AK provided the epitope-matched 331MGLTEFQAVK340 sequence as a common differentiating peptide for heat processing. These results provide new views on the structural effects of processing on major shrimp allergens and peptide candidates as processing biomarkers.

SWATH-MS-based proteomics reveals functional biomarkers of Th1/Th2 responses of tropomyosin allergy in mouse models.

Type-I food allergies are hypersensitive reactions compromising the immune organs and epithelial barriers. To investigate the organ-specific proteomic alterations of the allergy responses, the spleen and intestine of mice sensitized with high (shrimp and clam) and weak (fish) allergenic tropomyosins were analyzed using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS)-based proteomics. The results showed that Th1 and Th2 tropomyosin-induced responses in the spleen are characterized by the unique upregulation of innate (cochlin) and adaptive (Ig κ chain V-III region PC 7175) immune regulators, respectively. In the intestine, tropomyosin allergy concurred with the downregulation of 35 differentiating proteins featuring the overall impairment of metabolic pathways, absorption processes and ammonium ion responses. These data provide new functional biomarkers of tropomyosin-induced immune responses as well as candidate targets for intervention.

The structural intolerance of the PrP alpha-fold for polar substitution of the helix-3 methionines.

The conversion of the cellular prion protein (PrP(C)) into its disease-associated form (PrP(Sc)) involves a major conformational change and the accumulation of sulfoxidized methionines. Computational and synthetic approaches have shown that this change in the polarity of M206 and M213 impacts the C-terminal domain native alpha-fold allowing the flexibility required for the structural conversion. To test the effect in the full-length molecule with site-specificity, we have generated M-to-S mutations. Molecular dynamics simulations show that the replacement indeed perturbs the native state. When this mutation is placed at the conserved methionines of HaPrP(23-231), only substitutions at the Helix-3 impair the alpha-fold, stabilizing a non-native state with perturbed secondary structure, loss of native tertiary contacts, increased surface hydrophobicity, reduced thermal stability and an enhanced tendency to aggregate into protofibrillar polymers. Our work supports that M206 and M213 function as alpha-fold gatekeepers and suggests that their redox state regulate misfolding routes.

Discrimination between alternate membrane protein topologies in living cells using GFP/YFP tagging and pH exchange.

Membrane protein function is determined by the relative organization of the protein domains with respect to the membrane. We have experimentally verified the topology of a protein with diverse orientations arising from a single primary sequence (the cellular prion protein, PrP(C)), a novel somatostatin truncated receptor, and the Golgi-associated protein GPBP(91). Tagging with fluorescent proteins (FP) allows location of their expression at the plasma membrane or at endomembranes, but does not inform about their orientation. Exploiting the pH dependency of some FPs, we developed a pH exchange assay in which extracellularly exposed FPs are quenched by application of low pH buffer. We constructed standards to demonstrate and calibrate the assay, and the method was adapted for acidic organelle membrane proteins. This method can serve as a proof of concept, experimentally confirming and/or discriminating in living cells among theoretical topology predictions, providing the proportion of inside/outside orientation for proteins with multiple forms.

Fish muscle processing into seafood products reduces ß-parvalbumin allergenicity.

Fish is one of the eight major foods causing type-I food allergy, and the prevalence of its allergy is increasing in part due to changes in consumption habits. One of the main drivers for these changes has been the processing developments transforming the fish muscle into seafood products. Most fish allergic patients react to the Ca2+-binding protein ß-parvalbumin (ß-PV) abundant in muscle. Here we have analyzed the effect of processing in the content and allergenic properties of the ß-PV. We found that the transformation process decreases the ß-PV content (4.7 ± 0.3 mg/g muscle, 0.24 ± 0.03 mg/g surimi, ≤0.003 ± 0.001 mg/g in seafood products), reduces the specific-IgE binding and prevents allergy relevant properties such the protease resistance and amyloid aggregation. These results suggest seafood products as potentially tolerable foods for fish allergic patients, but milk and egg allergic patients should be aware of the presence relevant additives.

Identification of the Dominant T-Cell Epitopes of Lit v 1 Shrimp Major Allergen and Their Functional Overlap with Known B-Cell Epitopes.

Development of efficient peptide-based immunotherapy for shrimp allergy relies on the identification of the dominant T-cell epitopes of its major allergen, tropomyosin. In this study, immunoinformatic tools, T-cell proliferation, cytokine release, IgG/IgE binding, and degranulation assays were used to identify and characterize the T-cell epitopes in Lit v 1 in comparison with previously validated B-cell epitopes. The results showed that of the six in silico predicted T-cell epitopes only one (T2: VQESLLKANIQLVEK, 60-74) promoted T-cell proliferation, the release of IL-2, and upregulated secretion of Th2-associated cytokines in the absence of IgG/IgE binding and degranulation activities. These findings support T2 as a candidate for the development of an efficient peptide-based vaccine for the immunotherapy for shrimp-allergic patients.

Proteomics-Based Methodologies for the Detection and Quantification of Seafood Allergens.

Seafood is considered one of the main food allergen sources by the European Food Safety Authority (EFSA). It comprises several distinct groups of edible aquatic animals, including fish and shellfish, such as crustacean and mollusks. Recently, the EFSA recognized the high risk of food allergy over the world and established the necessity of developing new methodologies for its control. Consequently, accurate, sensitive, and fast detection methods for seafood allergy control and detection in food products are highly recommended. In this work, we present a comprehensive review of the applications of the proteomics methodologies for the detection and quantification of seafood allergens. For this purpose, two consecutive proteomics strategies (discovery and targeted proteomics) that are applied to the study and control of seafood allergies are reviewed in detail. In addition, future directions and new perspectives are also provided.

Transcriptomic Analysis Reveals the Wound Healing Activity of Mussel Myticin C.

Myticin C is the most studied antimicrobial peptide in the marine mussel Mytilusgalloprovincialis. Although it is constitutively expressed in mussel hemocytes and displays antibacterial, antiviral, and chemotactic functions, recent work has suggested that this molecule is mainly activated after tissue injury. Therefore, the main objective of this work was to characterize the hemocytes' transcriptomic response after a myticin C treatment, in order to understand the molecular changes induced by this cytokine-like molecule. The transcriptome analysis revealed the modulation of genes related to cellular movement, such as myosin, transgelin, and calponin-like proteins, in agreement with results of functional assays, where an implication of myticin C in the in vitro activation of hemocytes and migration was evidenced. This was also observed in vivo after a tissue injury, when hemocytes, with high concentrations of myticin C, migrated to the damaged area to heal the wound. All these properties allowed us to think about the biotechnological application of these molecules as wound healers. Human keratinocytes and larvae zebrafish models were used to confirm this hypothesis. Accelerated regeneration after a wound or tail fin amputation was observed after treatment with the myticin C peptide, supporting the chemotactic and healing activity of myticin C.

Detection of oxidized methionine in selected proteins, cellular extracts and blood serums by novel anti-methionine sulfoxide antibodies.

Methionine sulfoxide (MetO) is a common posttranslational modification to proteins occurring in vivo.These modifications are prevalent when reactive oxygen species levels are increased. To enable the detection of MetO in pure and extracted proteins from various sources, we have developed novel antibodies that can recognize MetO-proteins. These antibodies are polyclonal antibodies raised against an oxidized methionine-rich zein protein (MetO-DZS18) that are shown to recognize methionine oxidation in pure proteins and mouse and yeast extracts. Furthermore, mouse serum albumin and immunoglobulin (IgG)were shown to accumulate MetO as function of age especially in serums of methionine sulfoxide reductase A knockout mice. Interestingly, high levels of methionine-oxidized IgG in serums of subjects diagnosed with Alzheimer's disease were detected by western blot analysis using these antibodies. It is suggested that anti-MetO-DZS18 antibodies can be applied in the identification of proteins that undergo methionine oxidation under oxidative stress, aging, or disease state conditions.

Reconstruction of fish allergenicity from the content and structural traits of the component ß-parvalbumin isoforms.

Most fish-allergic patients have anti-ß-parvalbumin (ß-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the ß-PV load is considered a determinant for allergenicity, fish species express distinct ß-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their ß-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.

Methionine sulfoxides on PrPSc: a prion-specific covalent signature.

Prion diseases are fatal neurodegenerative disorders believed to be transmitted by PrP (Sc), an aberrant form of the membrane protein PrP (C). In the absence of an established form-specific covalent difference, the infectious properties of PrP (Sc) were uniquely ascribed to the self-perpetuation properties of its aberrant fold. Previous sequencing of the PrP chain isolated from PrP(27-30) showed the oxidation of some methionine residues; however, at that time, these findings were ascribed to experimental limitations. Using the unique recognition properties of alphaPrP mAb IPC2, protein chemistry, and state of the art mass spectrometry, we now show that while a large fraction of the methionine residues in brain PrP (Sc) are present as methionine sulfoxides this modification could not be found on brain PrP (C) as well as on its recombinant models. In particular, the pattern of oxidation of M213 with respect to the glycosylation at N181 of PrP (Sc) differs both within and between species, adding another diversity factor to the structure of PrP (Sc) molecules. Our results pave the way for the production of prion-specific reagents in the form of antibodies against oxidized PrP chains which can serve in the development of both diagnostic and therapeutic strategies. In addition, we hypothesize that the accumulation of PrP (Sc) and thereafter the pathogenesis of prion disease may result from the poor degradation of oxidized aberrantly folded PrP.

Are Amyloid Fibrils RNA-Traps? A Molecular Dynamics Perspective.

The self-assembly of proteins and peptides into amyloids is a key feature of an increasing number of diseases. Amyloid fibrils display a unique surface reactivity endowing the sequestration of molecules such as MicroRNAs, which can be the active moiety of the toxic action. To test this hypothesis we studied the recognition between a model RNA and two different steric zipper spines using molecular dynamics simulations. We found that the interaction occurs and displays peptide-sequence dependence. Interestingly, interactions with polar zipper surfaces such as the formed by SNQNNF are more stable and favor the formation of ß-barrel like complexes resembling the structures of toxic oligomers. These sequence-structure-recognition relationships of the two different assemblies may be exploited for the design of compounds targeting the fibers or competing with RNA-amyloid attachment.

Preparation of Amyloidogenic Aggregates from EF-Hand ß-Parvalbumin and S100 Proteins.

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca2+-bound or Ca2+-free states with distinct aggregation propensities. That is the case among ß-parvalbumins and S100 proteins, which under certain conditions undergo Ca2+-dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.