Review of the Greening Reaction by Thermal Treatment: New Insights Exploring the Structural Implications of Myoglobin.

Myoglobin is the main factor responsible for muscle pigmentation in tuna; muscle color depends upon changes in the oxidative state of myoglobin. The tuna industry has reported muscle greening after thermal treatment involving metmyoglobin (MetMb), trimethylamine oxide (TMAO), and free cysteine (Cys). It has been proposed that this pigmentation change is due to a disulfide bond between a unique cysteine residue (Cys10) found in tuna MetMb and free Cys. However, no evidence has been given to confirm that this reaction occurs. In this review, new findings about the mechanism of this greening reaction are discussed, showing evidence of how free radicals produced from Cys oxidation under thermal treatment participate in the greening of tuna and horse muscle during thermal treatment. In addition, the reaction conditions are compared to other green myoglobins, such as sulfmyoglobin, verdomyoglobin, and cholemyoglobin.

Proteomic analysis of chemically transformed NIH-3T3 cells reveals novel mechanisms of action of amaranth lunasin-like peptide.

Food-derived biopeptides can interact with genes and proteins to preserve health and prevent the development of diseases. Lunasin is a soybean cancer-preventive peptide that has been well characterized; however, few studies have been carried out to characterize the function of amaranth lunasin-like peptide (AhLun). The aim of this work was to analyze the proteomic profile changes in NIH-3T3 cells when they are chemically transformed with the carcinogen 3-methylcholanthrene (3MC) in the absence or presence of AhLun. The addition of AhLun into the culture medium did not affect the cell morphology; however, as a chemopreventive agent, it significantly reduced anisokaryosis formation when cells were treated with 3MC. Changes in protein accumulation in NIH-3T3 cells were evaluated by gel-based proteomics analysis. Differentially accumulated protein spots that exhibited at least a twofold change in spot intensity (p < 0.05), when compared with control cells, were analyzed by LC-MS/MS. Successfully identified proteins were grouped into six main categories according to their localization and function (nuclear, ribosomal, mitochondrial, metabolism, cytoskeletal, and miscellaneous). The gel-based proteomic approach for the evaluation of the chemopreventive potential of AhLun reveals novel pathways of action and provides new clues about the possible mechanisms of action of this bioactive peptide present in amaranth seeds.

Exploring the Milk-Clotting and Proteolytic Activities in Different Tissues of Vallesia glabra: a New Source of Plant Proteolytic Enzymes.

Proteolytic enzymes are widely distributed in nature, playing essential roles in important biological functions. Recently, the use of plant proteases at the industrial level has mainly increased in the food industry (e.g., cheesemaking, meat tenderizing, and protein hydrolysate production). Current technological and scientific advances in the detection and characterization of proteolytic enzymes have encouraged the search for new natural sources. Thus, this work aimed to explore the milk-clotting and proteolytic properties of different tissues of Vallesia glabra. Aqueous extracts from the leaves, fruits, and seeds of V. glabra presented different protein profiles, proteolytic activity, and milk-clotting activity. The milk-clotting activity increased with temperature (30-65 °C), but this activity was higher in leaf (0.20 MCU/mL) compared with that in fruit and seed extracts (0.12 and 0.11 MCU/mL, respectively) at 50 °C. Proteolytic activity in the extracts assayed at different pH (2.5-12.0) suggested the presence of different types of active proteases, with maximum activity at acidic conditions (4.0-4.5). Inhibitory studies indicated that major activity in V. glabra extracts is related to cysteine proteases; however, the presence of serine, aspartic, and metalloproteases was also evident. The hydrolytic profile of caseins indicated that V. glabra leaves could be used as a rennet substitute in cheesemaking, representing a new and promising source of proteolytic enzymes.

Proteomic analysis of the inhibitory effect of the butanolic fraction of Jacquinia macrocarpa on Fusarium verticillioides.

Jacquinia macrocarpa, a plant native to northwestern Mexico, has an inhibitory effect against phytopathogenic fungi. Previous studies have shown that the butanolic extract of J. macrocarpa causes retardation and atrophy in mycelial growth of Fusarium verticillioides. However, the action mechanism of this extract is unknown. We used a proteomics approach to understand the inhibitory effect of J. macrocarpa butanolic extract, based on differential protein accumulation in F. verticillioides. Proteins were extracted from F. verticillioides cultured in Czapek broth with and without 202.12 µg/mL (IC50) of butanolic extract of J. macrocarpa. Thirty-eight protein spots showing statistically significant changes (ANOVA, p < 0.01) and at least a 2-fold change in abundance between experimental conditions were analyzed by mass spectrometry. Identified proteins were grouped into different biological processes according to Gene Ontology, among them were amino acid metabolism, protein folding and stabilization, protein degradation, protein transport, carbohydrate metabolism, oxidative stress response, and miscellaneous. This work is the first report of changes in the proteomic profile of F. verticillioides exposed to the J. macrocarpa extract. This information provides new insights into the inhibitory mechanism of the extract and represents a starting point for dissection of the fungal response against the J. macrocarpa extract components.

The proteome map of the escamolera ant (Liometopum apiculatum Mayr) larvae reveals immunogenic proteins and several hexamerin proteoforms.

The larvae of escamolera ant (Liometopum apiculatum Mayr) have been considered a delicacy since Pre-Hispanic times. The increased demand for this stew has led to massive collection of ant nests. Yet biological aspects of L. apiculatum larvae remain unknown, and mapping the proteome of this species is important for understanding its biological characteristics. Two-dimensional gel electrophoresis (2-DE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to characterize the larvae proteome profile. From 380 protein spots analyzed, 174 were identified by LC-MS/MS and homology search against the Hymenoptera subset of the NCBInr protein database using the Mascot search engine. Peptide de novo sequencing and homology-based alignment allowed the identification of 36 additional protein spots. Identified proteins were classified by cellular location, molecular function, and biological process according to the Gene Ontology annotation. Immunity- and defense-related proteins were identified including PPIases, FK506, PEBP, and chitinases. Several hexamerin proteoforms were identified and the cDNA of the most abundant protein detected in the 2-DE map was isolated and characterized. L. apiculatum hexamerin (LaHEX, GeneBank accession no. MH256667) contains an open reading frame of 2199 bp encoding a polypeptide of 733 amino acid residues with a calculated molecular mass of 82.41 kDa. LaHEX protein is more similar to HEX110 than HEX70 from Apis mellifera. Down-regulation of LaHEX was observed throughout ant development. This work represents the first proteome map as well as the first hexamerin characterized from L. apiculatum larvae.

Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds.

Chan (Hyptis suaveolens) is a Mesoamerican crop highly appreciated since the pre-Hispanic cultures. Its proteins are a good source of essential amino acids; however, there are no reports on the properties of its individual proteins. In this study, the 11S globulin (Hs11S) was purified and biochemically characterized. The molecular weight of native Hs11S was about 150-300 kDa with isoelectric points of 5.0-5.3, composed by four monomers of 53.5, 52, 51.1 and 49.5 kDa, each formed by one acidic subunit and one basic subunit linked by a disulfide bond. Dynamic light scattering, size exclusion chromatography and native PAGE show that Hs11S is assembled in different oligomeric forms. LC-MS/MS analysis confirmed its identity. Hs11S presents antigenic determinants in common with lupin 11S globulin. Carbohydrate moieties or phosphate groups linked to Hs11S were not detected. This information is very useful in order to exploit and utilize rationally chan 11S globulin in food systems.

Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein.

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

Low temperature conditioning of garlic (Allium sativum L.) "seed" cloves induces alterations in sprouts proteome.

Low-temperature conditioning of garlic "seed" cloves substitutes the initial climatic requirements of the crop and accelerates the cycle. We have reported that "seed" bulbs from "Coreano" variety conditioned at 5°C for 5 weeks reduces growth and plant weight as well as the crop yields and increases the synthesis of phenolic compounds and anthocyanins. Therefore, this treatment suggests a cold stress. Plant acclimation to stress is associated with deep changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation. The aim of this work was to study the changes in the protein profiles of garlic "seed" cloves subjected to conditioning at low-temperature using proteomics approach. Two sets of garlic bulbs were used, one set was stored at room temperature (23°C), and the other was conditioned at low temperature (5°C) for 5 weeks. Total soluble proteins were extracted from sprouts of cloves and separated by two-dimensional gel electrophoresis. Protein spots showing statistically significant changes in abundance were analyzed by LC-ESI-MS/MS and identified by database search analysis using the Mascot search engine. The results revealed that low-temperature conditioning of garlic "seed" cloves causes alterations in the accumulation of proteins involved in different physiological processes such as cellular growth, antioxidative/oxidative state, macromolecules transport, protein folding and transcription regulation process. The metabolic pathways affected include protein biosynthesis and quality control system, photosynthesis, photorespiration, energy production, and carbohydrate and nucleotide metabolism. These processes can work cooperatively to establish a new cellular homeostasis that might be related with the physiological and biochemical changes observed in previous studies.

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress.

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC-MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C3-C4 photosynthetic plant.

Salt stress-induced alterations in the root proteome of Amaranthus cruentus L.

Salt stress is one of the major factors limiting crop productivity worldwide. Amaranth is a highly nutritious pseudocereal with remarkable nutraceutical properties; it is also a stress-tolerant plant, making it an alternative crop for sustainable food production in semiarid conditions. A two-dimensional electrophoresis gel coupled with a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approach was applied in order to analyze the changes in amaranth root protein accumulation in plants subjected to salt stress under hydroponic conditions during the osmotic phase (1 h), after recovery (24 h), and during the ionic phase of salt stress (168 h). A total of 101 protein spots were differentially accumulated in response to stress, in which 77 were successfully identified by LC-MS/MS and a database search against public and amaranth transcriptome databases. The resulting proteins were grouped into different categories of biological processes according to Gene Ontology. The identification of several protein isoforms with a change in pI and/or molecular weight reveals the importance of the salt-stress-induced posttranslational modifications in stress tolerance. Interestingly stress-responsive proteins unique to amaranth, for example, Ah24, were identified. Amaranth is a stress-tolerant alternative crop for sustainable food production, and the understanding of amaranth's stress tolerance mechanisms will provide valuable input to improve stress tolerance of other crop plants.