The role of cysteine in the formation of domain structures of papain and legumin in peas, involved in limited proteolysis

The limited use of plant proteins for food is explained by their low bioavailability and poor digestibility by enzymes of the gastrointestinal tract. Partially reproduced enzymatic processes of limited proteolysis that occur during seed germination are used to modify and improve the edibility characteristics of seed proteins. The present work discusses the possibility of reducing the duration of seed germination processes by optimising the conditions and parameters of limited proteolysis. To optimise manufacturing high-quality final product, enzymes (additional to the natural enzymes in the seed) and proteolysis conditions (in this case, temperature), as well as added substances (hydrolysis activators), were selected. The influence of cysteine on the formation of domain structures of proteins (enzymes and globulins) was evaluated. The proposed expressions can be used to determine those fragments of protein molecules that form stable domains and become unstructured when exposed to enzymes. Optimal conditions for limited proteolysis were identified based on the physical mechanism of action of papain-like proteolytic enzymes on pea legumin LegA (3KSC, CAA10722). It is shown that the decomposition of protein secondary structures takes 6–8 times longer, since the formed hydrogen bonds limit the access of enzymes to the corresponding amino-acid residues. It is also demonstrated that the decomposition of hydrogen bonds, e.g. by preliminary heat treatment of proteins, will broaden the prospects for limited proteolysis.

Mobilization of seed storage proteins is crucial to high vigor in common bean seeds

ABSTRACT: Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

AFP2 inhibits ABA responses during germination without ABI5 degradation but DWAs reduce desiccation tolerance

Overexpression of ABI5/ABF interacting proteins (AFPs) results in extreme ABA resistance of seeds and failure to acquire desiccation tolerance, at least in part through effects on chromatin modification. This study tests the hypothesis that the AFPs promote germination by also functioning as adapters for E3 ligases that ubiquitinate ABI5, leading to its degradation. Interactions between AFPs and two well-characterized classes of E3 ligases targeting ABI5, DWD HYPERSENSITIVE TO ABA (DWA)s and KEEP ON GOING (KEG), were analyzed by yeast two-hybrid, bimolecular fluorescence complementation, and genetic assays. Although the AFPs and E3 ligases showed weak direct interactions, loss of function for the E3 ligases did not impair ABA-resistance conferred by overexpression of the YFP-AFP2 fusion. Comparison of ABI5 and AFP2 levels in these lines showed that AFP2 accumulation increased during germination, but that ABI5 degradation followed germination, demonstrating that AFP2 controls ABA sensitivity during germination independently of ABI5 degradation. Surprisingly, AFP2 overexpression in the dwa1 dwa2 mutant background produced the unusual combination of extreme ABA resistance and desiccation tolerance, creating an opportunity to separate the underlying biochemical characteristics of ABA sensitivity and desiccation tolerance that we investigated by quantitative proteomics. Our analysis identified at least three-fold more differentially accumulated seed proteins than previous studies. Comparison of dry seed proteomes of the different genotypes allowed us to separate and refine the changes in protein accumulation patterns correlating with desiccation tolerance independently of ABA sensitivity, or vice versa, to a subset of cold-induced and defense stress-responsive proteins and signaling regulators.

A Molecular View of Amyotrophic Lateral Sclerosis Through the Lens of Interaction Network Modules

Background: Despite the discovery of familial cases with mutations in Cu/Zn-superoxide dismutase (SOD1), Guanine nucleotide exchange C9orf72, TAR DNA-binding protein 43 (TARDBP) and RNA-binding protein FUS as well as a number of other genes linked to Amyotrophic Lateral Sclerosis (ALS), the etiology and molecular pathogenesis of this devastating disease is still not understood. As proteins do not act alone, conducting an analysis of ALS at the system level may provide new insights into the molecular biology of ALS and put it into relationship to other neurological diseases.Methods: A set of ALS-associated genes/proteins were collected from publicly available databases and text mining of scientific literature. We used these as seed proteins to build protein-protein interaction (PPI) networks serving as a scaffold for further analyses. From the collection of networks, a set of core modules enriched in seed proteins were identified. The molecular biology of the core modules was investigated, as were their associations to other diseases. To assess the core modules’ ability to describe unknown or less well-studied ALS biology, they were queried for proteins more recently associated to ALS and not involved in the primary analysis.Results: We describe a set of 26 ALS core modules enriched in ALS-associated proteins. We show that these ALS core modules not only capture most of the current knowledge about ALS, but they also allow us to suggest biological interdependencies. In addition, new associations of ALS networks with other neurodegenerative diseases, e.g. Alzheimer’s, Huntington’s and Parkinson’s disease were found. A follow-up analysis of 140 ALS-associated proteins identified since 2014 reveals a significant overrepresentation of new ALS proteins in these 26 disease modules.Conclusions: Using protein-protein interaction networks offers a relevant approach for broadening the understanding of the biological context of known ALS-associated genes. Using a bottom-up approach for the analysis of protein-protein interaction networks is a useful method to avoid bias caused by over-connected proteins. Our ALS-enriched modules cover most known biological functions associated with ALS. The presence of recently identified ALS-associated proteins in the core modules highlights the potential for using these as a scaffold for identification of novel ALS disease mechanisms.

Silver-Protein Nanocomposites as Antimicrobial Agents

The use of nanomaterials alone or in composites with proteins is a promising alternative to inhibit pathogenic bacteria. In this regard, this study used seed proteins from both fenugreek (Trigonella foenum-graecum L.) (FNP) and mung bean (Viga radiate) (MNP), with silver nanoparticles (Ag-NPs) and nanocomposites of either Ag-NPs plus FNP (Ag-FNP) or Ag-NPs plus MNP (Ag-MNP) as inhibitory agents against pathogenic bacteria. FNP and MNP were isolated from fenugreek seeds and mung bean seeds, respectively, and fractionated using Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE). Both FNP and MNP were immobilized with Ag-NPs to synthesize the nanocomposites Ag-FNP and Ag-MNP, respectively. The physicochemical characteristics of Ag-NPs and their composites with proteins were studied by X-ray Diffraction (XRD), dynamic light scattering (DLS), the zeta potential, Scanning and Transmission Electron Microscopy (SEM and TEM, respectively), Atomic Force Microscopy (AFM), and the Brunauer–Emmett–Teller isotherm (BET), elucidating their structural parameters, size distribution, size charges, size surface morphology, particle shape, dimensional forms of particles, and specific surface area, respectively. The sole proteins, Ag-NPs, and their nanocomposites inhibited pathogenic Gram-positive and Gram-negative bacteria. The inhibitory activities of both nanocomposites (Ag-FNP and Ag-MNP) were more than those obtained by either Ag-NPs or proteins (FNP, MNP). Minimum inhibitory concentrations (MICs) of Ag-FNP were very low (20 and 10 µg mL−1) against Salmonellatyphimurium and Pseudomonasaerugenosa, respectively, but higher (162 µg mL−1) against E. coli and Listeriamonocytogenes. MICs of Ag-MNP were also very low (20 µg mL−1) against Staphylococcusaureus but higher (325 µg mL−1) against Listeriamonocytogenes. TEM images of Staphylococcusaureus and Salmonellatyphimurium, treated with Ag-FNP and Ag-MNP, at their MIC values, showed asymmetric, wrinkled exterior surfaces, cell deformations, cell depressions, and diminished cell numbers.

Production, Purification, and Potential Health Applications of Edible Seeds’ Bioactive Peptides: A Concise Review

Edible seeds play a significant role in contributing essential nutritional needs and impart several health benefits to improve the quality of human life. Previous literature evidence has confirmed that edible seed proteins, their enzymatic hydrolysates, and bioactive peptides (BAPs) have proven and potential attributes to ameliorate numerous chronic disorders through the modulation of activities of several molecular markers. Edible seed-derived proteins and peptides have gained much interest from researchers worldwide as ingredients to formulate therapeutic functional foods and nutraceuticals. In this review, four main methods are discussed (enzymatic hydrolysis, gastrointestinal digestion, fermentation, and genetic engineering) that are used for the production of BAPs, including their purification and characterization. This article’s main aim is to provide current knowledge regarding several health-promoting properties of edible seed BAPs in terms of antihypertensive, anti-cancer, antioxidative, anti-inflammatory, and hypoglycemic activities.

Comparative Study of the Structural and Functional Properties of Membrane-Isolated and Isoelectric pH Precipitated Green Lentil Seed Protein Isolates

The demand for isolated seed proteins continues to increase but functionality in food systems can be greatly dependent on the extraction method. In this work, we report the physicochemical and functional properties of lentil seed proteins isolated using various protocols. Lentil flour was defatted followed by protein extraction using isoelectric pH precipitation (ISO) as well as NaOH (MEM_NaOH) and NaCl (MEM_NaCl) extractions coupled with membrane ultrafiltration. The MEM_NaCl had significantly (p < 0.05) higher protein content (90.28%) than the ISO (86.13%) and MEM_NaOH (82.55%). At pH 3–5, the ISO was less soluble (2.26–11.84%) when compared to the MEM_NaOH (25.74–27.22%) and MEM_NaCl (27.78–40.98%). However, the ISO had higher yield and protein digestibility (48.45% and 89.82%) than MEM_NaOH (35.05% and 77.87%) and MEM_NaCl (13.35% and 77.61%), respectively. Near-UV circular dichroism spectra showed that the MEM_NaOH had loose tertiary conformation at pH 3, 5, 7 and 9 while ISO and MEM_NaCl had more compact structures at pH 7 and 9. The three protein isolates formed better emulsions (lower oil droplet sizes) at pH 7 and 9 when compared to pH 3 and 5. In contrast, foaming capacity was better at pH 5 than pH 3, 7, and 9.