Protein and Peptide Letters - Volume 29, Issue 5, 2022

Bioactive peptides with potential health benefits and metabolic functionality have been identified from plant-based food. The aim of this perspective is to report the recent progress in the research of plant-derived bioactive peptides using the combination of omics technologies and bioinformatics tools. Studies examining bioactive peptides with identified amino acid sequences and well-characterized biological functionalities are highlighted. Various software, webtools and workflows for analyzing and interpreting the biological data acquired from different omics approaches are discussed. The emerging evidence from the integration of proteomics and metabolomics data with advanced laboratory analytical methods supports more potential applications in the envisioned development of nutraceutical and therapeutic products. Notwithstanding, much works are mandatory to resolve those lied-ahead challenges before realizing the proposed applications of plant peptides.

Background: The Z-type variant of human α1-antitrypsin is involved in liver cirrhosis and pulmonary emphysema. Due to its slow folding characteristics, this variant accumulates folding intermediates and forms protein aggregates within hepatocytes. Misfolded proteins may induce oxidative stress and subsequent cell death. Objective: The potential application of antioxidant response signaling pathway and antioxidants to cope with Z-type α1-antitrypsin-induced oxidative stress was evaluated. Methods: Overexpression of Z-type α1-antitrypsin in Saccharomyces cerevisiae provoked oxidative stress and increased susceptibility to oxidative challenges such as hydrogen peroxide treatment. Deletion of antioxidant-response genes, including yap1, skn7, sod2, tsa1, and pst2, exacerbated the slow growth phenotype of Z-type α1-antitrypsin-expressing cells. Antioxidant treatment alleviated oxidative stress and cytotoxicity induced by Z-type α1-antitrypsin. Results: Our results show that cellular antioxidant capacity is crucial to protection against misfolded Z-type α1-antitrypsin. Conclusion: The information obtained here may be used to prevent oxidative stress caused by misfolded proteins, which are associated with several degenerative diseases, including amyotrophic lateral sclerosis and Parkinson’s disease.

Aims: This aimed to study the causative agent, epidemiology, clinical characteristics, and treatment strategy targeting the main protease in porcine epidemic diarrhea. Background: Porcine epidemic diarrhea (PED) is a contagious intestinal viral infection causing severe diarrhea, vomiting, and dehydration in pigs. High rates of mortalities and severe morbidities, approaching 100%, are reported in piglets infected with PEDV. In recent years, PED has been observed to influence the swine-farming nations in Europe, Asia, the USA, South Korea, and Canada. The PED virus (PEDV) transmission takes place through a faecal-oral route. Objective: The objective is to review the characteristics of PEDV and its role in the disease. In addition, we aim to outline some possible methods to combat PED infection, including targeting the main protease of coronavirus and their future perspectives. Methods: This study is a review of literature on the PED virus. Results: Apart from symptomatic treatment and supportive care, there is no available specific treatment for PEDV. Appropriate disinfectants and cleaning are pivotal for the control of PEDV. To date, apart from anti-PEDV inhibitors, there are no specific drugs available commercially to treat the disease. Therefore, 3C-like protease (3CLpro) in PEDV that has highly conserved structure and catalytic mechanism serves as an alluring drug as it plays a vital role during viral polyprotein processing at the time of infection. Conclusion: A well synchronized and collective effort of scientists, swine veterinarians, pork industry experts, and associated authorities is essential for the accomplishment of proper execution of these required measures.

Food-derived antihypertensive peptides are considered a natural supplement for controlling hypertension. Food protein serves as a macronutrient and acts as a raw material for the biosynthesis of physiologically active peptides. Food sources, like milk and milk products, animal proteins such as meat, chicken, fish, eggs, and plant-derived proteins from food products like soy, rice, wheat, mushroom, and pumpkins contain higher quantities of antihypertensive peptides. The food-derived antihypertensive peptides can suppress the action of renin and the angiotensinconverting enzyme (ACE), which are mainly involved in the regulation of blood pressure by RAS. ACE inhibitory peptides enhance endothelial nitric oxide's biosynthesis, which increases nitric oxide production in vascular walls and encourages vasodilation. The peptides also inhibit the interaction between angiotensin II and its receptor, which helps reduce hypertension. This review explores the novel sources and applications of food-derived peptides for the management of hypertension.

Background: In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gβ and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gβ subunit partners is still elusive. Objective: To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become necessary. Methods: In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E. coli were digested by proteases, which were also produced in the presented study. Results: Preliminary structural characterization studies without the Gβ partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant β-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes. Conclusion: The proposed optimized expression and purification protocol can contribute to investigations on the Gβγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical to shed light on the role(s) of different plant Gγ subunit types in biological processes.

Background: The identification of N-glycans in plant glycoproteins or plant-made pharmaceuticals is essential for understanding their structure, function, properties, immunogenicity, and allergenicity (induced by plant-specific core-fucosylation or xylosylation) in the applications of plant food, agriculture, and plant biotechnology. N-glycosidase A is widely used to release the Nglycans of plant glycoproteins because the core-fucosylated N-glycans of plant glycoproteins are hydrolyzed by N-glycosidase A but not by N-glycosidase F. However, the efficiency of Nglycosidase A activity in plant glycoproteins remains unclear. Objective: The aim of the study was to elucidate the efficient use of N-glycosidases to identify and quantify the N-glycans of plant glycoproteins; it aimed at identification of released N-glycans by Nglycosidase F and assessment of their relative quantities with a focus on unidentified N-glycans by N-glycosidase A in plant glycoproteins, Phaseolus vulgaris lectin (PHA) and horseradish peroxidase (HRP). Methods: Liquid chromatography–tandem mass spectrometry was used to analyze and compare the N-glycans of PHA and HRP treated with either N-glycosidase A or F under denaturing conditions. The relative quantities (%) of each N-glycan (>0.1%) to the total N-glycans (100%) were determined. Results: N-glycosidase A and F released 9 identical N-glycans of PHA, but two additional corefucosylated N-glycans were released by only N-glycosidase A, as expected. By contrast, in HRP, 8 N-glycans comprising 6 core-fucosylated N-glycans, 1 xylosylated N-glycan, and 1 mannosylated N-glycan were released by N-glycosidase A. Moreover, 8 unexpected N-glycans comprising 1 corefucosylated N-glycan, 4 xylosylated N-glycans, and 3 mannosylated N-glycans were released by Nglycosidase F. Of these, 3 xylosylated and 2 mannosylated N-glycans were released by only Nglycansodase F. Conclusion: These results demonstrate that N-glycosidase A alone is insufficient to release the Nglycans of all plant glycoproteins, suggesting that to identify and quantify the released N-glycans of the plant glycoprotein HRP, both N-glycosidase A and F treatments are required.

Background: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. Objective: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van’t Hoff enthalpy changes of denaturation ( ΔH^Ucal and ΔH^UvH ), as well as characterization of elements of secondary structure at three different pHs. Methods: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. Results: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔH^UvH U of 52.68 ºC and 484 kJ.mol^-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of β-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in β-structures that can contribute to the formation of protein aggregates. Conclusion: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.

Background: Protease inhibitors inhibit the activity of protease enzymes; hence, they are essentially involved in the regulation of the metabolic processes involving protease enzymes and the protection of the host organism against external damage due to proteases. These inhibitors are abundantly present in all living organisms but have not been much reported in mushrooms. Mushrooms are one of the major food components of humans, with delicious taste and high nutritional value. Mushrooms also have therapeutic and economic significance. The edible mushrooms with medicinal properties are much in commercial demand. To date, the presence of protease inhibitors has not been reported much in edible mushrooms. The present study reports the characterization of a protease inhibitor isolated from the common white button mushroom Agaricus bisporus. Objective: The objective of the present study is to characterize the novel protease inhibitor from Agaricus bisporus in order to determine its nature and activity under varying environmental conditions. Methods: The protease inhibitor was characterized through SDS PAGE, gel filtration chromatography, and de novo sequencing in order to determine its molecular mass and sequence, respectively. The optimum pH, temperature, and thermal stability were studied to determine the optimum working range of the protease inhibitor. The protease inhibitory activity (%) was determined in the presence of metal ions, surfactants, oxidizing agents, and reducing agents. The kinetic parameters and the type of inhibition exhibited by the protease inhibitor were determined using casein and trypsin protease enzyme. Results: The protease inhibitor was found to be a low molecular mass compound of 25 kDa. The de novo sequencing matched the inhibitor against a 227 amino acid containing peptide molecular mass of 24.6 kDa molecular mass. The protease inhibitory activity (%) was found highest at pH 7.0 and temperature 50 °C, and the inhibitor was stable from pH 4.0-9.0 and temperature 30-80 °C. In the presence of metal ions, the residual protease inhibitory activity (%) enhanced in the presence of Na+, Mg²⁺, and Fe³⁺. The residual activity increased in the presence of the surfactant SDS slightly in comparison to control, while it decreased in the case of Triton-X and Tween 20. The presence of oxidizing agents, hydrogen peroxide and dimethyl sulfoxide decreased the residual inhibitory activity. The protease inhibitor’s activity was unaffected by the reducing agents, dithiothreitol and β-mercaptoethanol, at up to 2mM concentration, but it decreased at higher concentrations. The inhibitor exhibited uncompetitive inhibition against trypsin with an inhibitory constant of 166 nM, indicating a strong affinity towards the protease, with a half-life of 93.90 minutes at 37 °C. Conclusion: Protease inhibitors isolated from mushrooms are generally small in size, more stable, and tolerant towards varying external conditions. The protease inhibitor isolated from Agaricus bisporus also exhibited similar characteristics.

Aim: This study aims to identify novel post-translational modifications in human serum albumin by mass spectrometry. Background: Serum albumin is the most abundant protein in plasma, has many physiological functions, and is in contact with most of the cells and tissues of the human body. Post-translational modifications (PTMs) may affect functions, stability, and localization of albumin. Methods: Human serum albumin (HSA) was used for tryptic digestion in-solution or in-gel. Mass spectrometry was applied to identify PTMs in HSA. 3-dimensional modeling was applied to explore the potential impact of PTMs on known functions of albumin. Results: Here, we report the identification of 61 novel PTMs of human serum albumin. Phosphorylation, glycosylation, nitrosylation, deamidation, methylation, acetylation, palmitoylation, geranylation, and farnesylation are some examples of the identified PTMs. Mass spectrometry was used for the identification of PTMs in a purified HSA and HSA from the human plasma. Threedimensional modeling of albumin with selected PTMs showed the location of these PTMs in the regions involved in albumin interactions with drugs, metals, and fatty acids. The location of PTMs in these regions may modify the binding capacity of albumin. Conclusion: This report adds 61 novel PTMs to the catalog of human albumin.