In Silico Analysis and Functional Characterization of Antimicrobial and Insecticidal Vicilin from Moth Bean (Vigna aconitifolia (Jacq.) Marechal) Seeds.

Vicilin has nutraceutical potential and different noteworthy medicative health-promoting biotic diversions, and it is remarkable against pathogenic microorganisms and insects. In this study, Vigna aconitifolia vicilin (VacV) has been identified and characterized from the seed of Vigna aconitifolia (Jacq.) Marechal (Moth beans). LC-MS/MS analysis of VacV provided seven random fragmented sequences comprising 238 residues, showing significant homology with already reported Vigna radiata vicilin (VraV). VacV was purified using ammonium sulfate precipitation (60%) followed by size exclusion chromatography on Hi-Load 16/60 Superdex 200 pg column and anion-exchange chromatography (Hi trap Q FF column). Purified VacV showed a major ~50 kDa band and multiple lower bands on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under both reduced and non-reduced conditions. After all, a three-dimensional molecular structure of VacV was predicted, which showed ß-sheeted molecular conformation similar to crystallographic structure of VraV. All Vicilins from V. aconitifolia and other plants were divided into six sub-groups by phylogenetic analysis, and VacV shared a high degree of similarity with vicilins of Vigna radiata, Pisum sativum, Lupinus albus, Cicer arietinum and Glycine max. Additionally, VacV (20 µg) has significant growth inhibition against different pathogenic bacteria along strong antifungal activity (50 µg). Likewise, VacV (3.0 mg) produced significant growth reduction in Rice Weevil Sitophilus oryzae larvae after 9 days compared with control. Furthermore, by using MMT assay, the cytotoxicity effect of VacV on the growth of HepG2 liver cancerous cells was tested. VacV showed cytotoxicity against the HepG-2 line and the acquired value was 180 µg after 48 h. Finally, we performed molecular docking against caspase-3 protein (PDB ID: 3DEI) for VacV bioactive receptor interface residues. Hence, our results reveal that VacV, has nutraceutical potential and moth beans can be used as a rich resource of functional foods.

Comparative Study to Characterise the Pharmaceutical Potential of Synthesised Snake Venom Bradykinin-Potentiating Peptides In Vivo.

BACKGROUND: Bradykinin-potentiating peptides (BPPs) are snake venom peptides inhibiting the angiotensin-converting enzyme (ACE). ACE plays an important role in the regulation of blood pressure. BPPs lead to the development of ACE inhibitors for the treatment of hypertension. OBJECTIVE: The objective of the present work was to carry out a comprehensive comparative study of four synthesised snake venom BPPs in vivo. METHODS: Four synthesised snake venom BPPs were administered to rats via the intraperitoneal route for 15 days at a fixed dose. Lisinopril was used as a comparative standard. Thirty male albino rats were divided into six groups: A, B, C, D, E (lisinopril), and F (control). Group F was maintained as the control group and given only saline. After 15 days, blood samples and tissues were removed for the study of selective biochemical parameters and histomorphometric analysis. Statistical evaluation of all results was also performed. RESULTS: The results indicated that peptide I, with the sequence ZSAPGNEAIPP, was highly toxic and adversely affected all the biochemical and histological parameters studied in this work. Peptide II (ZNWPHPQIPP) and peptide IV (ZQWAQGRAPHPP) showed lower toxicity. None of the BPPs raised the serum creatinine level and exhibited nephroprotective effects. Although lisinopril raised the creatinine level, it showed a protective role towards the pancreas and lungs in parallel. CONCLUSION: The present work shows that although there is a high sequence similarity between the four BPPs, their in vivo activity varies. The sequences of peptide II and peptide IV can be used to improve the design of current ACE inhibitors used for hypertension treatment.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Graphene quantum dots as cysteine protease nanocarriers against stored grain insect pests.

Storing grains remain vulnerable to insect pest attack. The present study developed a biopesticide using biomolecules and their encapsulation in nanoparticles. A 25 kDa cysteine protease extracted from seeds of Albizia procera (ApCP) was encapsulated in graphene quantum dots (GQDs). The insecticidal activity of ApCP, with or without GQDs, against two stored grain insect pests, Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius) was explored. Insects were exposed to three concentrations 7.0, 3.5 and 1.7 mg of ApCP per a gram of wheat flour and grains. The insecticidal activity of ApCP encapsulated with GQDs was improved compared to that of ApCP without GQDs for both insect pests. The number of eggs and larvae of T. castaneum was reduced by 49% and 86%, respectively. Larval mortality was increased to 72%, and adult eclosion of T. castaneum was reduced by 98% at a 7.0 mg/g concentration of ApCP with GQDs compared to that of ApCP without GQDs. Exposure to 7.0 mg/g ApCP with GQDs, the number of R. dominica eggs and larvae was reduced by 72% and 92% respectively, larval mortality was increased by 90%, and eclosion was reduced by 97%. The extraction, purification, characterization, quantification and encapsulation of ApCP with GQDs were also studied. Cysteine protease nanocarriers have the potential to control stored grain insect pests.

Snake Venom Peptides: Tools of Biodiscovery.

Nature endowed snakes with a lethal secretion known as venom, which has been fine-tuned over millions of years of evolution. Snakes utilize venom to subdue their prey and to survive in their natural habitat. Venom is known to be a very poisonous mixture, consisting of a variety of molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides. Proteins and peptides are the major constituents of the dry weight of snake venoms and are of main interest for scientific investigations as well as for various pharmacological applications. Snake venoms contain enzymatic and non-enzymatic proteins and peptides, which are grouped into different families based on their structure and function. Members of a single family display significant similarities in their primary, secondary and tertiary structures, but in many cases have distinct pharmacological functions and different bioactivities. The functional specificity of peptides belonging to the same family can be attributed to subtle variations in their amino acid sequences. Currently, complementary tools and techniques are utilized to isolate and characterize the peptides, and study their potential applications as molecular probes, and possible templates for drug discovery and design investigations.

MOLECULAR MODEL OF CYTOTOXIN-1 FROM NAJA MOSSAMBICA MOSSAMBICA VENOM IN COMPLEX WITH CHYMOTRYPSIN.

Snake venom is a myriad of biologically active proteins and peptides. Three finger toxins are highly conserved in their molecular structure, but interestingly possess diverse biological functions. During the course of evolution the introduction of subtle mutations in loop regions and slight variations in the three dimensional structure, has resulted in their functional versatility. Cytotoxin-1 (UniProt ID: P01467), isolated from Naja mossambica mossambica, showed the potential to inhibit chymotrypsin and the chymotryptic activity of the 20S proteasome. In the present work we describe a molecular model of cytotoxin-1 in complex with chymotrypsin, prepared by the online server ClusPro. Analysis of the molecular model shows that Cytotoxin-1 (P01467) binds to chymotrypsin through its loop I located near the N-terminus. The concave side of loop I of the toxin fits well in the substrate binding pocket of the protease. We propose Phe10 as the dedicated P1 site of the ligand. Being a potent inhibitor of the 20S proteasome, cytotoxin-1 (P01467) can serve as a potential antitumor agent. Already snake venom cytotoxins have been investigated for their ability as an anticancer agent. The molecular model of cytotoxin-1 in complex with chymotrypsin provides important information towards understanding the complex formation.