Buffalo colostrum peptide mitigates Parkinson's disease pathophysiology through Cullin-3 inhibition.

Colostrum/Milk is a chief repertoire of antioxidant peptides. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a viable target for Parkinson's Disease (PD), as this pathway deduced to be impaired in PD. Cullin-3 is one of the crucial E3 ligase responsible for its regulation. The present study screened peptide libraries of buffalo colostrum & milk peptides for Cullin-3 inhibition, thus ensuing activation of Nrf2 to alleviate the molecular etiopathology in PD using the C. elegans as a model. The structure was modelled, binding sites analyzed and peptide-interactions analyzed by docking. Among the 55 sequences (≤1 kDa), the peptide SFVSEVPEL having the highest dock score (-16.919) was synthesized and evaluated for its effects on oxidative stress markers, antioxidant enzymes, neurochemical marker and Nrf2/Skn-1 levels. The lead peptide alleviated the oxidative pathophysiology and behavioural deficits associated with PD in C. elegans.

Milk Peptides as Novel Multi-Targeted Therapeutic Candidates for SARS-CoV2.

The COVID-19, an acute respiratory syndrome caused by SARS-CoV2 is a major catastrophic event of the twenty first century. Relentless efforts for the development of effective pharmaco-therapeutics are in progress but the respite is the development of effective vaccines. However, monotherapy might not always exhibit complete efficacy and may culminate in the rapid evolution of drug-resistant viral strains. Hence, simultaneous modulation of multiple druggable targets not only enhances therapeutic efficacy but also quell the prospects of mutant viruses. Currently, milk peptides have bloomed beyond just being a quintessential part of nutrition to prominent therapeutic implications in human health and diseases. Hence, we have focused on colostrum/milk peptides as they have already been acknowledged for their high potency, target specificity with significantly low or no side effects and bio-toleration. The results presented provide a conceptual strategy for the rational designing of prospective multitargeted peptide inhibitors for SARS-CoV2.

Discovery, synthesis, and in vitro evaluation of a novel bioactive peptide for ACE and DPP-IV inhibitory activity.

Biologically active or bioactive peptides are unique amino acid sequences found encrypted in food proteins. These peptides, upon hydrolysis, can exert positive physiological effects on human health, different from that of their native protein. These effects are brought about by their interaction with specific targets in the body, thereby, mimicking physiologically relevant peptides. Peptides are derived from food proteins, they are popular natural alternatives for the management of common metabolic disorders. In the present study, we aimed to identify bioactive peptide sequences (less than 3 kDa) from fat globule membrane protein (FGMP) hydrolysates of buffalo colostrum using a combination of empirical, computational and in vitro methods. The empirical approach aided in the identification of 89 FGMP peptides (m/z-415 to 2939) which were annotated and profiled for bioactivity. Few lead peptides were analyzed by molecular docking for the inhibitory potential of Angiotensin Converting Enzyme (ACE) and Dipeptidyl Peptidase-IV (DPP-IV). A heptapeptide (m/z-723.3) synthesized was found to inhibit ACE (IC50: 74.27 µM) and DPP-IV (IC50: 3.83 mM).

A new pyrrole based small molecule from Tinospora cordifolia induces apoptosis in MDA-MB-231 breast cancer cells via ROS mediated mitochondrial damage and restoration of p53 activity.

Approximately 15% of globally diagnosed breast cancers are designated as triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular calcium, mitochondrial membrane potential (ΔΨm), MPTP, cardiolipin peroxidation and caspase activity were determined fluorometrically. BAX, BCL-2, cytochrome c, caspases, and p53 protein expressions were determined by immunoblotting. Further, the effect of TCCP on DNA and cell death was determined by DNA fragmentation assay, annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with pifithrin-µ. This led to the downstream altered expression of Bcl-2 and Bax proteins, mitochondrial membrane depolarization, MPTP, and cardiolipin peroxidation. TCCP induced cytochrome c release into the cytosol, caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the phosphatidylserine externalization and increase in sub G1 population. The in vivo Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231 cells and also induced EAT cell death in vivo thereby inhibiting tumor proliferation.

A pyrrole-based natural small molecule mitigates HSP90 expression in MDA-MB-231 cells and inhibits tumor angiogenesis in mice by inactivating HSF-1.

Heat shock proteins (HSPs), molecular chaperones, are crucial for the cancer cells to facilitate proper functioning of various oncoproteins involved in cell survival, proliferation, migration, and tumor angiogenesis. Tumor cells are said to be "addicted" to HSPs. HSPs are overexpressed in many cancers due to upregulation of transcription factor Heat-shock factor 1 (HSF-1), the multifaceted master regulator of heat shock response. Therefore, pharmacological targeting of HSPs via HSF-1 is an effective strategy to treat malignant cancers like triple negative breast cancer. In the current study, we evaluated the efficacy of a pyrrole derivative [bis(2-ethylhexyl)1H-pyrrole-3,4-dicarboxylate], TCCP, purified from leaves of Tinospora cordifolia for its ability to suppress heat shock response and angiogenesis using MDA-MB-231 cells and the murine mammary carcinoma: Ehrlich ascites tumor model. HSP90 was downregulated by TCCP by inactivation of HSF-1 resulting in inhibition of tumor cell proliferation, VEGF-induced cell migration, and concomitant decrease in tumor burden and neo-angiogenesis in vivo. The mechanism of suppression of HSPs involves inactivation of PI3K/Akt and phosphorylation on serine 307 of HSF-1 by the activation of ERK1. HSF-1 and HSP90 and 70 localization and expression were ascertained by immunolocalization, immunoblotting, and qPCR experiments. The anti-angiogenic effect of TCCP was studied in vivo in tumor-bearing mice and ex vivo using rat corneal micro-pocket assay. All the results thus corroborate the logic behind inactivating HSF-1 using TCCP as an alternative approach for cancer therapy.

Comprehensive characterization of bioactive peptides from Buffalo (Bubalus bubalis) colostrum and milk fat globule membrane proteins.

Milk fat is dispersed in milk as small spherical globules stabilized in the form of emulsion by its surrounding membrane, often referred to as fat globule membrane (FGM). Buffalo, a major milking mammal of Asia and second most milking mammal across the globe presents physicochemical features different from that of other ruminant species containing higher content of lipids and proteins. The present study describes characterization of FGM proteins isolated from both buffalo milk and colostrum. A detailed proteomic analysis of peptides generated by in vitro gastrointestinal simulation digestion of buffalo milk and colostrum FGM fractions was performed by nLC-ESI MS/MS. The peptide based clustering of FGM proteins unravelled association of membrane proteins in fat transport, enzymatic activity, general transport, defence, cell signalling, membrane/protein trafficking protein synthesis/binding/folding including unknown functions. Gene annotation, STRING and YLoc analyses provided putative insights into major secretory pathways in milk and colostrum FGM peptides, interactive protein networks including their sub cellular localization. The peptides of milk and colostrum FGM offered cellular protection as powerful antioxidants indicated their promising perspectives in commercial formulations and nutraceuticals.

Empirical and bioinformatic characterization of buffalo (Bubalus bubalis) colostrum whey peptides & their angiotensin I-converting enzyme inhibition.

Whey based peptides are well known for their nutritional and multifunctional properties. In this context, whey proteins from buffalo colostrum & milk were digested by in vitro simulation digestion and analyzed by nano-LC-MS/MS. Functional protein association networks, gene annotations and localization of identified proteins were carried out. An ACE inhibitory peptide sorted from the library was custom synthesized and an in vitro ACE assay was performed. The study led to the identification of 74 small peptides which were clustered into 5 gene functional groups and majority of them were secretory proteins. Among the identified peptides, majority of them were found identical to angiotensin I-converting enzyme (ACE) inhibitors, antioxidant, antimicrobial, immunomodulatory and opioidal peptides. An octapeptide (m/z - 902.51, IQKVAGTW) synthesized was found to inhibit ACE with an IC50 of 300±2µM. The present investigation thus establishes newer vista for food derived peptides having ACE inhibitory potential for nutraceutical or therapeutic applications.

Inhibition of Snake Venom Metalloproteinase by ß-Lactoglobulin Peptide from Buffalo (Bubalus bubalis) Colostrum.

Bioactive peptide research has experienced considerable therapeutic interest owing to varied physiological functions, efficacy in excretion, and tolerability of peptides. Colostrum is a rich natural source of bioactive peptides with many properties elucidated such as anti-thrombotic, anti-hypertensive, opioid, immunomodulatory, etc. In this study, a variant peptide derived from ß-lactoglobulin from buffalo colostrum was evaluated for the anti-ophidian property by targeting snake venom metalloproteinases. These are responsible for rapid local tissue damages that develop after snakebite such as edema, hemorrhage, myonecrosis, and extracellular matrix degradation. The peptide identified by LC-MS/MS effectively neutralized hemorrhagic activity of the Echis carinatus venom in a dose-dependent manner. Histological examinations revealed that the peptide mitigated basement membrane degradation and accumulation of inflammatory leucocytes at the venom-injected site. Inhibition of proteolytic activity was evidenced in both casein and gelatin zymograms. Also, inhibition of fibrinolytic and fibrinogenolytic activities was seen. The UV-visible spectral study implicated Zn2+ chelation, which was further confirmed by molecular docking and dynamic studies by assessing molecular interactions, thus implicating the probable mechanism for inhibition of venom-induced proteolytic and hemorrhagic activities. The present investigation establishes newer vista for the BLG-col peptide with anti-ophidian efficacy as a promising candidate for therapeutic interventions.

Glycome characterization of immunoglobulin G from buffalo (Bubalus bubalis) colostrum.

Immunoglobulin G (IgG) is a major glycoprotein in ruminant colostrum. First day buffalo colostrum protein was purified on Sephadex G-100 and its mass was determined by MALDI-TOF as 147.848 KDa. The PMF data of protein subunits revealed its homology to IgG, which was supported by the identification of peptide sequences LLIYGATSR and VYNEYLPAPIVR corresponding to light and heavy chains of IgG by CID MS/MS analysis. The N-glycan microheterogeneity was established based on chemoselective glycoblotting technique with the identification of high mannose, neutral complex/hybrid and sialylated complex/hybrid glycans. A complete structural assignment of 54 N-linked oligosaccharides were identified and the ratio of sialyl oligosaccharides was found to be higher compared to neutral saccharides. The fucosylation observed in more than 20 oligosaccharides, high mannose and trisialyl oligosaccharides were present in diminutive amount. The high non-fucosyl and sialyl oligosaccharides in buffalo colostrum IgG provide ample scope for its utilization in targeted therapies to elicit effective ADCC and anti-inflammatory responses.

A variant peptide of buffalo colostrum ß-lactoglobulin inhibits angiotensin I-converting enzyme activity.

ß-lactoglobulin is a rich source of bioactive peptides. The LC-MS separated tryptic peptides of buffalo colostrum ß-lactoglobulin (BLG-col) were computed based on MS-MS fragmentation for de novo sequencing. Among the selected peptides (P1-P8), a variant was detected with methionine at position 74 instead of glutamate. The sequences of two peptides were identical to hypocholesterolemic peptides whereas the remaining peptides were in accordance with buffalo milk ß-lactoglobulin. Comparative sequence analysis of BLG-col to milk ß-lactoglobulin was carried out using CLUSTALW2 and a molecular model for BLG-col was constructed (PMDB ID-PM0076812). The synthesized variant pentapeptide (IIAMK, m/z-576 Da) was found to inhibit angiotensin I-converting enzyme (ACE) with an IC(50) of 498 ± 2 µM, which was rationalized through docking simulations using Molgrow virtual docker.

Silkworm thermal biology: a review of heat shock response, heat shock proteins and heat acclimation in the domesticated silkworm, Bombyx mori.

Heat shock proteins (HSPs) are known to play ecological and evolutionary roles in this postgenomic era. Recent research suggests that HSPs are implicated in cardiovascular biology and disease development, proliferation and regulation of cancer cells, cell death via apoptosis, and several other key cellular functions. These activities have generated great interest amongst cell and molecular biologists, and these biologists are keen to unravel other hitherto unknown potential functions of this group of proteins. Consequently, the biological significance of HSPs has led to cloning and characterization of genes encoding HSPs in many organisms including the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). However, most of the past investigations in B. mori were confined to expression of HSPs in tissues and cell lines, whereas information on their specific functional roles in biological, physiological, and molecular processes is scarce. Naturally occurring or domesticated polyvoltines (known to be the tropical race) are more resistant to high temperatures and diseases than bi- or univoltines (temperate races). The mechanism of ecological or evolutionary modification of HSPs during the course of domestication of B. mori - particularly in relation to thermotolerance in geographically distinct races/strains - is still unclear. In addition, the heat shock response, thermal acclimation, and hardening have not been studied extensively in B. mori compared to other organisms. Towards this, recent investigations on differential expression of HSPs at various stages of development, considering the concept of the whole organism, open ample scope to evaluate their biological and commercial importance in B. mori which has not been addressed in any of the representative organisms studied so far. Comparatively, heat shock response among different silkworm races/strains of poly-, bi-, and univoltines varies significantly and thermotolerance increases as the larval development proceeds. Hence, this being the first review in this area, an attempt has been made to collate all available information on the heat shock response, HSPs expression, associated genes, amino acid sequences, and acquired/unacquired thermotolerance. The aim is to present this as a valuable resource for addressing the gap in knowledge and understanding evolutionary significance of HSPs between domesticated (B. mori) and non-domesticated insects. It is believed that the information presented here will also help researchers/breeders to design appropriate strategies for developing novel strains for the tropics.

Isolation and characterization of a sialo-glycopeptide from buffalo colostrum.

A sialoglycopeptide was isolated from buffalo colostrum in pure form by chromatography on Sephadex G-25 and QAE-Sephadex A-25. This was found to be homogeneous by cellulose acetate membrane electrophoresis and reverse phase HPLC. It consisted of fucose, galactose, mannose, N-acetyl glucosamine and N-acetyl neuraminic acid in the ratio 1:2:3:4:1, and aspartic acid, serine, threonine, proline and glutamic acid were the major amino acids. Glycine was identified as the N-terminal amino acid residue. The structure elucidation of the carbohydrate moiety was carried out by methylation analysis, mass spectrometry. 1H-NMR spectroscopy and the probable structure was revealed to be that of a complex biantennary type.