ABSTRACT
BACKGROUND: Parkinson's disease ranks second, after Alzheimer's as the major neurodegenerative disorder, for which no cure or disease-modifying therapies exist. Ample evidence indicate that PD manifests as a result of impaired anti-oxidative machinery leading to neuronal death wherein Cullin-3 has ascended as a potential therapeutic target for diseases involving damaged anti-oxidative machinery. OBJECTIVE: The design of target specific inhibitors for the Cullin-3 protein might be a promising strategy to increase the Nrf2 levels and to decrease the possibility of "off-target" toxic properties. METHODS: In the present study, an integrated computational and wet lab approach was adopted to identify small molecule inhibitors for Cullin-3. The rational drug designing process comprised homology modeling and derivation of the pharmacophore for Cullin-3, virtual screening of Zinc natural compound database, molecular docking and Molecular dynamics based screening of ligand molecules. In vivo validations of an identified lead compound were conducted in the PD model of C. elegans. RESULTS AND DISCUSSION: Our strategy yielded a potential inhibitor; (Glide score = -12.31), which was evaluated for its neuroprotective efficacy in the PD model of C. elegans. The inhibitor was able to efficiently defend against neuronal death in PD model of C. elegans and the neuroprotective effects were attributed to its anti-oxidant activities, supported by the increase in superoxide dismutase, catalase and the diminution of acetylcholinesterase and reactive oxygen species levels. In addition, the Cullin-3 inhibitor significantly restored the behavioral deficits in the transgenic C. elegans. CONCLUSION: Taken together, these findings highlight the potential utility of Cullin-3 inhibition to block the persistent neuronal death in PD. Further studies focusing on Cullin-3 and its mechanism of action would be interesting.
Subject(s)
Caenorhabditis elegans Proteins/antagonists & inhibitors , Cullin Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Neuroprotective Agents/chemistry , Oxidative Stress/drug effects , Parkinson Disease/drug therapy , Parkinson Disease/prevention & control , Acetylcholinesterase/metabolism , Animals , Antioxidants/pharmacology , Caenorhabditis elegans , Databases, Pharmaceutical , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Humans , Models, Animal , Molecular Docking Simulation , Molecular Dynamics Simulation , NF-E2-Related Factor 2/metabolism , Neuroprotective Agents/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
ß-lactoglobulin (ß-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as ß-lg. The sequences IIVTQ f[1-5] and LSFNPTQLEEQCHV f(149-162) of m/z 933(+) and 851(2+) were found to match N- and C-extreme of ß-lg while IDALNENK f(84-91) and TPEVDDEALEKFDK f(125-138) sequences deduced for m/z 916(+) and 818(2+) were in compliance to buffalo milk ß-lg. Considering the sequence similarity of ß-lg to glycodelin, a proven angiogenic protein, similar role for ß-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled ß-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.
Subject(s)
Buffaloes , Colostrum/chemistry , G-Protein-Coupled Receptor Kinase 4/metabolism , Lactoglobulins/metabolism , Lactoglobulins/pharmacology , Neovascularization, Physiologic/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Humans , Lactoglobulins/chemistry , Male , Protein Binding , Proteomics , RatsABSTRACT
Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90-, 84-, 62-, 60-, 52- and 33-kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2-DE with MALDI-TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1-DE gel was identified as HSP83 by MALDI-TOF-MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock-induced protein spots were excised from 2-DE gels for MALDI-TOF-MS analysis. The Mascot search results are for HSP68, HSC70-1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC-3 and HSP83 in NB(4)D(2). Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB(4)D(2). Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.
