RESUMO
BACKGROUND: N-ras protein is encoded by the NRAS gene and operates as GDP-GTP-controlled on/off switching. N-ras interacts with cellular signaling networks that regulate various cellular activities including cell proliferation and survival. The nonsynonymous single nucleotide polymorphism (nsSNPs)-mediated alteration can substantially disrupt the structure and activity of the corresponding protein. N-ras has been reported to be associated with numerous diseases including cancers due to the nsSNPs. A comprehensive study on the NRAS gene to unveil the potentially damaging and oncogenic nsSNPs is yet to be accomplished. Hence, this extensive in silico study is intended to identify the disease-associated, specifically oncogenic nsSNPs of the NRAS gene. RESULTS: Out of 140 missense variants, 7 nsSNPs (I55R, G60E, G60R, Y64D, L79F, D119G, and V152F) were identified to be damaging utilizing 10 computational tools that works based on different algorithms with high accuracy. Among those, G60E, G60R, and D119G variants were further filtered considering their location in the highly conserved region and later identified as oncogenic variants. Interestingly, G60E and G60R variants were revealed to be particularly associated with lung adenocarcinoma, rhabdomyosarcoma, and prostate adenocarcinoma. Therefore, D119G could be subjected to detailed investigation for identifying its association with specific cancer. CONCLUSION: This in silico study identified the deleterious and oncogenic missense variants of the human NRAS gene that could be utilized for designing further experimental investigation. The outcomes of this study would be worthwhile in future research for developing personalized medicine.
RESUMO
Galectin-8 is a ß-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-ß-d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3'-O-sialylated lactose (3'-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8â µm, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.
Assuntos
Galactosídeos/metabolismo , Galectinas/metabolismo , Arginina/química , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Galactosídeos/síntese química , Galactosídeos/química , Galectinas/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios ProteicosRESUMO
The protein α-synuclein (αSN) aggregates to form fibrils in neuronal cells of Parkinson's patients. Here we report on the effect of neutral (zwitterionic) nanoliposomes (NLPs), supplemented with cholesterol (NLP-Chol) and decorated with PEG (NLP-Chol-PEG), on αSN aggregation and neurotoxicity. Both NLPs retard αSN fibrillization in a concentration-independent fashion. They do so largely by increasing lag time (formation of fibrillization nuclei) rather than elongation (extension of existing nuclei). Interactions between neutral NLPs and αSN may locate to the N-terminus of the protein. This interaction can even perturb the interaction of αSN with negatively charged NLPs which induces an α-helical structure in αSN. This interaction was found to occur throughout the fibrillization process. Both NLP-Chol and NLP-Chol-PEG were shown to be biocompatible in vitro, and to reduce αSN neurotoxicity and reactive oxygen species (ROS) levels with no influence on intracellular calcium in neuronal cells, emphasizing a prospective role for NLPs in reducing αSN pathogenicity in vivo as well as utility as a vehicle for drug delivery.
Assuntos
Lipossomos/química , Nanopartículas/química , Neurônios/efeitos dos fármacos , Doença de Parkinson/terapia , alfa-Sinucleína/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animais , Cálcio/metabolismo , Colesterol/química , Humanos , Células PC12 , Polietilenoglicóis/química , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.