Identification of zebrafish fumarate hydratase active site by molecular docking and simulation studies.

Fumarate hydratase (FH), one of the members of TCA cycle, acts as a catalyte for the synthesis of malate from fumarate. FH has been proposed to play as a tumour suppressor leading to the pathogenicity of leiomyomas, renal cell carcinoma and paraganglioma. Mutations in the active site of FH lead to alteration in the protein structure. Similarly, binding of several chemical inhibitors to the active site also leads to the disruption of protein structural integrity thereby leading to protein dysfunction. Therefore, in order to address this mechanism leading to cancer, the binding efficiency of potential human FH inhibitor citrate to zebrafish fh has been extensively analysed in this study by molecular docking and simulation experiments followed by quantification of fumarate hydratase enzyme activity to validate and confirm the findings. Molecular docking revealed stronger interaction of zebrafish fh protein with inhibitor citrate when compared to natural substrate fumarate. Study on the dynamics of docked structures further confirmed that citrate was found to possess more binding affinity than fumarate. In vitro biochemical analysis also revealed concentration dependent potential inhibitory effect of citrate on zebrafish fh, thus confirming the findings of the in-silico experiments.Communicated by Ramaswamy H. Sarma.

Allura red rapidly induces amyloid-like fibril formation in hen egg white lysozyme at physiological pH.

Allura red (AR) is an artificial azo dye mostly used in food industries and has potential health risks. We examined the role of AR in amyloidogenesis using hen egg white lysozyme (HEWL) at pH 7.0. The amyloidogenic induction properties of AR in HEWL were identified by circular dichroism (CD), turbidity, intrinsic fluorescence, light scattering, transmission electron microscopy (TEM), and molecular dynamic simulation studies. Turbidity and light scattering measurements showed that HEWL becomes aggregated in the presence of 0.03-15.0 mM of AR at pH 7.0 but not at very low AR concentrations (0.01-0.28 mM). However, AR-induced aggregation is a kinetically rapid process, with no observable lag phase and saturation within 6 s. The kinetics results suggested that the HEWL aggregation induced by AR is very rapid. The CD results demonstrated that the total ß-sheet content of HEWL was increased in the AR treated samples. The TEM results are established that AR-induced aggregates had amyloid-like structures. Molecular dynamics simulations analysis showed that the bound AR-HEWL structures were highly favored compared to unbound structures. The mechanism of AR-induced amyloid fibril formation may involve electrostatic, hydrogen bonding, and hydrophobic interactions.

Prediction of promiscuous epitopes in the e6 protein of three high risk human papilloma viruses: a computational approach.

A najor current challenge and constraint in cervical cancer research is the development of vaccines against human papilloma virus (HPV) epitopes. Although many studies are done on epitope identification on HPVs, no computational work has been carried out for high risk forms which are considered to cause cervical cancer. Of all the high risk HPVs, HPV 16, HPV 18 and HPV 45 are responsible for 94% of cervical cancers in women worldwide. In this work, we computationally predicted the promiscuous epitopes among the E6 proteins of high risk HPVs. We identified the conserved residues, HLA class I, HLA class II and B-cell epitopes along with their corresponding secondary structure conformations. We used extremely precise bioinformatics tools like ClustalW2, MAPPP, NetMHC, EpiJen, EpiTop 1.0, ABCpred, BCpred and PSIPred for achieving this task. Our study identified specific regions 'FAFR(K)DL' followed by 'KLPD(Q)LCTEL' fragments which proved to be promiscuous epitopes present in both human leukocyte antigen (HLA) class I, class II molecules and B cells as well. These fragments also follow every suitable character to be considered as promiscuous epitopes with supporting evidences of previously reported experimental results. Thus, we conclude that these regions should be considered as the important for design of specific therapeutic vaccines for cervical cancer.