ABSTRACT
Saccharomyces cerevisiae Ypr147cp was found localized to lipid droplets but the physiological role of Ypr147cp remains unknown. Sequence analysis of Ypr147cp revealed an α/ß hydrolase domain along with the conserved GXSXG lipase motif. Recombinant Ypr147cp showed both triacylglycerol lipase and ester hydrolase activities. Knock out of YPR147C led to accumulation of TAG in ypr147cΔ when compared to wild type (WT). In addition, transmission electron microscopic analysis of ypr147cΔ cells revealed a greater number of lipid bodies, justifying the increase in TAG content, and the phenotype was rescued upon overexpression of YPR147C in ypr147cΔ. Moreover, the lipid profiling confirmed the accumulation of fatty acids derived from neutral and phospholipids in ypr147cΔ cells. Based on these results, Ypr147cp is identified as a lipid droplet associated triacylglycerol lipase along with an ester hydrolyzing capacity.
Subject(s)
Esterases/metabolism , Lipase/metabolism , Lipid Metabolism/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Databases, Factual , Esterases/genetics , Lipase/genetics , Lipid Droplets/enzymology , Lipid Droplets/ultrastructure , Phylogeny , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Triglycerides/metabolismABSTRACT
In this study, we have developed an electrochemical immunosensor for the detection of prostate-specific antigen (PSA) based on gold nanoparticles (AuNPs) and a chitosan (CHI) nanocomposite film coated on a screen printed electrode (SPE). The modified SPE was characterized using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immunosensor was fabricated by sequential immobilization of anti-PSA primary antibody, PSA antigen and HRP-tagged secondary antibody on the surface of AuNPs/CHI/SPE. When this immunosensor was placed in 2.5mM H2O2, as HRP substrate, and 1mM methylene blue (MB), as redox mediator, greatly amplified immunosensing signals were observed. Immunosensing signals for the reduction of H2O2 were monitored in phosphate buffer (pH 7.0) by CV and square wave voltammetry (SWV). Under optimized conditions steady state current increased linearly with PSA concentration over the range 1-18ng/ml with a detection limit of 0.001ng/ml. The fabricated immunosensor demonstrated excellent sensitivity, stability and reproducibility and therefore was successfully applied to analyse PSA in biological samples. Finally, the results indicate that the proposed immunosensor has potential in clinical screening of cancer biomarkers.
Subject(s)
Biosensing Techniques/instrumentation , Prostate-Specific Antigen/analysis , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/immunology , Chitosan/chemistry , Electrochemical Techniques/instrumentation , Equipment Design , Gold/chemistry , Humans , Male , Metal Nanoparticles/chemistry , Nanocomposites/chemistryABSTRACT
Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G(205)XS(207)XG(209) and H(120)XXXXD(125) Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser(207) of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser(207) and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase.
