Quantitative aspects of endocytic activity in lipid-mediated transfections

AbstractVariation in transfection efficiency observed in different cell-types is poorly understood. To investigate the influence of endocytic activity on lipid-mediated transfections, we have monitored both the processes in 12 different cell-types. The endocytic activity shows a strong positive correlation (P<0.01), with transfection efficiency. Treatment with wortmannin resulted in cell-type-dependent inhibition of transfection. Studies on M-phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell-types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid-mediated transgene expression

Selective enrichment of omega-3 fatty acids in oils by phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release &omega;-3 fatty acids from oils for preparing enriched &omega;-3 fatty acid supplements. However, use of lipases in enrichment of &omega;-3 fatty acids is limited due to their insufficient specificity for &omega;-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of &omega;-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of &omega;-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained &omega;-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against &omega;-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof &omega;-3 fatty acids

Role of Active Site Rigidity in Activity: MD Simulation and Fluorescence Study on a Lipase Mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site

Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity.

Enrichment of omega-3 fatty acids (ɷ-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ɷ-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the ɷ-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity

A computational search for lipases that can preferentially hydrolyze long-chain omega-3 fatty acids from fish oil triacylglycerols

Consumption of long-chain omega-3 fatty acids is known to decrease the risk of major cardiovascular events. Lipases, a class of triacylglycerol hydrolases, have been extensively tested to concentrate omega-3 fatty acids from fish oils, under mild enzymatic conditions. However, no lipases with preference for omega-3 fatty acids selectivity have yet been discovered or developed. In this study we performed an exhaustive computational study of substrate-lipase interactions by docking, both covalent and non-covalent, for 38 lipases with a large number of structured triacylglycerols containing omega-3 fatty acids. We identified some lipases that have potential to preferentially hydrolyze omega-3 fatty acids from structured triacylglycerols. However omega-3 fatty acid preferences were found to be modest. Our study provides an explanation for absence of reports of lipases with omega-3 fatty acid hydrolyzing ability and suggests methods for developing these selective lipases

In vitro evolved non-aggregating and thermostable lipase: structural and thermodynamic investigation

Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named “6B” using multiple rounds of in vitro evolution. T_m and optimum activity temperature of 6B is 78 °C and 65 °C, respectively, which is ∼ 22 °C and 30 °C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance

Novel non-glycerol-based cytofectins with lactic acid-derived head groups

We report herein the design, synthesis, and transfection biology of a novel series of non-glycerol-based cationic lipids with lactic acid-derived head groups The synthetic procedure adopted herein for preparing 1-hydroxy-prop-2-yl head-group-based monocationic transfection lipids 1-7 is fairly straightforward and potentially applicable in designing other cationic lipids with lactic acid-derived head groups. A striking anchor-length dependency was observed in NIH3T3 cells in the sense that except lipid 4, all the other lipids were essentially transfection-inefficient. Ethidium bromide assay for the lipid:DNA interactions is consistent with the general observation that significant lipid:DNA interactions do not guarantee on improved transfection efficiency cationic lipid mediated gene delivery. Given its remarkable transfection properties and low cellular toxicity, lipid 4 is likely to find future use in the area of liposomal gene delivery

Active site dynamics of wild type and 6B lipase.

<p>(A) RMSD of C_α atoms of wild type and 6B lipases from their energy minimized crystal structures as a function of MD simulation time. For clarity, single simulation data is shown for both wild type and 6B lipase while others are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s003" target="_blank">Fig. S2A</a>. (B) RMSF of C_α atoms of individual residues during 2–20 ns simulation time. Active site residues positions are shown as solid spheres. (C) Typical time-resolved fluorescence anisotropic decay profiles of acrylodan attached to C77 in wild type and 6B lipase background.</p

Active site in transition state bound and free form.

<p>Structural overlap of active site of the free wild type lipase and in complex with covalently attached transition state analog (chain A of PDB id: 1I6W and 1R4Z). Transition state analog is O-[(R)-1,2-O-isopropylidene-sn-glycerol]6-hexenyl phosphonate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188-Labeikovsky1" target="_blank">[34]</a>. Free enzyme is shown in green while complex is shown in elemental color. Side chains are shown as sticks while backbone as lines. Stereo figure is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035188#pone.0035188.s007" target="_blank">Fig. S6</a>.</p

Catalytic mechanism of ester hydrolysis by <i>B. subtilis</i> lipase.

<p>Catalytic mechanism of ester hydrolysis by <i>B. subtilis</i> lipase.</p