Solution Nuclear Magnetic Resonance Studies of Sterol Carrier Protein 2 Like 2 (SCP2L2) Reveal the Insecticide Specific Structural Characteristics of SCP2 Proteins in <i>Aedes aegypti</i> Mosquitoes

Sterol carrier protein 2 like 2 from <i>Aedes aegypti</i> (<i>Ae</i>SCP2L2) plays an important role in lipid transport in mosquitoes for its routine metabolic processes. Repeated unsuccessful attempts to crystallize ligand free SCP2L2 prompted us to undertake nuclear magnetic resonance (NMR) spectroscopy to determine its three-dimensional structure. We report here the three-dimensional structures and dynamics of apo-<i>Ae</i>SCP2L2 and its complex with palmitate. The ¹⁵N heteronuclear single-quantum coherence spectrum of apo-<i>Ae</i>SCP2L2 displayed multiple peaks for some of the amide resonances, implying the presence of multiple conformations in solution, which are transformed to a single conformation upon formation of the complex with plamitate. The three-dimensional structures of apo-<i>Ae</i>SCP2L2 and palmitated <i>Ae</i>SCP2L2 reveal an α/β mixed fold, with five β-strands and four α-helices, very similar to the other SCP2 protein structures. Unlike the crystal structure of palmitated <i>Ae</i>SCP2L2, both solution structures are monomeric. It is further confirmed by the rotational correlation times determined by NMR relaxation times (<i>T</i>₁ and <i>T</i>₂) of the amide protons. In addition, the palmitated <i>Ae</i>SCP2L2 structure contains two palmitate ligands, bound in the binding pocket, unlike the three palmitates bound in the dimeric form of <i>Ae</i>SCP2L2 in the crystals. The relaxation experiments revealed that complex formation significantly reduces the dynamics of the protein in solution

IPA analysis of fluvastatin mediated differentially regulated proteins.

<p>The most significant networks, pathways and molecular functions are summarized.</p><p>IPA analysis of fluvastatin mediated differentially regulated proteins.</p

Protein sub networks of fluvastatin mediated differentially regulated proteins.

<p>Protein-protein physical/functional interaction global complex network (A) and sub network (B) generated by Ingenuity Pathway Analysis tool. Grey filled boxes are the differentially expressed proteins. Only significant sub networks are represented.</p

Fluvastatin induces cell death in MDA-MB-231 breast cancer cells: Effect of mevalonate.

<p>A, MDA-MB-231 cells were treated with various concentrations of fluvastatin (0–20 µM) for 48 h and B shows cells treated for different time points with 10 µM fluvastatin. At the end of the treatments, cell viability was measured by trypan blue exclusion method. C, MCF-10A cells were treated with fluvastatin (0–20 µM) for a period of 24 and 48 h and cell death was measured as described earlier. D, MDA-MB-231 cells were treated with fluvatstatin (10 µM) in presence or absence of mevalonate (25 µM) for a period of 48 h and cell viability was measured by trypan blue exclusion assay. Data represented is Mean±SD from at least three independent experiments. *, significantly different compared to untreated conditions; #, significantly different compared to mevalonate and fluva+mev condition. Statistical significance was tested at P<0.05 level by two-tailed, unpaired, Student's t-test.</p

Fluvastatin causes controlled proteolysis of vimentin in breast cancer cells.

<p>A, shows the transcript levels of vimentin in presence and absence of fluvastatin (10 µM) for 24 h in MDA-MB-231 cells. B, shows the identified peptide sequence coverage of two different MW's of truncated vimentin protein spots appeared in fluvastatin (10 µM) treated MDA-MB-231 cells for 24 h. C, MDA-MB-231 cells were treated with various concentrations of statin (0–20 µM) for a period of 24 h and at the end of the treatments, vimentin protein levels were measured by Western blot analysis. D is same as C except that cells were treated with fluvastatin (10 µM) for various time points (0–24 h) and vimentin protein levels were measured by Western analysis. E, BT-549 cells were treated with fluvastatin (20 µM) for 24 h and vimentin levels were measured by Western blot analysis. F, MCF-10A cells were either untreated or treated with fluvastatin (10 µM) for a period of 24 h and vimentin protein levels were measured by Western analysis. G, MDA-MB-231 cells were treated in presence or absence of mevalonate (25 µM) for a period of 24 h and vimentin protein levels were measured by Western analysis. Data represented are Mean±SD from at least three independent experiments. *, significantly different compared to untreated conditions; #, significantly different compared to mevalonate and fluva+mev condition. Statistical significance was tested at P<0.05 level by two-tailed, unpaired, Student's t-test.</p

Caspase-3 inhibitor but not proteasomal inhibitor alters fluvastatin-mediated regulation of vimentin levels in MDA-MB-231 cells.

<p>A, MDA-MB-231 cells were treated with various concentrations of fluvastatin (0–20 µM) for a period of 24 h and at the end of the treatments trypsin-like and chymotrypsin-like activities of the 26S proteasome were measured using respective fluorogenic substrates as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108890#s2" target="_blank">materials and methods</a> section. B, is same as A except that cells were treated with fluvastatin (10 µM) for various time points (0–24 h). C, MDA-MB-231 cells were treated in the presence or absence of MG-132 (1 µM) for a period of 24 h and at the end of the treatments, vimentin and β-catenin protein levels were measured by Western blot analysis. D, MDA-MB-231 cells were pre-treated with caspase-3 inhibitor (50 µM) for a period of 6 h in the presence or absence of fluvastatin (10 µM) for a period of 24 h and vimentin protein levels were measured by Western analysis. Data represented are Mean±SD from at least three independent experiments. *, significantly different compared to untreated conditions; #, significantly different compared to MG-132 and fluva+MG-132 condition; **, significantly different compared to fluvastatin alone treated condition. Statistical significance was tested at P<0.05 level by two-tailed, unpaired, Student's t-test.</p

Enlarged view of fluvastatin mediated differentially regulated proteins of the differentially regulated proteins in MDA-MB-231 cells.

<p>A and B shows enlarged view of statin mediated up regulated (A) and down regulated (B) spots respectively. The results presented in C–F are from 3 independent experiments. C and D represent the correlation coefficient of untreated MDA-MB-231 cell lysate. C represents the comparison between gel 1 versus gel 2 and D represents gel 1 versus gel 3. E and F represent the correlation coefficient of statin treated MDA-MB-231 cell lysate. E represents the comparison between gel 1 versus gel 2 and F represents gel 1 versus gel 3.</p

Identification of proteins from differentially regulated protein spots by mass spectrometry using LTQ-FTICR.

<p>Database IPI human v3.12; MS/MS ion search, peptide mass tolerance: ±10 ppm, fragment mass tolerance: ±0.8 Da, enzyme: trypsin, variable modifications: carbamidomethyl (C), oxidation (M), max missed cleavages: 2; Appeared upon treatment (AT).</p><p>Identification of proteins from differentially regulated protein spots by mass spectrometry using LTQ-FTICR.</p

Proteome map of fluvastatin regulated proteins in MDA-MB-231 breast cancer cells.

<p>MDA-MB-231 cells were either untreated or treated with fluvastatin (10 µM) for a period of 24 h and then both untreated and treated cell lysates were subjected to iso-electric focusing and second dimension was resolved with 12% SDS-PAGE. Differentially regulated protein spots were indicated and marked. Gel shown is a representative of three independent experiments.</p

Complete analysis of data from screening for antitubercular activity and physico chemical properties of the hit compounds.

<p>Rifampicin and Isoniazid were taken as controls. MIC values for controls against <i>M</i>. <i>smegmatis</i> are Rif-2.43±0.02μM and Inh-11.03±0.05 μM and <i>H37Rv</i> are Rif-0.08±0.01μM and Inh-0.22±0.3 μM.</p