In silico elucidation of novel anticancer lead molecules targeting human prostate specific gene-1 protein

In silico elucidation of novel anticancer lead molecules targeting human prostate specific gene-1 protei

Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant <i>Leishmania donovani</i>

<div><p>Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS) of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in <i>Leishmania donovani</i>. In this study, TryS gene of <i>L. donovani</i> (LdTryS) was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0–8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of <i>L. major</i> are conserved in <i>L. donovani</i>. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼2.0-folds) than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H₂O₂ treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H₂O₂. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in <i>L. donovani</i> and involvement of TryS during oxidative stress to help the parasites survival.</p></div

Effect of H₂O₂ on growth inhibition of <i>L. donovani</i> parasites.

<p>(A) <i>L. donovani</i> promastigotes (1×10⁶ cells/ml) culture was treated with increasing concentration of H₂O₂ (0–200 µM) up to 15 hrs and growth inhibitory effect of H₂O₂ determined by MTT assay at 3 hr intervals. The cell viability after exposure with increasing concentration of H₂O₂ was determined to optimize time of exposure and dose. (B) The intracellular ROS level was determined by quantification of DCF fluorescence. Results were normalized with cell numbers and presented relative to untreated control cells. (C) To confirm intracellular ROS production a quenching study was performed. The parasites treated with H₂O₂ in the presence of 20 µM <i>N</i>-acetyl-L-cysteine (NAC) ROS scavenger reversed the effect of H₂O_2. The experiments were repeated three times and graphs represent the mean ± SD.</p

Subcellular localization of LdTryS.

<p>(A) Differential digitonin permeabilization of stationary phase promastigotes with increasing concentrations of digitonin. Supernatant and pellet fractions were run on 10% SDS-PAGE and transferred on to nitrocellulose membrane for western blot analysis using anti-LdTryS (1∶3000), anti LdcTXN (1∶4000), and anti LdIscS (1∶2000). cTXN and IscS served as cytosolic and mitochondrial markers, respectively. (B) Immunofluorescence microscopy of <i>L. donovani</i> promastigote with anti-LdTryS sera: phase contrast image, DAPI stained nucleus (N) and kinetoplast (K), Mitotracker stained mitochondria, anti-TryS labeled promastigote along with its merged image with DAPI is showing TryS localization in the cytoplasm.</p

Expression and purification of recombinant LdTryS protein.

<p>(A): LdTryS was expressed as a fusion protein containing the amino-terminal histidine tag and purified with the Ni²⁺-NTA column as described in “Materials and Methods”. The total cell lysate and samples at each purification step were electrophoresed on 10% SDS-PAGE gel and stained with coomassie brilliant blue. Lane M, protein marker; lane 1, an <i>E. coli</i> transformant with pET-15b empty vector as control; lane 2, total lysate of cells expressing LdTryS gene; lane 3, supernatant of lane 2 after centrifugation at 21,000 rpm; lane 4, unbound fraction of the Ni²⁺-NTA column; lane 5, 6 wash from the Ni²⁺-NTA column with 10, 50 mM imidazole; lane 7, 8 and 9 elutes from the Ni²⁺-NTA column with 100 mM, 200 mM and 300 mM imidazole, respectively. (B): Coomassie staining of undigested and thrombin digested rLdTryS protein. Lane 10, undigested rLdTryS; lane 11, thrombin digested rLdTryS. (C): Western blot with anti-LdTryS sera. Lane 12, represents 10 ng of rLdTryS protein and lane 13, represent 20 µg total cell lysates of <i>L. donovani</i>, lane 14 and 15, represent 20 µg supernatant and pellet fractions, respectively.</p

Enzymatic analysis of recombinant purified LdTryS.

<p>(A) pH profile using a coupled assay in a mixed buffer system. Activity is expressed as a percentage relative to the maximum activity observed with LdTryS. Kinetic properties of LdTryS with substrates GSH (B) and Spd (C) were analysed. <i>K</i>_m values were determined for each substrate by fitting data into Michaelis Menten equation and resulting Lineweaver-Burk plots. The experiments were performed three times in duplicate and data presents the mean ± SD.</p

Up regulation of LdTryS in <i>L. donovani</i> promastigotes in response to H₂O₂ treatment.

<p>(A) TryS expression in <i>L. donovani</i> parasites in the presence of H₂O₂ (10–200 µM) was analysed by western blot. H₂O₂ treated parasites showed increased expression level of LdTryS, whereas the β-actin expression level did not change significantly. The experiments were repeated twice in duplicates and quantitation was done by densitometric analysis using Quantity One (Bio-Rad). Band intensity is presented as fold increase/decrease of LdTryS expression. (B) LdTryS expression level was analyzed by semiquantitative RT-PCR, and PCR product stained with ethidium bromide and photographed. PCR of α-tubulin was used as housekeeping control that showed uniform expression pattern irrespective of H₂O₂ concentration.</p

Multiple sequence alignments of deduced amino acid sequences of TryS from <i>L. donovani</i> and other organisms.

<p>Protein sequences were aligned using the CLUSTAL W program (<a href="http://www.ebi.ac.uk/clustalw/" target="_blank">www.ebi.ac.uk/clustalw/</a>). Sequences are <i>L. donovani</i> (CAD23679), <i>L. infantum</i> (XP_001466426), <i>L. major</i> (XP_003721994), <i>L. amazonensis</i> (ABQ57409), <i>L. braziliensis</i> (XP_001565955), <i>C. fasciculata</i> (AAT99012), <i>T. brucei</i> (CAC87573), and <i>T. cruzi</i> (XP_816076). <i>Asterisks</i> indicate identical amino acids. <i>Dots</i> and <i>colons</i> indicate conserved amino acid substitutions. <i>Dashes</i> indicate gaps. <i>Closed boxes</i> at the amino terminus indicate conserved amino acids involved in amidase activity in all <i>Leishmania</i> species except <i>L. braziliensis</i>. <i>Closed dashed boxes</i> at the central region (on the 6^th, 7^th, and 8^th rows) interact with GSH. <i>Gray boxes</i> at the central region and C-terminal region indicate amino acids involved in synthetase activity and <i>gray closed</i> boxes show a. a. involved in binding triphosphate moiety of ATP. <i>Dashed lines</i> above the alignment indicate the linker regions between amidase and synthetase domain at N- and C-terminal region of the protein. A <i>Solid line</i> above the alignment indicates unique insertion in TryS of eukaryotes which is absent in prokaryotes TryS.</p

Determination of expression level of LdTryS in sensitive (S) vs. drug resistant (R) strains of <i>L. donovani.</i>

<p>(A) Semiquantitative RT-PCR analysis of LdTryS transcript in Amp B sensitive vs. resistant isolates. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both Amp B sensitive and resistant promastigotes. (B) Bar graph represents quantitative real time PCR analysis of LdTryS expression level in Amp B sensitive vs. resistant isolates. Data are normalized by the target/reference ratio of the calibrator. (C) The total <i>Leishmania</i> lysates (30 µg) were electrophoresed on 10% SDS-PAGE gel and stained with coomassie brilliant blue. Lane 1 represents, protein marker; lane 2 represents, sensitive strain (S); lanes 3, and 4 represent, resistant isolates (R1, & R2). (D) Shows western blot of same coomassie gel using anti-LdTryS (1∶3000). The image was analyzed by densitometrically. Data was normalized and β-actin was used as control. The experiments were repeated twice and graphs represent the mean ± SD.</p

Nbp35-Cfd1 complex structure showing the regions involved in interaction.

<p>(A) Complex structure of Nbp35-Cfd1 where the molecular contacts are highlighted (blue–Nbp35) and (green-Cfd1) with mentioned residues involved in interaction. (B) Porcupine plots for Nbp35-Cfd1 complex showing backbone fluctuation from simulation time course. The regions involved in contact are highlighted with dotted region.</p