Aparna Laskar

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7.

BACKGROUND Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses. METHODS In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~30nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD50 doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM. RESULTS This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis. CONCLUSIONS A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells. GENERAL SIGNIFICANCE CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.

Modeling and structural analysis of PA clan serine proteases

BackgroundSerine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques.ResultsOur predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species.ConclusionsEncompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S1 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, this analysis is beneficial for future molecular modeling strategies and structural analysis of serine protease models.

Exploring the structure of opioid receptors with homology modeling based on single and multiple templates and subsequent docking: A comparative study

Opioid receptors are the principal targets for opioids, which have been used as analgesics for centuries. Opioid receptors belong to the rhodopsin family of G-protein coupled receptors (GPCRs). In the absence of crystal structures of opioid receptors, 3D homology models have been reported with bovine rhodopsin as a template, though the sequence homology is low. Recently, it has been reported that use of multiple templates results in a better model for a target having low sequence identity with a single template. With the objective of carrying out a comparative study on the structural quality of the 3D models based on single and multiple templates, the homology models for opioid receptors (mu, delta and kappa) were generated using bovine rhodopsin as single template and the recently deposited crystal structures of squid rhodopsin, turkey β-1 and human β-2 adrenoreceptors along with bovine rhodopsin as multiple templates. In this paper we report the results of comparison between the refined 3D models based on multiple sequence alignment (MSA) and models built with bovine rhodopsin as template, using validation programs PROCHECK, PROSA, Verify 3D, Molprobity and docking studies. The results indicate that homology models of mu and kappa with multiple templates are better than those built with only bovine rhodopsin as template, whereas, in many aspects, the homology model of delta opioid receptor with single template is better with respect to the model based on multiple templates. Three nonselective ligands were docked to both the models of mu, delta and kappa opioid receptors using GOLD 3.1. The results of docking complied well with the pharamacophore, reported for nonspecific opioid ligands. The comparison of docking results for models with multiple templates and those with single template have been discussed in detail. Three selective ligands for each receptor were also docked. As the crystallographic structures are not yet known, this comparison will help in choosing better homology models of opioid receptors for studying ligand receptor interactions to design new potent opioid antagonists.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.

Transient overexpression of Werner protein rescues starvation induced autophagy in Werner syndrome cells.

Reduced autophagy may be associated with normal and pathological aging. Here we report a link between autophagy and Werner protein (WRNp), mutated in Werner syndrome, the human premature aging Werner syndrome (WS). WRN mutant fibroblast AG11395 and AG05229 respond weakly to starvation induced autophagy compared to normal cells. While the fusion of phagosomes with lysosome is normal, WS cells contain fewer autophagy vacuoles. Cellular starvation autophagy in WS cells is restored after transfection with full length WRN. Further, siRNA mediated silencing of WRN in the normal fibroblast cell line WI-38 results in decreased autophagy and altered expression of autophagy related proteins. Thus, our observations suggest that WRN may have a role in controlling autophagy and hereby cellular maintenance.

Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.

Cyclophilin-mediated reactivation pathway of inactive adenosine kinase aggregates.

Monomeric adenosine kinase (AdK), a pivotal salvage enzyme of the purine auxotrophic parasite, Leishmania donovani, tends to aggregate naturally or selectively in presence of ADP, leading to inactivation. A cyclophilin (LdCyP) from the parasite reactivated the enzyme by disaggregating it. We studied the aggregation pathway of AdK with or without ADP. Transmission electron microscopy revealed that ADP-induced aggregates, as opposed to annular or torus-shaped natural aggregates, were mostly amorphous with protofibril-like structures. Interestingly, only the natural aggregates bound thioflavin T with a KD of 3.33 μM, indicating cross β-sheet structure. Dynamic light scattering experiments indicated that monomers formed aggregates either upon prolonged storage or ADP exposure. ADP-aggregates were disaggregated by LdCyP with concomitant reactivation of the enzyme. The activity revived with decrease in the aggregate size. Displacement of ADP from the ADP-aggregated enzyme by LdCyP resulted in reactivation. CD-spectral studies suggested that, like the natural aggregates, ADP induced formation of β-sheet structure in the ADP-aggregates. However, unlike the natural aggregate, it could be reconverted to α-helical conformation upon addition of LdCyP. Based on the results, a regulatory mechanism through interplay of ADP and/or LdCyP interaction with the enzyme is envisaged and a pathway of AdK reactivation by LdCyP-chaperone is proposed.

Three-Dimensional Molecular Modeling of a Diverse Range of SC Clan Serine Proteases

Serine proteases are involved in a variety of biological processes and are classified into clans sharing structural homology. Although various three-dimensional structures of SC clan proteases have been experimentally determined, they are mostly bacterial and animal proteases, with some from archaea, plants, and fungi, and as yet no structures have been determined for protozoa. To bridge this gap, we have used molecular modeling techniques to investigate the structural properties of different SC clan serine proteases from a diverse range of taxa. Either SWISS-MODEL was used for homology-based structure prediction or the LOOPP server was used for threading-based structure prediction. The predicted models were refined using Insight II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core, differences in overall amino acid distribution, and unique surface electrostatic potential patterns between species. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on SC clan serine proteases.

Formulation and antitumorigenic activities of nanoencapsulated nifetepimine: A promising approach in treating triple negative breast carcinoma

Triple negative breast cancer (TNBC) is one of the most common invasive malignancies among women, associated with poor prognosis. Standard chemotherapy targets all dividing cells, resulting in dose-limiting toxicities. In this study, we demonstrated a strategy of encapsulating a hydrophobic synthetic compound, nifetepimine, having anticancer properties, in poly (lactic-co-glycolic acid) nanoparticles to increase selectivity of drug to cancerous cells with minimum toxicity towards normal cells. Nanoencapsulated nifetepimine (30-100nm) having loading and encapsulation efficiency of 7.45% and 75% respectively, was successfully internalized inside TNBC cells upon sustained release resulting in apoptosis. An in vivo bio-distribution study indicated that nanonifetepimine selectively accumulated into breast tumor sites of mice, primarily due to prolonged blood circulation time and binding of nifetepimine to epidermal growth factor receptor that remains overexpressed in most of the TNBC tumors. Moreover, we observed significant reduction in breast tumor volume with improved survival implying high tumor targetability of nanonifetepimine.