Asim Poddar

Structure and Activity of Lysozyme on Binding to ZnO Nanoparticles

The interaction between ZnO nanoparticles (NPs) and lysozyme has been studied using calorimetric as well as spectrophotometric techniques, and interpreted in terms of the three-dimensional structure. The circular dichroism spectroscopic data show an increase in alpha-helical content on interaction with ZnO NPs. Glutaraldehyde cross-linking studies indicate that the monomeric form occurs to a greater extent than the dimer when lysozyme is conjugated with ZnO NPs. The enthalpy-driven binding between lysozyme and ZnO possibly involves the region encompassing the active site in the molecule, which is also the site for the dimer formation in a homologous structure. The enzyme retains high fraction of its native structure with negligible effect on its activity upon attachment to NPs. Compared to the free protein, lysozyme-ZnO conjugates are more stable in the presence of chaotropic agents (guanidine hydrochloride and urea) and also at elevated temperatures. The possible site of binding of NP to lysozyme has been proposed to explain these observations. The stability and the retention of a higher level of activity in the presence of the denaturing agent of the NP-conjugated protein may find useful applications in biotechnology ranging from diagnostic to drug delivery.

Curcumin recognizes a unique binding site of tubulin.

Although curcumin is known for its anticarcinogenic properties, the exact mechanism of its action or the identity of the target receptor is not completely understood. Studies on a series of curcumin analogues, synthesized to investigate their tubulin binding affinities and tubulin self-assembly inhibition, showed that: (i) curcumin acts as a bifunctional ligand, (ii) analogues with substitution at the diketone and acetylation of the terminal phenolic groups of curcumin are less effective, (iii) a benzylidiene derivative, compound 7, is more effective than curcumin in inhibiting tubulin self-assembly. Cell-based studies also showed compound 7 to be more effective than curcumin. Using fluorescence spectroscopy we show that curcumin binds tubulin 32 Å away from the colchicine-binding site. Docking studies also suggests that the curcumin-binding site to be close to the vinblastine-binding site. Structure-activity studies suggest that the tridented nature of compound 7 is responsible for its higher affinity for tubulin compared to curcumin.

Stable and Potent Analogues Derived from the Modification of the Dicarbonyl Moiety of Curcumin

Curcumin has shown promising therapeutic utilities for many diseases, including cancer; however, its clinical application is severely limited because of its poor stability under physiological conditions. Here we find that curcumin also loses its activity instantaneously in a reducing environment. Curcumin can exist in solution as a tautomeric mixture of keto and enol forms, and the enol form was found to be responsible for the rapid degradation of the compound. To increase the stability of curcumin, several analogues were synthesized in which the diketone moiety of curcumin was replaced by isoxazole (compound 2) and pyrazole (compound 3) groups. Isoxazole and pyrazole curcumins were found to be extremely stable at physiological pH, in addition to reducing atmosphere, and they can kill cancer cells under serum-depleted condition. Using molecular modeling, we found that both compounds 2 and 3 could dock to the same site of tubulin as the parent molecule, curcumin. Interestingly, compounds 2 and 3 also show better free radical scavenging activity than curcumin. Altogether, these results strongly suggest that compounds 2 and 3 could be good replacements for curcumin in future drug development.

Binding of Indanocine to the Colchicine Site on Tubulin Promotes Fluorescence, and Its Binding Parameters Resemble Those of the Colchicine Analogue AC

Indanocine, a synthetic indanone, has shown potential antiproliferative activity against several tumor types. It is different from many other microtubule-disrupting drugs, because it displays toxicity toward multidrug resistance cells. We have examined the interaction of indanocine with tubulin and determined their binding and thermodynamic parameters using isothermal titration calorimetry (ITC). Indanocine is weakly fluorescent in aqueous solution, and the binding to tubulin enhances fluorescence with a large blue shift in the emission maxima. Indanocine binds to the colchicine site of tubulin, although it bears no structural similarity with colchicine. Nevertheless, like colchicine analogue AC, indanocine is a flexible molecule in which two halves of the molecule are connected through a single bond. Also, like AC, indanocine binds to the colchicine binding site of tubulin in a reversible manner and the association reaction occurs at a faster rate compared to that of colchicine-tubulin binding. The binding kinetics was studied using stopped-flow fluorescence. The association process follows biphasic kinetics similar to that of the colchicine-tubulin interaction. The activation energy of the reaction was 10.5 +/- 0.81 kcal/mol. Further investigation using ITC revealed that the enthalpy of association of indanocine with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -175.1 cal mol(-1) K(-1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen bonding forces. Finally, we conclude that even though indanocine possesses no structural similarity with colchicine, it recognizes the colchicine binding site of tubulin and its binding properties resemble those of the colchicine analogue AC.

Role of the carboxy‐termini of tubulin on its chaperone‐like activity

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of α‐crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone‐like activity as demonstrated by prevention of DTT‐induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, βγ‐crystallin, and other proteins. We have shown that the tubulin lost its chaperone‐like activity upon digestion of its negatively charged C‐termini. In this article, the role of the C‐terminus of individual subunits has been investigated. We observe that the digestion of C‐terminus of β‐subunit with subtilisin causes loss of chaperone‐like activity of tubulin. The contribution of C‐terminus of α‐subunit is difficult to establish directly as subtilisin cleaves C‐terminus of β‐subunit first. This has been ascertained indirectly using a 14‐residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C‐terminus of α‐subunit. We have shown that the binding of P2 peptide to αβ‐tubulin causes complete loss of its chaperone‐like activity. NMR and gel‐electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C‐terminus of α‐subunit compared to that of β‐subunit. Thus, we conclude that both the C‐termini are necessary for the chaperone‐like activity of tubulin. Implications for the chaperone functions in vivo have been discussed. Proteins 2001;44:262–269.

BisANS binding to tubulin: Isothermal titration calorimetry and the site‐specific proteolysis reveal the GTP‐induced structural stability of tubulin

Interactions of bisANS and ANS to tubulin in the presence and absence of GTP were investigated, and the binding and thermodynamic parameters were determined using isothermal titration calorimetry. Like bisANS binding to tubulin, we observed a large number of lower affinity ANS binding sites (N1 = 1.3, K1 = 3.7 × 105 M−1, N2 = 10.5, K2 = 7 × 104/M−1) in addition to 1–2 higher affinity sites. Although the presence of GTP lowers the bisANS binding to both higher and lower affinity sites (N1 = 4.3, N2 = 11.7 in absence and N1 = 1.8, N2 = 3.6 in presence of GTP), the stoichiometries of both higher and lower affinity sites of ANS remain unaffected in the presence of GTP. BisANS‐induced structural changes on tubulin were studied using site‐specific proteolysis with trypsin and chymotrypsin. Digestion of both α and β tubulin with trypsin and chymotrypsin, respectively, has been found to be very specific in presence of GTP. GTP has dramatic effects on lowering the extent of nonspecific digestion of β tubulin with trypsin and stabilizing the intermediate bands produced from both α and β. BisANS‐treated tubulin is more susceptible to both trypsin and chymotrypsin digestion. At higher bisANS concentration (>20 μM) both α and β tubulins are almost totally digested with enzymes, indicating bisANS‐induced unfolding or destabilization of tubulin structure. Again, the addition of GTP has remarkable effect on lowering the bisANS‐induced enhanced digestion of tubulin as well as stabilizing effect on intermediate bands. These results of isothermal titration calorimetry, proteolysis and the DTNB‐kinetics data clearly established that the addition of GTP makes tubulin compact and rigid and hence the GTP‐induced stabilization of tubulin structure. No such destabilization of tubulin structure has been noticed with ANS, although, like bisANS, ANS possesses a large number of lower affinity binding sites. On the basis of these results, we propose that the unique structure of bisANS, which in absence of GTP can bind tubulin as a bifunctional ligand (through its two ANS moieties), is responsible for the structural changes of tubulin. Proteins 2003;50:283–289.

Chaperone‐mediated inhibition of tubulin self‐assembly

Molecular chaperones are known to play an important role in facilitating the proper folding of many newly synthesized proteins. Here, we have shown that chaperone proteins exhibit another unique property to inhibit tubulin self‐assembly efficiently. Chaperones tested include α‐crystallin from bovine eye lenses, HSP16.3, HSP70 from Mycobacterium tuberculosis and α s‐casein from milk. All of them inhibit polymerization in a dose‐dependent manner independent of assembly inducers used. The critical concentration of MTP polymerization increases with increasing concentration of HSP16.3. Increase in chaperone concentration lowers the extent of polymerization and increases the lag time of self‐assembly reaction. Although the addition of a chaperone at the early stage of elongation phase shows no effect on polymerization, the same concentration of chaperone inhibits polymerization completely when added before the initiation of polymerization. Bindings of HSP16.3 and α s‐casein to tubulin have been confirmed using isothermal titration calorimetry. Affinity constants of tubulin are 5.3 ×× 104 and 9.8 ×× 105 M−1 for HSP16.3 and α s‐casein, respectively. Thermodynamic parameters indicate favourable entropy and enthalpy changes for both chaperones‐tubulin interactions. Positive entropy change suggests that the interaction is hydrophobic in nature and desolvation occurring during formation of tubulin‐chaperone complex. On the basis of thermodynamic data and observations made upon addition of chaperone at early elongation phase or before the initiation of polymerization, we hypothesize that chaperones bind tubulin at the protein‐protein interaction site involved in the nucleation phase of self‐assembly. Proteins 2007.

The B‐ring substituent at C‐7 of colchicine and the α‐C‐terminus of tubulin communicate through the “tail–body” interaction

The carboxy terminals of αβ‐tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B‐ring analogs (with C‐7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C‐7 substituents is pH independent and has lower activation energy. β‐C‐terminus‐truncated tubulin (αβs) shows similar pH sensitivity and activation energy to native tubulin (αβ). Removal of the C‐termini of both subunits of tubulin (αsβs) or the binding of a basic peptide P2 to the negatively charged α‐C‐terminus of tubulin causes a colchicine–tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C‐terminal α‐tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the α‐C‐terminus with the main body of tubulin indirectly affects the colchicine–tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail–body interaction, a B‐ring substituent makes contact with the α‐tubulin and induces significant conformational changes in α‐tubulin. Proteins 2004.

MAP2c prevents arachidonic acid-induced fibril formation of tau: Role of chaperone activity and phosphorylation

Tau has long been associated with Alzheimers disease, where it forms neurofibrillary tangles. Here we show for the first time by electron microscopy that MAP2c prevents arachidonic acid-induced in vitro aggregation of tau. However, phosphorylated MAP2c failed to prevent the same. Previously we reported that MAP2c possesses chaperone-like activity while tau does not (Sarkar et al., 2004, Eur J Biochem., 271(8), 1488-96). Here we demonstrate that phosphorylation severely impaired the chaperone activity of MAP2c, implying a crucial role of chaperone in preventing tau fibrillation. Additionally, the ability of MAP2c to induce microtubule polymerization was abolished completely upon phosphorylation. As tau and MAP2c possess highly homologous C-termini, we speculated that the N-terminus of MAP2c might account for its chaperone activity. Nevertheless, experiments showed that N-terminus of MAP2c alone is inactive as a chaperone. Our preliminary findings suggest that MAP2c/MAP2 could be one of the regulators maintaining tau homeostasis in the cell.

Cytotoxic biphenyl-4-carboxylic acid targets the tubulin–microtubule system and inhibits cellular migration in HeLa cells

Abstract Two structurally similar biphenyl compounds, biphenyl-2-carboxylic acid (B2C) and biphenyl-4-carboxylic acid (B4C), were selected to assess their cellular cytotoxic and antimitotic behaviour in the quest of a potent anticancer compound. The HeLa and MCF-7 cell lines were used to determine the cytotoxic effect of the two biphenyl compounds using the MTT assay. Confocal microscopy was performed to analyze the degree of nuclear condensation and fragmentation associated with depolymerized microtubules that result from inhibiting in vitro tubulin polymerization. Circular dichroism spectroscopy along with DTNB kinetics were conducted to predict alterations in the tubulin secondary structure and global conformational changes in the tertiary structure of the protein, respectively. Finally, a wound healing assay was employed to assess whether cellular migration was inhibited in the treated HeLa cells. B4C imparted more cellular cytotoxicity in the HeLa and MCF-7 cell lines (IC50 ∼ 4 μM). B4C inhibited in vitro tubulin polymerization into microtubules with an IC50 of 50 μM. Confocal microscopy of treated cell indicated presumptive apoptosis, exhibiting fragmented nuclei with significant microtubular disruption, which was confirmed by Western blot analysis. Circular dichroism revealed a significant reduction in the α-helix content of treated tubulin. DTNB kinetics showed that approximately six –SH groups were buried in the structure. The wound healing assay revealed that B4C prevented cellular invasion. B4C had greater cytotoxic and antimitotic effects against HeLa cells than B2C. To delineate the specific mechanism of action of B4C and its derivatives, further research is warranted.