Leila Hassani

Inhibition of amyloid fibrillation of hen egg-white lysozyme by the natural and synthetic curcuminoids

Amyloid fibrillogenesis of almost twenty different proteins followed by deposition of amyloid fibrils in organs results in several neurodegenerative disorders such as Alzheimers, Parkinsons, Huntingtons diseases and type II diabetes. Natural polyphenolic compounds are introduced as appropriate candidates for inhibition of amyloid formation. In the present study, the inhibitory activities of curcumin (CUR) as a natural polyphenolic compound and diacetylcurcumin (DAC) as a synthetic derivative of CUR on the amyloid fibrillation of hen egg white lysozyme (HEWL) as an appropriate model protein for in vitro fibrillation were investigated using thioflavin T (ThT) fluorescence, atomic force microscopy (AFM) and circular dichroism spectroscopy. Also, the binding interaction evaluations were carried out using fluorescence quenching, fluorescence resonance energy transfer (FRET), synchronous fluorescence, circular dichroism spectroscopy and molecular docking approaches. The binding interaction evaluations revealed that two hydrogen bonds between CUR and HEWL formed via one of the phenolic OH groups and the carbonyl of the β-diketone moiety of CUR with Arg-112 and Leu-56 of HEWL, whereas, two hydrogen bonds formed via two acetyl groups of DAC with Asn-39 and Trp-63 of HEWL. Both amyloid fibrillation and binding results indicated that interaction of DAC with HEWL is stronger than that of CUR, and amyloid fibrillation of HEWL was inhibited more effectively by DAC than CUR. It can be inferred from these results that the acetyl groups of DAC can play a similar role to the hydroxyl groups of CUR in the interaction with amino acid residues of protein and interrupting amyloid structure. The present results demonstrate that as one of the strategies for synthesis of novel amyloid inhibitors, chemical modification of the natural curcuminoids framework can be introduced.

Spectroscopic investigation on the interaction of copper porphyrazines and phthalocyanine with human telomeric G-quadruplex DNA.

The G-quadruplex DNA is a novel target for anticancer drug discovery and many scientific groups are investigating interaction of small molecules with G-quadruplex DNA to discover therapeutic agents for cancer. Here, interaction of a phthalocyanine (Cu(PcTs)) and two tetrapyridinoporphyrazines ([Cu(2,3-tmtppa)](4+) and [Cu(3,4-tmtppa)](4+)) with Na(+) and K(+) forms of human telomeric G-quadruplex DNA has been investigated by spectroscopic techniques. The results indicated that interaction of the cationic porphyrazines is remarkably stronger than the anionic phthalocyanine and they presumably bind to the G-quadruplex DNA through end-stacking. Fluorescent intercalator displacement assay implied the displacement ability of the complexes with thiazole orange. In addition, circular dichroism spectra of both quadruplex forms converge to the Na(+) isoform after binding to the porphyrazines. In conclusion, the porphyrazines as the complexes that bind to the G-quadruplex DNA, could be suitable candidates for further investigations about inhibition of telomerase enzyme.

Modification of Lysine Residues of Horseradish Peroxidase and Its Effect on Stability and Structure of the Enzyme

Biotechnology is consistently seeking improved enzyme stability. Enzymes have great properties, although their marginal stability limits their applications. Among the strategies for improving stability of the enzymes, chemical modification is a simple and effective technique. In the present study, chemical modification of horseradish peroxidase (HRP) was carried out with 2,3-dichloromaleic anhydride and 2,3-dimethylmaleic anhydride. HRP is an important heme-containing enzyme. It is widely applied in pharmacological, chemical, and medical industries. Here, thermal stability of HRP was investigated at different temperatures. In addition, the enzyme stability was evaluated in urea, DMSO, alkaline pH, and hydrogen peroxide solutions by spectroscopic techniques. Structural investigation indicated that the both anhydrides slightly decrease compactness of the enzyme structure. The results also indicated that 2,3-dichloromaleic anhydride increases thermal stability of the enzyme and its stability in urea and DMSO solutions, but 2,3-dimethylmaleic anhydride only stabilizes HRP in urea solution. Furthermore, the experiments implied that none of the modifiers are effective on the stability of HRP in extreme pH and oxidative condition. Catalytic efficiency and activation energy did not change remarkably following reaction of the enzyme with the both carboxylic anhydrides. Consequently, improvement in the stability of HRP depends on not only the type of modifier but also denaturing condition.

A spectroscopic investigation of the interaction between c-MYC DNA and tetrapyridinoporphyrazinatozinc(II)

The c-MYC gene plays an important role in the regulation of cell proliferation and growth and it is overexpressed in a wide variety of human cancers. Around 90% of c-MYC transcription is controlled by the nuclease-hypersensitive element III1 (NHE III1), whose 27-nt purine-rich strand has the ability to form a G-quadruplex structure under physiological conditions. Therefore, c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Here, the interaction of water-soluble tetrapyridinoporphyrazinatozinc(II) with 27-nt G-rich strand (G/c-MYC), its equimolar mixture with the complementary sequence (GC/c-MYC) and related C-rich oligonucleotide (C/c-MYC) is investigated. Circular dichroism (CD) measurements of the G-rich 27-mer oligonucleotide in 150 mM KCl, pH 7 demonstrate a spectral signature consistent with parallel G-quadruplex DNA. Furthermore, the CD spectrum of the GC rich oligonucleotide shows characteristics of both duplex and quadruplex structures. Absorption spectroscopy implies that the complex binding of G/c-MYC and GC/c-MYC is a two-step process; in the first step, a very small red shift and hypochromicity and in the second step, a large red shift and hyperchromicity are observed in the Q band. Emission spectra of zinc porphyrazine are quenched upon addition of three types of DNA. According to the results of spectroscopy, it can be concluded the dominant binding mode is probably, outside binding and end stacking.

Interaction of aurein 1.2 and its analogue with DPPC lipid bilayer

Antibacterial peptides have potential as novel therapeutic agents for bacterial infections. Aurein 1.2 is one of the smallest antibacterial peptides extracted from an anuran. LLAA is a more active analogue of aurein 1.2. Antibacterial peptides usually accomplish their function by interacting with bacterial membrane selectively. In this study, we tried to find the reasons for the stronger antibacterial activity of LLAA compared with aurein 1.2. For this purpose, the interaction of aurein 1.2 and LLAA with dipalmitoylphosphatidylcholine (DPPC) was investigated by molecular dynamics (MD) simulation. In addition, the structure of peptides and their antibacterial activity were investigated by circular dichroism (CD) and dilution test method, respectively. MD results showed that LLAA is more flexible compared with aurein 1.2. Furthermore, LLAA loses its structure more than aurein 1.2 in the DPPC bilayer. A higher amount of water molecules penetrate into bilayer in the presence of LLAA relative to aurein 1.2. According to the antibacterial result that indicated LLAA is remarkably more active than aurein 1.2, it can be concluded that flexibility of the peptide is a determining factor in antibacterial activity. Probably, flexibility of the peptides facilitates formation of effective pores in the lipid bilayer.

Effect of artemin on structural transition of β-lactoglobulin

Encysted embryos of Artemia are exceptionally resistant to severe environmental stress. This resistance is thought to depend in part on the existence of a protein termed artemin. There is only little information about the function of artemin. It has been reported artemin is a thermostable protein with RNA-binding ability. In addition, it reduces the extent of aggregation significantly and enhances the efficiency of refolding and activity recovery of carbonic anhydrase and horseradish peroxidase. In this study, the effect of artemin purified from Artemia urmiana on bovine β-lactoglobulin (BLG) and its α-helical intermediate state has been evaluated by circular dichroism and intrinsic and extrinsic fluorescence spectroscopy. The results obtained in aqueous buffer show, artemin decreases the compactness of BLG structure and causes to the exposure of some hydrophobic groups. The results also indicate artemin has an inhibitory effect on β-sheet→α-helix transition in the secondary structure of β-lactoglobulin. Since this transition occurs during unfolding of β-lactoglobulin, it seems artemin influences on the folding pathway of β-lactoglobulin. This structural effect of artemin can result from its high surface hydrophobicity. Consequently, it is expected that artemin has chaperoning potency because of its effect on the folding of BLG.

The biological effects of vanadyl curcumin and vanadyl diacetylcurcumin complexes: the effect on structure, function and oxidative stability of the peroxidase enzyme, antibacterial activity and cytotoxic effect

Abstract Curcumin has multiple pharmacological effects, but it has poor stability. Complexation of curcumin with metals improves its stability. Here, the effects of vanadyl curcumin and vanadyl diacetylcurcumin on the function and structure of horseradish peroxidase enzyme were evaluated by spectroscopic techniques. Cytotoxic effect of the complexes was also assessed on MCF-7 breast cancer, bladder and LNCaP prostate carcinoma cell line. The results showed that the complexes improve catalytic activity of HRP, and also increase its tolerance against the oxidative condition. The result also indicated that the affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism and fluorescence spectroscopies showed that compactness of the enzyme structure around the catalytic heme group and the distance between the heme group and tryptophan residue decreases after the binding. The antibacterial and cytotoxic results indicated that the complexes have anticancer potential, but they have no considerable antibacterial activity.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.Graphical AbstractCurcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

Kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water and the mechanism by which glycerol obstructs the enzyme emitter site

Renilla Luciferase is a bioluminescent enzyme which is broadly implemented as protein reporter in biology-related researches. In this study, new evidences on the kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water using MD simulation along with experimental procedures including fluorescence and CD spectroscopy were obtained. The results indicated that the Renilla luciferase activity decreased at 0.8 and 1.2 M of glycerol through the obstruction of enzyme emitter site. The present study may describe a new molecular mechanism of decreasing enzyme activity in the presents of glycerol.

An inter-subunit disulfide bond of artemin acts as a redox switch for its chaperone-like activity

Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman’s assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.