In-vitro and in-silico investigation of protective mechanisms of crocin against E46K α-synuclein amyloid formation.

α-Synuclein is a presynaptic neuronal protein that is abundant in the human brain and is linked genetically and neuropathologically to Parkinson's disease (PD). The E46K mutation of the α-synuclein gene has been linked to autosomal dominant early-onset of PD. Crocin is a carotenoid chemical compound of saffron that has been shown antioxidant and neural protective activity. This study examined the effect of Crocin in preventing the amyloid fibril in the E46K α-synuclein, through in vitro studies and computational simulations. The result demonstrated that Crocin acts as a molecular chaperone to prevent amyloid fibril formation of E46K α-synuclein in a concentration-dependent manner. In fact, Crocin redirects E46K α-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway or at least reduce its aggregation tendency. Combined results from molecular dynamics and docking studies indicate that the inhibitory effect of the Crocin may be due to binding of the Crocin with the hydrophobic region (contact interface) of the α-synuclein which has the propensity to form amyloid aggregate. The results indicated Crocin can potentially bind to the C-terminal and mainly NAC (central hydrophobic region) domain of the E46K α-synuclein, and stabilizes the protein by masking the polymerization hotspot and consequently converting the protein into amyloid fibrils. These results support that Crocin is a effective inhibitor of E46K α-synuclein fibrillization and it could be considered as a potential therapeutic agent in the treatment of Parkinson disease.

Prevention of α-crystallin glycation and aggregation using l-lysine results in the inhibition of in vitro catalase heat-induced-aggregation and suppression of cataract formation in the diabetic rat.

The principle role of α-crystallin is chaperoning activity that protect s other proteins against different stresses. High glucose concentration induces the osmotic stress and results in biomacromolecules glycation, which is subsequently cause their conformational and functional changes. Here, the roles of l-lysine (Lys) on the prevention of α-crystallin glycation in both in vitro and in vivo conditions are investigated. The catalase (CAT) activity was considered as a marker of α-crystallin functionality in both conditions. Streptozotocin-induced diabetic rats were treated with 0.1% of the Lys in drinking water. The purified α-crystallin was also incubated with glucose, in the presence or absence of the Lys and its structure-function was compared. The results showed that the visual cataract score was significantly lower in the diabetic rats treated with Lys. After Lys treatment, CAT, superoxide dismutase, aldose reductase and other biochemical parameters in the lens and serum of the diabetic rats returned to the normal value. Formation of the advanced glycation endproducts (AGEs), protein cross-linking, and the hydrophobicity of α-crystallin were changed due to glycation, but they were reversed by Lys treatment. The glycated α-crystallin lost its chaperone activity against heat denatured-CAT, but in the presence of Lys, it preserved its activity and prevented CAT aggregation. In conclusion, Lys treatment significantly inhibited the progression of diabetic cataract in rats. These effects were due to the Lys antiglycating and antioxidant effects, in addition to its protective effect against α-crystallin chaperoning activity.

Synthesis of new ultrasonic-assisted palladium oxide nanoparticles: an in vitro evaluation on cytotoxicity and DNA/BSA binding properties.

Better solubility and improved toxicity of palladium complexes compared with cisplatin were major reasons for synthesis of novel Pd(II) complex, [Pd(8Q)(bpy)]NO3 (8Q=8-hydroxyquinolinate, bpy=2,2'-bipyridine). Interaction between the [Pd(8Q)(bpy)]NO3 complex and calf thymus DNA in aqueous solution has been investigated by circular dichroism (CD), UV-Visible absorption and fluorescence spectroscopic techniques. These experiments showed that prepared Pd(II) complex can effectively intercalate into CT-DNA and weakly bind to BSA in which the bovine serum albumin molecule was unfolded slightly. The cytotoxicity of the prepared complex has been evaluated on the MCF-7 and DU145 cell lines by MTT and TUNEL assay. The MTT results were showed that in DU145, the CC50 values of [Pd(8Q)(bpy)]NO3 and cisplatin are very close together (10.4 and 8.3 µM, respectively), unlike MCF-7. Accordingly, TUNEL assay was performed on DU145 and apoptosis was clearly obvious by 43% DNA fragmentation in the treated cell lines. So, we can suggest the [Pd(8Q)(bpy)]NO3 as alternative drug for cisplatin in the future which has great potential in DNA denaturation and apoptosis specially on prostate cancer. PdO nanoparticles were successfully prepared without supported any surfactants via sonochemical approach. The synthesized PdONPs were characterized using UV-Vis and FTIR spectroscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Communicated by Ramaswamy H. Sarma.

Palladium(II) complexes of biorelevant ligands. Synthesis, structures, cytotoxicity and rich DNA/HSA interaction studies.

In this work, a pair of new palladium(II) complexes, [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)], (where Gly is glycine, Phe is phenylalanine, and Tyr is tyrosine) were synthesized and characterized by UV-Vis, FT-IR, elemental analysis, 1H-NMR, and conductivity measurements. The detailed 1H NMR and infrared spectral studies of these Pd(II) complexes ascertain the mode of binding of amino acids to palladium through nitrogen of -NH2 and oxygen of -COO- groups as bidentate chelates. The Pd(II) complexes have been tested for in vitro cytotoxicity activities against cancer cell line of K562. Interactions of these Pd(II) complexes with CT-DNA and human serum albumin were identified through absorption/emission titrations and gel electrophoresis which indicated significant binding proficiency. The binding distance (r) between these synthesized complexes and HSA based on Forster's theory of non-radiation energy transfer were calculated. Alterations of HSA secondary structure induced by complexes were confirmed by FT-IR measurements. The results of emission quenching at three temperatures have revealed that the quenching mechanism of these Pd(II) complexes with CT-DNA and HSA were the static and dynamic quenching mechanism, respectively. Binding constants (Kb), binding site number (n), and the corresponding thermodynamic parameters were calculated and revealed that the hydrogen binding and hydrophobic forces played a major role when Pd(II) complexes interacted with DNA and HSA, respectively. We bid that [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)] complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of [Pd(Gly)(Tyr)] > [Pd(Gly)(Phe)] with CT-DNA- and HSA-binding.

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA.

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO3 (I) and [Zn(phen)(Gly)]NO3 (II) (where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV-vis, FT-IR, and 1H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV-vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV-vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (Kb), the Stern-Volmer constant (Ksv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow's site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.

The effect of green synthesis silver nanoparticles (AgNPs) from Pulicaria undulata on the amyloid formation in α-lactalbumin and the chaperon action of α-casein.

The formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Molecular chaperones are a family of proteins that are believed to have the ability to inhibit protein aggregation. The present study examines the effect of different concentrations of green synthesis silver nanoparticles (AgNPs) from Pulicaria undulata L. on the aggregation of α-lactalbumin (α-LA) and the chaperone action of αs-casein. The effects of the AgNPs were determined by measuring light scattering absorption, fluorescence (ThT assay, intrinsic fluorescence assay and ANS binding assay), TEM, CD spectroscopy and SDS-PAGE. The results showed that AgNPs have the ability to prevent the aggregation of α-LA in a concentration-dependent manner. In fact, by increasing the concentration of AgNPs within a specified range, the adsorption and interaction between AgNPs and protein have increased and protein conformational changes and self-association decreased, thus amyloid aggregation is prevented. Our results also showed that α-casein effectively prevented the aggregation of the α-lactalbumin which increased in the presence of the AgNPs. Standard experimental results, however, proved that nanoparticles had no effect on the structure and hence the chaperone ability of α-casein. Our findings showed that AgNPs can prevent protein aggregation and have no effect on the chaperone ability of αs-casein. In the main, results of this study show that biosynthesized AgNPs mediated by Pulicaria undulata L. has the capability in inhibiting amyloid fibril formation and thus could be consider as a therapeutic agent in the treatment of amyloidosis disorders.

The potential inhibitory effect of ß-casein on the aggregation and deposition of Aß1-42 fibrils in Alzheimer's disease: insight from in-vitro and in-silico studies.

Aß1-40 and Aß1-42 have been shown to be the main components of the amyloid plaques found in the extracellular environment of neurons in Alzheimer's disease. ß-Casein, a milk protein, has been shown to display a remarkable chaperone ability in preventing the aggregation of proteins. In this study, the ability of ß-casein to suppress the amyloid fibril formation of Aß1-42 has been examined through in vitro studies and molecular docking simulation. The results demonstrate the inhibitory effect of ß-casein on fibril formation in Aß1-42, in a concentration dependent manner, suggesting that the chaperone binds to the Aß1-42 and prevents amyloid fibril formation. Molecular docking results show that the inhibitory effect of the ß-casein may be due to binding of the chaperone with the aggregation-prone region of the Aß1-42 mainly via hydrophobic interactions. ß-Casein probably binds to the CHC and C-terminal domain of the Aß1-42, and stabilizes proteins by inhibiting the conversion of monomeric Aß1-42 into fibrils. Thus our data suggests that the hydrophobic interactions between ß-casein and Aß1-42 play an important role in the burial of the hydrophobic part of the Aß1-42. This means that ß-casein maybe considered for use in preventing amyloid fibril formation in degenerative diseases such as Alzheimer.

Inhibitory Effect of Crocin(s) on Lens α-Crystallin Glycation and Aggregation, Results in the Decrease of the Risk of Diabetic Cataract.

The current study investigates the inhibitory effect of crocin(s), also known as saffron apocarotenoids, on protein glycation and aggregation in diabetic rats, and α-crystallin glycation. Thus, crocin(s) were administered by intraperitoneal injection to normal and streptozotocin-induced diabetic rats. The cataract progression was recorded regularly every two weeks and was classified into four stages. After eight weeks, the animals were sacrificed and the parameters involved in the cataract formation were measured in the animal lenses. Some parameters were also determined in the serum and blood of the rats. In addition, the effect of crocin(s) on the structure and chaperone activity of α-crystallin in the presence of glucose was studied by different methods. Crocin(s) lowered serum glucose levels of diabetic rats and effectively maintained plasma total antioxidants, glutathione levels and catalase activity in the lens of the animals. In the in vitro study, crocin(s) inhibited α-crystallin glycation and aggregation. Advanced glycation end products fluorescence, hydrophobicity and protein cross-links were also decreased in the presence of crocin(s). In addition, the decreased chaperone activity of α-crystallin in the presence of glucose changed and became close to the native value by the addition of crocin(s) in the medium. Crocin(s) thus showed a powerful inhibitory effect on α-crystallin glycation and preserved the structure-function of this protein. Crocin(s) also showed the beneficial effects on prevention of diabetic cataract.

The effect of Arg on the structure perturbation and chaperone activity of α-crystallin in the presence of the crowding agent, dextran.

α-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses. It has a molecular weight of 20 kDa and is composed of two subunits: αA and αB. α-Crystallin is a member of the small heat shock protein (sHsps) family that has been shown to prevent protein aggregation. Small molecules are organic compounds that have low molecular weight (<800 Da). Arginin (Arg) is a small molecule and has been shown to prevent protein aggregation through interaction with partially folded intermediates. In this study, the effect of Arg on the chaperone activity of α-crystallin in the presence of dextran, as a crowding agent, against ordered and disordered aggregation of different target proteins (α-lactalbumin, ovotransferrin, and catalase) has been investigated. The experiments were done using visible absorption spectroscopy, ThT-binding assay, fluorescence spectroscopy, and CD spectroscopy. The results showed that in amorphous aggregation and amyloid fibril formation, both in the presence and absence of dextran, Arg had a positive effect on the chaperone action of α-crystallin. However, in the presence of dextran, the effect of Arg on the chaperone ability of α-crystallin was less than in its absence. Thus, our result suggests that crowding interior media decreases the positive effect of Arg on the chaperone ability of α-crystallin. This is a very important issue, since we are trying to find a mechanism to protect living cells against the toxic effect of protein aggregation.

Chaperone potential of Pulicaria undulata extract in preventing aggregation of stressed proteins.

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.

The Effects of Extending of Co-planarity in a Series of Structurally Relative Polypyridyl Palladium(II) Complexes on DNA-binding and Cytotoxicity Properties.

In depth interaction studies between calf thymus deoxyribonucleic acid (CT-DNA) and a series of four structurally relative palladium(II) complexes [Pd(en)(HB)](NO3)2 (a-d), where en is ethylenediamine and heterocyclic base (HB) is 2,2'-bipyridine (bpy, a); 1,10-phenanthroline (phen, b); dipyridoquinoxaline (dpq, c) and dipyridophenazine (dppz, d) (Figure 1), were performed. These studies have been investigated by utilizing the electronic absorption spectroscopy, fluorescence spectra and ethidium bromide (EBr) displacement and gel filtration techniques. a-d complexes cooperatively bind and denature the DNA at low concentrations. Their concentration at midpoint of transition, L1/2, follows the order a >> b > c > d. Also the g, the number of binding sites per 1000 nucleotides, follows the order a >> b ~ c > d. EBr and Scatchard experiments for a-d complexes suggest efficient intercalative binding affinity to CT-DNA giving the order: d > c > b > a. Several binding and thermodynamic parameters are also described. The biological activity of these cationic and water soluble palladium complexes were tested against chronic myelogenous leukemia cell line, K562. b, c and d complexes show cytotoxic concentration (Cc50) values much lower than cisplatin.

The protective effect of crocin on the amyloid fibril formation of Aß42 peptide in vitro.

Aß is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer's disease. There are two common isoforms of Aß: the more common form, Aß40, and the less common but more amyloidogenic form, Aß42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aß42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aß42 amyloids. To further investigate the relationship between crocin and Aß42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aß42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aß. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aß42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and ß-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aß amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

Glycine therapy inhibits the progression of cataract in streptozotocin-induced diabetic rats.

PURPOSE: The purpose of this paper was to investigate the effect of the oral administration of L-glycine (Gly) on the development of diabetic cataract induced by streptozotocin (STZ) in rats. METHODS: Two groups of male Wistar rats were intraperitoneally injected with a single dose of STZ (65 mg/kg bodyweight). Then, one group of diabetic rats and a control group were administered with 1% of Gly in drinking water for three months, ad libitum. Cataract development was monitored biweekly through ophthalmoscope inspection and was classified into four stages. At the end of 12 weeks, the animals were sacrificed and some biochemical parameters were determined in their lenses. The parameters include advanced glycation end products (AGEs), glycated proteins, total and soluble protein, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), aldose reductase (AR), and sorbitol dehydrogenase (SDH). Some parameters were also determined in the serum and blood of the rats. RESULTS: Diabetic cataract gradually progressed in the STZ-administered group with no other treatment. Consequently, up to the end of the experiment, 2/3 of the animals in this group reached to the last stage of the cataract (mature cataract). The progress of this process was much slower in the diabetic group that was treated with Gly. At the end of the study, the visual cataract score was significantly lower in the diabetic group treated with Gly compared to those administered with STZ. Some lens parameters, including glycated proteins, AGEs, SOD, and AR activities, were increased while some others, including soluble and total protein, GSH level, and CAT activity, were decreased due to diabetes induction. After Gly treatment, all the above-named parameters had reverse changes except for the CAT activity. The SDH activity in the lenses had no changes due to diabetes or treatment. In addition, this treatment significantly decreased the amount of serum glucose (Glc), serum AGEs, and glycated hemoglobin (HbA1c) in the diabetic rats. Gly also increased the ferric reducing antioxidant power (FRAP) in the serum of diabetic rats. However, the decreased bodyweight of animals due to diabetes induction was not compensated by Gly administration. It is important to note that Gly had no effect on normal rat parameters. CONCLUSIONS: The results indicated that the oral administration of Gly significantly delayed the onset and the progression of diabetic cataract in rats. These effects were due to its antiglycating action and to a lesser extent, due to the inhibition of oxidative stress and polyol pathway.

The effects of molecular crowding on the amyloid fibril formation of alpha-lactalbumin and the chaperone action of alpha-casein.

Amyloid fibrils arise from the slow aggregation of intermediately folded protein states. In this study the kinetics of the protein fibril formation of alpha-lactalbumin and its prevention by alphaS-casein in the presence and absence of the crowding agent, dextran (68 kDa), have been compared using a thioflavin T binding assay. It was found that alphaS-casein, a molecular chaperone found in bovine milk, is a potent in vitro inhibitor of alpha-lactalbumin fibrillization. The effect of alphaS-casein in preventing fibril formation was significant, although less than it is in the absence of the crowding agent, dextran. The interaction between the chaperone and the alpha-lactalbumin and structural change in the target protein are also shown using intrinsic fluorescence intensity, an ANS binding assay, CD spectroscopy and size-exclusion HPLC. In summary, alpha-casein interacts with alpha-lactalbumin and prevents amyloid formation but not as well as it does when the crowding agent, dextran, not present.

The effect of dextran on subunit exchange of the molecular chaperone alphaA-crystallin.

Alpha-crystallin, a member of small heat shock protein (sHsp) family, is comprised of alphaA and alphaB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The alphaA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, alpha-crystallin elicits its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that alpha-crystallin and alphaA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the alphaA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to alphaA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of alphaA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the alphaA-crystallin subunit exchange rate is not temperature-dependent. In the absence of dextran, the exchange rate of alphaA-crystallin subunits correlates with its chaperone efficiency, i.e. the chaperone ability of alphaA-crystallin increases with temperature. However in the presence of dextran, the temperature dependence of the chaperone ability of alphaA-crystallin is eliminated.