Sheraz Ahmad Bhat

Employing in vitro analysis to test the potency of methylglyoxal in inducing the formation of amyloid-like aggregates of caprine brain cystatin

AbstractThiol protease inhibitors (cystatins) are implicated in various disease states from cancer to neurodegenerative conditions and immune responses. Cystatins have high amyloidogenic propensity and they are prone to form fibrillar aggregates leading to amyloidosis. Particularly challenging examples of such disorders occur in type 2 diabetes, Alzheimer’s and Parkinson’s diseases. The aim of the present study is to find an interaction between the compound methylglyoxal (MG) which is particularly elevated in type 2 diabetes with caprine brain cystatin (CBC). Results have shown that elevated concentration of MG forms amyloid aggregates of CBC. This was achieved by allowing slow growth in a solution containing moderate to high concentrations of MG. When analysed with microscopy, the protein aggregate present in the sample after incubation consisted of extended filaments with ordered structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit increased Thioflavin T fluorescence.

Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation.

Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40 and 50% TFE to proceed α to β transition, suggesting the formation of an intermolecular β-structure. AFM images confirmed the nature of Fib aggregates at 40 and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular β-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intramolecular hydrogen bonds over intermolecular hydrogen bonds leading to helix formation. To conclude, protein aggregation is accompanied by the formation of β-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the β-sheet and α-helix conformation.

Conformational behaviour and aggregation of chickpea cystatin in trifluoroethanol: effects of epicatechin and tannic acid.

Conformational alterations and aggregates of chickpea cystatin (CPC) were investigated upon sequential addition of trifluoroethanol (TFE) over a range of 0-70% v/v. CPC on 30% and 40% v/v TFE addition exhibited non-native β-sheet, altered intrinsic fluorescence, increased thioflavin T fluorescence, prominent red shifted shoulder peak in Congo red absorbance, and enhanced turbidity as well as Rayleigh scattering, suggesting the aggregate formation. TEM results confirmed the formation of fibrillar aggregates at 30% and 40% v/v TFE. On increasing concentration of TFE to 70% v/v, CPC showed retention of native-like secondary structure, increased intrinsic and ANS fluorescence. Thus our results show that favourable condition for fibrillation of CPC is in the range of 30-40% TFE. Moreover, anti-aggregational effects of polyphenols, epicatechin (EC) and tannic acid (TA) were analysed using ThT binding assay and other biophysical assays. EC and TA produced a concentration dependent decline in ThT fluorescence suggesting inhibition of the fibril formation. Furthermore, TA in comparison to EC, served as a more effective inhibitor against amyloid fibril formation of CPC. This work supports the universality of the amyloid-like aggregation not restricted to some special categories of protein and the fact that this aggregation can be prevented.

Conformational transitions induced by in vitro macromolecular crowding lead to the amyloidogenesis of buffalo heart cystatin

The biological cells and extracellular matrix exhibit a highly crowded environment, called as macromolecular crowding. Crowding significantly influences protein structure and may lead to its aggregation. In the present study, buffalo heart cystatin (BHC), after purification from buffalo heart tissue, has been used as a model protein for studying effect of macromolecular crowding in the presence of high concentrations of bovine serum albumin (BSA), poly‐ethylene glycol‐1000 (PEG‐1000), and poly‐ethylene glycol‐4000 (PEG‐4000). Cystatins are thiol protease inhibitors and found to be involved in various important physiological processes. Functional inactivation of BHC was observed upon crowding, which varied as a function of concentration and molecular weight of crowding agents as well as incubation time. Structural changes of BHC at tertiary and secondary level were detected with the help of fluorescence and CD spectroscopy. CD analysis showed changes of α‐helix to β‐sheet, which could be due to aggregation. The ANS‐fluorescence study suggested the unfolding and presence of some partially folded intermediates. Increase in ThT‐fluorescence and absorption of Congo red spectra with red shift, confirmed the amyloid type aggregation of BHC in the presence of various crowding agents. Finally, electron microscopy provided the physical evidence about the formation of amyloid fibrils. Results suggested that among the various crowding agents used, amyloidogenesis of BHC was maximal in case of BSA followed by PEG‐4000 and least for PEG‐1000. The present work makes an important contribution in crowding mediated protein aggregation, which can have implications of potential interest. Copyright

Deciphering the interaction of bovine heart cystatin with ZnO nanoparticles: Spectroscopic and thermodynamic approach

ZnO-NPs have been widely used in biomedical fields such as therapeutics, cellular imaging, and drug delivery. However, the risk of exposure of nanoparticles to the biological system is not well understood. Nanoparticle-protein interaction is pivotal to understand their biological behavior and predict nanoparticle toxicity that is crucial for its safer applications. In the present study zinc oxide nanoparticles (ZnO-NPs) were synthesized and subjected to interact with buffalo heart cystatin (BHC), purified from buffalo heart, to assess the effect(s) of ZnO-NPs on the structure and function of BHC. In vitro toxicity assessments revealed that BHC, upon interaction with ZnO-NPs, led to the altered protein conformation and perturbed function. A decrease in the anti-papain activity of BHC was observed. Spectroscopic studies demonstrated that formation of BHC-ZnO-NPs complex accompanied by structural changes in BHC along with a significant decrease in its α-helical content. ITC determined the thermodynamic parameters of binding between ZnO-NPs and BHC quantitatively. Increased surface hydrophobicity (change in the tertiary structure) was observed by ANS fluorescence that demonstrated the formation of molten globular intermediates that were found to be stable without any signs of aggregation as depicted by ThT fluorescence. TEM images gave the physical evidence of the formation of ZnO-NPs-BHC corona.

Denaturation induced aggregation in α-crystallin: differential action of chaotropes.

α‐Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α‐crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α‐crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α‐crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α‐crystallin at 216 nm, suggesting gain in β‐sheet structure in the presence of GdnHCl (0.5–1 M) followed by unfolding at higher concentration (2–6 M). However, only minor changes in the secondary structure of α‐crystallin were observed in the presence of urea. Moreover, 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α‐crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α‐crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α‐crystallin in the presence of GdnHCl, whereas only aggregate‐like structures were observed in α‐crystallin treated with urea. Our results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright

Evaluation of polyphenols as possible therapeutics for amyloidoses: Comparative analysis of Kaempferol and Catechin

Several mammalian proteins fold abnormally under non physiological conditions, to form pathological deposits that are associated with many degenerative diseases. In vitro variation of solvent conditions and pH can lead to partial unfolding and subsequent fibril formation. In the present study, we examined the effects of low pH on goat brain cystatin (GBC) with a focus on amyloid fibril formation. The results demonstrate that GBC can form amyloid like fibrils at pH 3.0. Moreover this study is aimed at exploring the inhibitory activity of polyphenols, Kaempferol (KM) and Catechin (CA) against the fibrillation of GBC. Using fluorescence spectroscopic analysis with Thioflavin T, CD and electron microscopic studies, anti-fibrillation effects of polyphenols, KM and CA were analyzed. The study also revealed that KM and CA produced a concentration dependent anti-fibrillogenic effects with KM producing more pronounced effect compared to CA. The study proposed a mechanistic approach assuming structural constraints and specific aromatic interactions of polyphenols with β sheets of GBC fibrils.

In vitro disintegration of goat brain cystatin fibrils using conventional and gemini surfactants: Putative therapeutic intervention in amyloidoses.

Many protein misfolding diseases in mammalian system are characterised by the accumulation of protein aggregates in amyloid fibrillar forms. Several therapeutic approaches include reduction in the production of the amyloidogenic form of proteins, increase in the clearance rate of misfolded or aggregated proteins, and direct inhibition of the self-assembly process have been explained. One of the possible remedial treatments for such disorders may be to identify molecules which are capable of either preventing formation of fibrils or disintegrating the formed fibrils. In this work, we have studied the effect of conventional surfactants; sodium dodecylsulphate (SDS), cetyl trimethylammonium bromide (CTAB) and dicationic gemini (16-4-16) surfactant on the disintegration of the goat brain cystatin (GBC) fibrils above their critical micelle concentrations (CMC) using ThT fluorescence, CD, TEM, Congo red and turbidity approaches. The results obtained are significant and showing the best disintegrating potency on GBC fibrils with gemini surfactant. The outcome from this work will aid in the development and/or design of potential inhibitory agents against amyloid deposits associated with amyloid diseases.

Synthesis, molecular docking and biological evaluation of new steroidal 4H-pyrans.

A series of new steroidal 4H-pyrans (4-6) have been synthesized from steroidal α, β-unsaturated ketones (1-3). The products (4-6) were characterized by IR, (1)H NMR, (13)C NMR, MS and analytical data. The interaction studies of compounds (4-6) with DNA were carried out by employing gel electrophoresis, UV-vis and fluorescence spectroscopy. The gel electrophoresis pattern revealed that compounds (4-6) bind to DNA and also demonstrated that the compound 6 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322. The compounds 4 and 5 bind to DNA preferentially through electrostatic and hydrophobic interactions with Kb values found to be 5.3×10(3) and 3.7×10(3) M(-1), respectively, indicating the higher binding affinity of compound 4 towards DNA. The docking study suggested the intercalation of compounds in between the nucleotide base pairs. The cytotoxicity and genotoxicity of the newly synthesized compounds were checked by MTT and comet assay, respectively during which compound 6 showed potential behaviour.

Glycation induced conformational transitions in cystatin proceed to form biotoxic aggregates: A multidimensional analysis

Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant β structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.