Sumit Kumar Chaturvedi

SDS Can Be Utilized as an Amyloid Inducer: A Case Study on Diverse Proteins

Sodium dodecyl sulphate (SDS), an anionic surfactant that mimics some characteristics of biological membrane has also been found to induce aggregation in proteins. The present study was carried out on 25 diverse proteins using circular dichroism, fluorescence spectroscopy, dye binding assay and electron microscopy. It was found that an appropriate molar ratio of protein to SDS readily induced amyloid formation in all proteins at a pH below two units of their respective isoelectric points (pI) while no aggregation was observed at a pH above two units of pI. We also observed that electrostatic interactions play a leading role in the induction of amyloid. This study can be used to design or hypothesize a molecule or drug, which may counter act the factor responsible for amyloid formation.

Vitamin k3 inhibits protein aggregation: Implication in the treatment of amyloid diseases

Protein misfolding and aggregation have been associated with several human diseases such as Alzheimer’s, Parkinson’s and familial amyloid polyneuropathy etc. In this study, anti-fibrillation activity of vitamin k3 and its effect on the kinetics of amyloid formation of hen egg white lysozyme (HEWL) and Aβ-42 peptide were investigated. Here, in combination with Thioflavin T (ThT) fluorescence assay, circular dichroism (CD), transmission electron microscopy and cell cytotoxicity assay, we demonstrated that vitamin k3 significantly inhibits fibril formation as well as the inhibitory effect is dose dependent manner. Our experimental studies inferred that vitamin k3 exert its neuro protective effect against amyloid induced cytotoxicity through concerted pathway, modifying the aggregation formation towards formation of nontoxic aggregates. Molecular docking demonstrated that vitamin k3 mediated inhibition of HEWL and Aβ-42 fibrillogenesis may be initiated by interacting with proteolytic resistant and aggregation prone regions respectively. This work would provide an insight into the mechanism of protein aggregation inhibition by vitamin k3; pave the way for discovery of other small molecules that may exert similar effect against amyloid formation and its associated neurodegenerative diseases.

Biophysical insight into the anti-amyloidogenic behavior of taurine

In this work, we investigated the inhibitory ability of taurine on the aggregation of Human serum albumin (HSA) and also examined how it controls the kinetic parameters of the aggregation process. We demonstrated the structural alterations in the HSA after binding to the taurine at 65 °C by exploiting various biophysical techniques. UV-vis spectroscopy was used to check the turbidometric changes in the protein. Thioflavin T fluorescence kinetics was subjected to explore kinetic parameters comparing the amyloid formation in the presence of varying concentration of taurine. Further, Congo red binding and ANS binding assays were performed to determine the inhibitory effect of taurine on HSA fibrillation process and surface hydrophobicity modifications occurring before and after the addition of taurine with protein, respectively. Far UV CD and Dynamic Light Scattering (DLS) confirmed that taurine stabilized the protein α-helical structure and formed complex with HSA which is further supported by differential scanning calorimetry (DSC). Moreover, microscopic imaging techniques were also done to analyze the morphology of aggregation formed. Taurine is also capable of altering the cytotoxicity of the proteinaceous aggregates. Molecular docking study also deciphered the possible residues involved in protein and drug interaction.

Comparative insight into surfactants mediated amyloidogenesis of lysozyme

Electrostatic and hydrophobic interactions have an important role in the protein aggregation. In this study, we have investigated the effect of charge and hydrophobicity of oppositely charged surfactants i.e., anionic (AOT and SDS) and cationic (CTAB and DTAB) on hen egg white lysozyme at pH 9.0 and 13.0, respectively. We have employed various methods such as turbidity measurements, Rayleigh light scattering, ThT, Congo red and ANS dye binding assays, far-UV CD, atomic force microscopy, transmission electron and fluorescence microscopy. At lower molar ratio, both anionic and cationic surfactants promote amyloid fibril formation in lysozyme at pH 9.0 and 13.0, respectively. The aggregation was proportionally increased with respect to protein concentration and hydrophobicity of surfactant. The morphology of aggregates at both the pH was fibrillar in structure, as visualized by dye binding and microscopic imaging techniques. Initially, the interaction between surfactants and lysozyme was electrostatic and then hydrophobic as investigated by ITC. This study demonstrates the crucial role of charge and hydrophobicity during amyloid fibril formation.

Anti-amyloidogenic behavior and interaction of Diallylsulfide with Human Serum Albumin

In this work, binding of garlic component-Diallysulfide (DAS) with major human blood transport protein, Human Serum Albumin (HSA) and its anti- amyloidogenic behavior has been studied by utilizing various spectroscopic and molecular docking strategies. The HSA exhibit significant reduction in fluorescence intensity upon interaction with DAS. DAS quenches the fluorescence of HSA in concentration dependent manner with binding affinity of 1.14×103M-1. UV-visible spectroscopy results confirm the formation of DAS-HSA complex and secondary structure of HSA get stabilized upon complexation with DAS as observed by far UV CD spectroscopy and Differential Scanning Calorimetry. The topology of HSA in absence and presence of DAS was monitored through Dynamic Light Scattering (DLS) technique, inferred that protein becomes more compact in presence of DAS. Further, molecular docking study shows that DAS bind to the nearby site II in subdomain III of HSA. Moreover, effect of DAS was studied on HSA fibrillation process. ThT binding, ANS fluorescence assay, CD measurement, DLS and Transmission Electron Microscopy (TEM) results altogether confirm the anti-amyloidogenic property of DAS. This work will provide biophysical insight into the interaction of DAS with HSA and will help in designing more potential therapeutic strategies against protein aggregation by exploiting other related compounds.

Ascorbic acid inhibits human insulin aggregation and protects against amyloid induced cytotoxicity

Protein aggregation into oligomers and fibrils are associated with many human pathophysiologies. Compounds that modulate protein aggregation and interact with preformed fibrils and convert them to less toxic species, expect to serve as promising drug candidates and aid to the drug development efforts against aggregation diseases. In present study, the kinetics of amyloid fibril formation by human insulin (HI) and the anti-amyloidogenic activity of ascorbic acid (AA) were investigated by employing various spectroscopic, imaging and computational approaches. We demonstrate that ascorbic acid significantly inhibits the fibrillation of HI in a dose-dependent manner. Interestingly ascorbic acid destabilise the preformed amyloid fibrils and protects human neuroblastoma cell line (SH- SY5Y) against amyloid induced cytotoxicity. The present data signifies the role of ascorbic acid that can serve as potential molecule in preventing human insulin aggregation and associated pathophysiologies.

Attenuation of amyloid fibrillation in presence of Warfarin: A biophysical investigation

Protein misfolding and aggregation are associated with more than twenty diseases, such as neurodegenerative diseases. The amyloid oligomers and fibrils may induce cell membrane disruption and lead to cell apoptosis. A great number of studies have focused on discovery of amyloid inhibitors which may prevent or treat amyloidosis. In this study, we used human serum albumin (HSA) as an amyloid model to test the anti-amyloid effects of warfarin (WFN), a very well-known drug for treatment of thrombosis and also used by biophysicists to characterize the specific binding site on HSA (site I of subdomain IIA). We have used a combination of different biophysical, spectroscopic and imaging techniques to prove the anti-amyloidogenic behavior of WFN. Our results demonstrated that WFN is capable enough to inhibit the HSA fibrillation. Exposed HSA surface hydrophobicity was decreased by 50% as judged by ANS analysis. Moreover, anti-amyloidegenic behavior of WFN was found to be concentration dependent as supported by decreased ThT fluorescence by 22.4% and 46% at WFN concentrations of 500 and 1000μM, respectively. Circular dichroism technique showed the change in secondary structure of native HSA as well as in presence of WFN. These results suggests that WFN is capable of inhibiting amyloid aggregation, hence, WFN related compounds may thus be further explored for designing effective anti-amyloidosis compounds.

Vitamin B12 offers neuronal cell protection by inhibiting Aβ-42 amyloid fibrillation

Protein misfolding and aggregation has been implicated as the cause of more than 20 diseases in humans such as Alzheimers and Parkinsons and systemic amyloidosis. Retardation of Aβ- 42 aggregation is considered as a promising and challenging strategy for developing effective therapeutics against Alzheimers disease. Herein, we demonstrated the effect of vitamin B12 (VB) on inhibiting amyloid formation by employing ThT fluorescence assay, circular dichroism, ANS fluorescence assay, dynamic light scattering measurements and transmission electron microscopy and cell viability assay. Our results demonstrate that vitamin B12 (VB), inhibits Aβ- 42 aggregation in a concentration dependent manner. Further VB also provide protection against amyloid induced cytotoxicity in human neuronal cell line. This study points towards a promising strategy to combat Aβ- 42 aggregation and may have broader implication for targeting other neurological disorders whose distinct hallmark is also amyloid formation.

Elucidating the mode of action of urea on mammalian serum albumins and protective effect of sodium dodecyl sulfate.

The effect of sodium dodecyl sulfate (SDS) on human, bovine, porcine, rabbit and sheep serum albumins were investigated at pH 3.5 by using various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence and dynamic light scattering (DLS). In the presence of 4.0mM SDS the secondary structure of all the albumins were not affected as measured by CD but fluorescence spectra revealed 8.0 nm blue shift in emission maxima. We further checked the stability of albumins in the absence and presence of 4.0mM SDS by urea and temperature at pH 3.5. In the absence of SDS, urea starts unfolding both secondary as well as tertiary structural elements of the all the albumins at approximately 2.0M urea but in the presence of 4.0mM SDS, urea was unable to unfold even up to 9.0M. The albumins were thermally less stable at pH 3.5 with decrease in Tm but in the presence of 4.0mM SDS, the Tm was increased. From this study, it was concluded that SDS is showing a protective effect against urea as well as thermal denaturation of albumins. This behavior may be due to electrostatic as well as the hydrophobic interaction of SDS with albumins. Further, we have proposed the mechanism of action of urea. It was found that urea interacted with proteins directly when proteins are in charged form. Indirect interaction may be taking place when the environment is more hydrophobic.

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer’s dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aβ-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.