The inhibitory effect of Sunset Yellow on thermally induced Human Serum Albumin aggregates: Possible role in naturopathy.

Intensive research in the field of protein aggregation confirmed that the deposition of amyloid fibrils of proteins are the major cause for the development of various neurotoxic and neurodegenerative diseases, which could be controlled by ensuring the efficient inhibition of aggregation using anti aggregation strategies. Herein, we elaborated the anti amyloidogenic potential of Sunset Yellow (SY) dye against Human Serum Albumin (HSA) fibrillogenesis utilising different biophysical, computational and microscopic techniques. The inhibitory effect of sunset yellow was confirmed by Rayleigh Light Scattering (RLS) measurements along with different dye binding assays (ANS, ThT and CR) by showing concentration dependent reduction in scattering intensity and fluorescence intensity respectively. Further, destabilization and anti fibrillation activity of HSA aggregates were characterized through spectroscopic techniques like Circular Dichroism (CD) and other microscopic techniques like Transmission Electron Microscopy (TEM) for elucidating the structural properties. The SDS-PAGE was also carried out that render the disaggregation effect of the dye on the protein. Moreover, Molecular Docking studies revealed the binding parameters justifying the stable protein-dye complex. Simulation studies were also performed accordingly. Thus, this dye which is used as food additive can serve as a potential aggregation inhibiting agent that can aid in the prevention of amyloidogenic diseases.

Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity.

Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.

Surfactant-mediated amyloidogenesis behavior of stem bromelain; a biophysical insight.

Neurodegenerative disorders are mainly associated with amyloid fibril formation of different proteins. Stem bromelain (SB), a cysteine protease, is known to exist as a molten globule state at pH 10.0. It passes through the identical surrounding (pH 10.0) in the gut epithelium of intestine upon oral administration. Protein-surfactant complexes are widely employed as drug carriers, so the nature of surfactant toward protein is of great interest. The present work describes the effect of cationic surfactants (CTAB & DTAB) and their hydrophobic behavior toward amyloidogenesis behavior of SB at pH 10.0. Multiple approaches including light scattering, far UV-CD, turbidity measurements, and dye binding assay (ThT, Congo red and ANS) were performed to measure the aggregation propensity of SB. Further, we monitored the hydrodynamic radii of aggregates formed using dynamic light scattering technique. Structure of fibrils was also visualized through fluorescence microscopy as well as TEM. At pH 10.0, low concentration of CTAB (0-200 µM) induced amyloid formation in SB as evident from a prominent increase in turbidity and light scattering, gain in ß-sheet content, and enhanced ThT fluorescence intensity. However, further increase in CTAB concentration suppressed the fibrillation phenomenon. In contrast, DTAB did not induce fibril formation at any concentration used (0-500 µM) due to lower hydrophobicity. Net negative charge developed on protein at high pH (10.0) might have facilitated amyloid formation at low concentration of cationic surfactant (CTAB) due to electrostatic and hydrophobic interactions.

Amyloidogenic behavior of different intermediate state of stem bromelain: A biophysical insight.

Stem bromelain, a cysteine proteases from Ananas comosus is a widely accepted therapeutic drug with broad medicinal application. It exists as intermediate states at pH 2.0 and 10.0, where it encountered in gastrointestinal tract during adsorption (acidic pH) and in gut epithelium (alkaline pH), respectively. In this study, we monitored the thermal aggregation/amyloid formation of SB at different pH intermediate states. Thermal treatment of stem bromelain at pH 10.0 favors the fibrillation in which the extent of aggregation increases with increase in protein concentration. However, no fibril formation in stem bromelain at pH 2.0 was found at all the concentration used at pH 10.0. The fibril formation was confirmed by various techniques such as turbidity measurements, Rayleigh light scattering, dye binding assays and far UV circular dichroism. The Dynamic light scattering confirmed the formation of aggregates by measuring the hydrodynamic radii pattern. Moreover, microscopic techniques were performed to analyze the morphology of fibrils. The aggregation behavior may be due to variation in number of charged amino acid residues. The less negative charge developed at pH 10.0 may be responsible for aggregation. This work helps to overcome the aggregation related problems of stem bromelain during formulations in pharmaceutical industry.

Concentration-dependent antagonistic persuasion of SDS and naphthalene derivatives on the fibrillation of stem bromelain.

Sodium dodecyl sulfate, a biological membrane mimetic, can be used to study the conversion of globular proteins into amyloid fibrils in vitro. Using multiple approaches, the effect of SDS was examined on stem bromelain (SB), a widely recognized therapeutic protein. SB is known to exist as a partially folded intermediate at pH 2.0, situation also encountered in the gastrointestinal tract (its site of absorption). In the presence of sub-micellar SDS concentration (500-1000 µM), this intermediate was found to exhibit great propensity to form large-sized ß-sheeted aggregates with fibrillar morphology, the hall marks of amyloid structure. We also observed inhibition of fibrillation by two naphthalene-based compounds, ANS and bis-ANS. While bis-ANS significantly inhibited fibril formation at 50 µM, ANS did so at relatively higher concentration (400 µM). Alcohols, but not salts, were found to weaken the inhibitory action of these compounds suggesting the possible involvement of hydrophobic interactions in their binding to protein. Besides, isothermal titration calorimetry and molecular docking studies suggested that inhibition of fibrillation by these naphthalene derivatives is mediated not just through hydrophobic forces, but also by disruption of π-π interactions between the aromatic residues together with the inter-polypeptide chain repulsion among negatively charged ANS/bis-ANS bound SB.