Samreen Amani

Deciphering structural intermediates and genotoxic fibrillar aggregates of albumins: a molecular mechanism underlying for degenerative diseases.

The misfolding and aggregation of proteins is involved in some of the most prevalent neurodegenerative disorders. The importance of human serum albumin (HSA) stems from the fact that it is involved in bio-regulatory and transport phenomena. Here the effect of acetonitrile (ACN) on the conformational stability of HSA and by comparison, ovalbumin (OVA) has been evaluated in the presence and absence of NaCl. The results show the presence of significant amount of secondary structure in HSA at 70% ACN and in OVA at 50% ACN, as evident from far-UV Circular Dichroism (CD) and Attenuated Total Reflection Fourier transformed infra red spectroscopy (ATR-FTIR). Tryptophan and 8-Anilino-1-Naphthalene-Sulphonic acid (ANS) fluorescence indicate altered tryptophan environment and high ANS binding suggesting a compact “molten globule”-like conformation with enhanced exposure of hydrophobic surface area. However, in presence of NaCl no intermediate state was observed. Detection of aggregates in HSA and OVA was possible at 90% ACN. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and ATR-FTIR. These aggregates exhibit increase Thioflavin T (Th T) fluorescence with a red shift of Congo red (CR) absorption spectrum. X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM) analysis confirmed the presence of fibrillar aggregates. Single cell gel electrophoresis (SCGE) assay of these fibrillar aggregates showed the DNA damage resulting in cell necrosis confirming their genotoxic nature. Some proteins not related to any human disease form fibrils in vitro. In the present study ACN gives access to a model system to study the process of aggregation.

Acetonitrile can promote formation of different structural intermediate states on aggregation pathway of immunoglobulin G from human and bovine.

A sequential addition of acetonitrile to human and bovine immunoglobulin G induces molten globule-like state at 50% (v/v) and 60% (v/v) respectively having secondary structure similar to native protein as evident from far-UV circular dichroism and Fourier transform infra red spectroscopy. Further addition of acetonitrile up to 80% forms aggregate of IgG as confirmed by increase in thioflavin T, loss of signals in near-UV CD spectra, decrease in ANS and tryptophan fluorescence. Thus at high acetonitrile concentration, a relatively large amount of partially unfolded intermediates of IgG are present which result in aggregates formation.

Equilibrium studies of cellulase aggregates in presence of ascorbic and boric acid.

The aggregate formation of cellulase was detected at 300 and 10 mM ascorbic and boric acid respectively. These aggregates showed reduced enzyme activity, loss in near-UV signal, decrease tryptophan and ANS fluorescence. They possess increase in non-native β-sheet structure as evident from far-UV CD and FTIR spectra, large hydrodynamic radii, increase thioflavin T fluorescence and shift in Congo red. Cellulase at 90 mM ascorbic acid exists as molten globule with retention of secondary structure, altered tryptophan environment, high ANS binding and loss in tertiary structure. Ascorbic acid acts as an antioxidant up to 90 mM and beyond this as a pro-oxidant.

Detection and analysis of amorphous aggregates and fibrils of cytochrome c in the presence of phenolic acids

Cytochrome c (cyt c) exists as a partially unfolded intermediate at 45 mM gallic acid (GA) possessing disrupted secondary structure, altered Trp environment and high ANS binding. Increasing the concentration of either GA or ferulic acid (FA) up to 50 mM results in cyt c aggregation as confirmed by shift in Congo red, increase thioflavin T, decrease ANS and Trp fluorescence. SEM confirmed the formation of fibrils and amorphous aggregates of cyt c in presence of 50 mM FA and GA respectively. Single cell gel electrophoresis establishes very less probability of this noble protein to cause misfolding and aggregation-prone diseases.

Potassium bromate causes cell lysis and induces oxidative stress in human erythrocytes

In the present study, we have studied the effect of KBrO3 on human erythrocytes under in vitro conditions. Erythrocytes were isolated from the blood of healthy nonsmoking volunteers and incubated with different concentrations of KBrO3 at 37°C for 60 min. This resulted in marked hemolysis in a KBrO3‐concentration dependent manner. Lysates were prepared from KBrO3‐treated and control erythrocytes and assayed for various parameters. KBrO3 treatment caused significant increase in protein oxidation, lipid peroxidation, hydrogen peroxide levels, and decrease in total sulfhydryl content, which indicates induction of oxidative stress in human erythrocytes. Methemoglobin levels and methemoglobin reductase activity were significantly increased while the total antioxidant power of lysates was greatly reduced upon KBrO3 treatment. Intracellular production of reactive oxygen species increased in a dose dependent manner. Exposure of erythrocytes to KBrO3 also caused decrease in the activities of catalase, glutathione peroxidase, thioredoxin reductase, glucose 6‐phosphate dehydrogenase and glutathione reductase whereas the activities of Cu‐Zn superoxide dismutase and glutathione‐S‐transferase were increased. These results show that KBrO3 induces oxidative stress in human erythrocytes through the generation of reactive oxygen species and alters the cellular antioxidant defense system.

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 104 M−1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes.