Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase.

Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.

Investigating Chaperone like Activity of Green Silver Nanoparticles: Possible Implications in Drug Development.

Protein aggregation and amyloidogenesis have been associated with several neurodegenerative disorders like Alzheimer's, Parkinson's etc. Unfortunately, there are still no proper drugs and no effective treatment available. Due to the unique properties of noble metallic nanoparticles, they have been used in diverse fields of biomedicine like drug designing, drug delivery, tumour targeting, bio-sensing, tissue engineering etc. Small-sized silver nanoparticles have been reported to have anti-biotic, anti-cancer and anti-viral activities apart from their cytotoxic effects. The current study was carried out in a carefully designed in-vitro to observe the anti-amyloidogenic and inhibitory effects of biologically synthesized green silver nanoparticles (B-AgNPs) on human serum albumin (HSA) aggregation taken as a model protein. We have used different biophysical assays like thioflavin T (ThT), 8-Anilino-1-naphthalene-sulphonic acid (ANS), Far-UV CD etc. to analyze protein aggregation and aggregation inhibition in vitro. It has been observed that the synthesized fluorescent B-AgNPs showed inhibitory effects on protein aggregation in a concentration-dependent manner reaching a plateau, after which the effect of aggregation inhibition was significantly declined. We also observed meaningful chaperone-like aggregation-inhibition activities of as-synthesized florescent B-AgNPs in astrocytes.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Biophysical insight reveals tannic acid as amyloid inducer and conformation transformer from amorphous to amyloid aggregates in Concanavalin A (ConA).

The aggregation phenomenon (amyloid and amorphous) is associated with several pathological complications in human, such as Alzheimer's, Parkinson's, Huntington, Cataract diseases, and Diabetes mellitus type 2. In the present study we are offering evidence and breaking the general belief with regard to the polyphenols action as protein aggregate inhibitors. Herein we confirm that tannic acid (TA) is not only an amyloid inducer, but also it switches one type of conformation, ultimately morphology, into another. We ascertain based on our findings that aggregates are not rigid structures and the stability can be challenged under certain conditions. This study also confirms that unfolded and amorphous aggregates can serve as precursors of amyloids and TA interactions with unordered aggregates (amorphous) bringing orderliness in the conformation via amyloidosis. The shifting of unordered conformation toward orderliness is governed by the modulation in surface hydrophobic patches in Concanavalin A (ConA). Hence, a degree of exposed hydrophobic cluster can be claimed as a strong parameter to detect and distinguish the native, amorphous and both types of amyloids. Turbidity and Rayleigh light scattering measurements followed similar pattern while Thioflavin T and 1-anilino-8-naphthalene sulfonate fluorescence assays of the binding with amorphous and amyloid followed an inverse relation. Electron microscopic studies revealed the morphological variation in the ConA at 65°C as amorphous while the ConA treated with TA followed by heat treatment at 65°C was defined as amyloid in nature. Interestingly for the first time we are reporting the slight agglutination activity by the ConA amyloids.

Biophysical insight into the interaction mechanism of plant derived polyphenolic compound tannic acid with homologous mammalian serum albumins.

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼104 to 106M-1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in ß-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.

Polyols (Glycerol and Ethylene glycol) mediated amorphous aggregate inhibition and secondary structure restoration of metalloproteinase-conalbumin (ovotransferrin).

Under physical or chemical stress, proteins tend to form aggregates either highly ordered (amyloid) or unordered (amorphous) causing many pathological disorders in human and loss of proteins functionality in both laboratory conditions and industries during production and storage at commercial level. We investigated the effect of increasing temperature on Conalbumin (CA) and induced aggregation at 65°C. The enhanced Thioflavin T (ThT) and ANS (1-anilinonaphtalene 8-sulfonic acid) fluorescence intensity, show no shift on Congo red binding, additionally, transmission and scanning electron microscopy (TEM) (SEM) reveal amorphous morphology of the aggregate. Our investigation clearly demonstrated that polyols namely Glycerol (GL) and Ethylene glycol (EG) are so staunch to inhibit amorphous aggregates via restoring secondary conformation. Addition of polyols (15% GL and 35% EG) significantly decrease the turbidity, Rayleigh scattering ThT and ANS fluorescence intensity. The dynamic light scattering (DLS) data show that hydrodynamic radii (Rh) of the aggregates is ∼20 times higher than native CA while nearly similar for GL and EG protected CA due to condensation of core size with little difference.

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study.

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.

Secondary structural changes in guanidinium hydrochloride denatured mammalian serum albumins and protective effect of small amounts of cationic gemini surfactant pentanediyl-α,ω-bis(cetyldimethylammonium bromide) and methyl-ß-cyclodextrin: A spectroscopic study.

In the present study the cationic gemini surfactant assisted refolding of guanidinium hydrochloride (GdCl) denatured mammalian serum albumins viz. sheep serum albumin (SSA), rat serum albumin (RSA) and porcine serum albumin (PSA) using a combination of cationic gemini surfactants, pentanediyl-α,ω-bis(cetyldimethylammonium bromide) (C16H33(CH3)2N(+)-(CH2)5-N(+)(CH3)2C16H33)⋅2Br(-) designated as G5 and methyl-ß-cyclodextrin in the artificial chaperone assisted two step method, is attempted. The studies were carried out in an aqueous medium (pH 7.4) using dynamic light scattering (DLS), circular dichroism (CD), and fluorescence spectroscopy. A perusal of DLS data indicates that against the native hydrodynamic radius (Rh) of 4.3nm in SSA, 3.9nm in PSA and 3.5nm in RSA, the Rh of the said proteins, when refolding is attempted by simple dilution, increases to 21.7nm, 36.6nm and 37.2nm, respectively. Hydrodynamic radii very near to the native protein, i.e., 4.0nm, 4.1nm and 4.4nm for RSA, PSA and SSA respectively, is obtained on the sequential addition of G5 and methyl-ß-cyclodextrin to the denatured protein. Circular dichroism studies corroborate with the DLS data. The results obtained from the multi-technique approach are ascribed to the presence of two charged head-groups and two hydrocarbon tails in the gemini surfactants resulting in a very strong electrostatic and hydrophobic interactions. Based on the present study it is suggested that the gemini surfactants may be utilized in the protein refolding studies and thus may address one of the most pressing demand of biotechnology industry for the development of efficient and inexpensive folding aides.

Interaction of 5-fluoro-5'-deoxyuridine with human serum albumin under physiological and non-physiological condition: a biophysical investigation.

Uridine analogs 5'dFUrd (a cytotoxic metabolite of a prodrug capecitabine that enzymatically converted into 5'dFUrd) commonly used in the treatment of advanced human cancers, especially gastrointestinal tract, ovary, colorectal, breast cancers etc. Drugs/metabolites are transported in the blood by transporter proteins like human serum albumin (HSA). Here we investigate the interaction of 5'dFUrd to HSA by spectroscopic and calorimetric techniques at physiological (pH 7.4) and non-physiological (pH 9.0) conditions. The binding constant (Kb), enthalpy change (ΔH°), entropy change (ΔS°) and Gibbs free energy change (ΔG°) were also calculated under both conditions. The secondary structure of HSA showed greater alteration in helicity at physiological pH. ITC measurement reveals that HSA have high binding affinity at physiological pH as compares to non-physiological conditions. The thermostability of HSA alone as well of the HSA-drug complex was found to be higher at physiological pH. The binding study was also explored through molecular docking studies which revealed that 5'dFUrd was bound to subdomain IIA of Sudlow's site I through multiple mode of interaction. These results suggest that 5'dFUrd have high binding affinity at physiological condition or "N" isoform so lower drug concentrations are required in compare to non-physiological or "B" isoform of HSA to completely occupied the binding site of the protein.

Interaction of the 5-fluorouracil analog 5-fluoro-2'-deoxyuridine with 'N' and 'B' isoforms of human serum albumin: a spectroscopic and calorimetric study.

Drugs and metabolites are transported in the blood by plasma proteins, such as human serum albumin (HSA). The uridine analog 2'dFUrd, which is a cytotoxic prodrug metabolite of capecitabine, has remarkable activity against solid tumors when administered orally. We report the results of an in vitro experimental study on the interactions of 2'-dFUrd with the N-isoform (at pH 7.4) and B-isoform (at pH 9.0) of HSA, investigated using fluorescence spectroscopy, circular dichroism (CD), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and molecular docking. The binding constant (Kb) was higher for the N-isoform than for the B-isoform. Thermodynamic parameters, such as enthalpy change (ΔH°), entropy change (ΔS°), and Gibbs free energy change (ΔG°), were also calculated for both isoform interactions using calorimetric techniques. The thermostabilities of HSA and the HSA-2'dFUrd complex were found to be higher for the N-isoform. The interaction of 2'dFUrd with HSA was also explored in molecular docking studies, which revealed that 2'dFUrd was bound to the Sudlow site I in subdomain IIA through multiple modes of interaction, such as hydrophobic interactions and hydrogen bonding. These results suggest that 2'dFUrd has higher binding affinity for the N-isoform of HSA.