A quercetin-based flavanoid (rutin) reverses amyloid fibrillation in ß-lactoglobulin at pH 2.0 and 358 K.

ß-lactoglobulin (BLG) is a well characterized milk protein and a model for folding and aggregation studies. Rutin is a quercetin based-flavanoid and a famous dietary supplement. It is a potential protector from coronary heart disease, cancers, and inflammatory bowel disease. In this study, amyloid fibrillation is reported in BLG at pH 2.0 and temperature 358 K. It is inhibited to some extent by rutin with a rate of 99.3 h-1 M-1. Amyloid fibrillation started taking place after 10 h of incubation and completed near 40 h at a rate of 16.6 × 10-3 h-1, with a plateau during 40-108 h. Disruption of tertiary structure of BLG and increased solvent accessibility of hydrophobic core seem to trigger intermolecular assembly. Increase in 7% ß-sheet structure at the cost of 10% α-helical structures and the electron micrograph of BLG fibrils at 108 h further support the formation of amyloid. Although it could not block amyloidosis completely, and even the time required to reach plateau remains the same, a decrease of growth rate from 16.6 × 10-3 to 13.5 × 10-3 h-1 was observed in the presence of 30.0 µM rutin. Rutin seems to block solvent accessibility of the hydrophobic core of BLG. A decrease in the fibril population was observed in electron micrographs, with the increase in rutin concentration. All evidences indicate reversal of fibrillation in BLG in the presence of rutin.

Unraveling the molecular mechanism of the effects of sodium dodecyl sulfate, salts, and sugars on amyloid fibril formation in camel IgG.

Sodium dodecyl sulfate (SDS) is an anionic surfactant that can be used to stimulate protein fibrillation in vitro. Here, we investigated the effects of SDS on camel IgG aggregation at pH 3.5 and 7.4. SDS-induced amyloid fibril formation in camel IgG was examined by turbidity measurements, Rayleigh scattering, Thioflavin T (ThT) fluorescence, intrinsic fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM). The results suggest that low SDS concentrations (0.2-2.0 mM) induce amyloid-like aggregates of camel IgG at pH 3.5, indicating an SDS/camel IgG ratio below 1000. However, in the presence of higher concentrations of SDS (2.5-10.0 mM), amyloid fibril formation was not observed. Furthermore, at the higher concentrations, the ß-sheet structure of camel IgG was transformed into a α-helical structure. The amyloid fibril formation was not observed in the presence of SDS at pH 7.4. Additionally, the role of salts and sugars was evaluated in the SDS-induced aggregation process. Interestingly, in the presence of 0.15 N of NaCl and (NH4)2SO4, SDS promoted camel IgG aggregation up to very high concentrations of SDS (0.2-10.0 mM; SDS/camel IgG ratio, 95-4750) and no suppression was observed. Moreover, osmoprotectants (trehalose and sucrose) were ineffective, neither promoting nor inhibiting the SDS-induced aggregation process. However, at pH 3.5, electrostatic and hydrophobic interactions, and hydrogen bonds were the major contributing factors in SDS-induced fibrillation. However, no aggregation was observed at pH 7.4 due to electrostatic repulsion between SDS and camel IgG because both of these molecules have overall similar charges.

BA9 lineage of respiratory syncytial virus from across the globe and its evolutionary dynamics.

Respiratory syncytial virus (RSV) is an important pathogen of global significance. The BA9 is one of the most predominant lineages of the BA genotype of group B RSV that has acquired a 60bp duplication in its G protein gene. We describe the local and global evolutionary dynamics of the second hyper variable region in the C- terminal of the G protein gene of the BA9 lineage. A total of 418 sequences (including 31 study and 387 GenBank strains) from 29 different countries were used for phylogenetic analysis. This analysis showed that the study strains clustered with BA (BA9 and BA8) and SAB4 genotype of group B RSV. We performed time-scaled evolutionary clock analyses using Bayesian Markov chain Monte Carlo methods. We also carried out glycosylation, selection pressure, mutational, entropy and Network analyses of the BA9 lineage. The time to the most recent common ancestor (tMRCA) of the BA genotype and BA9 lineage were estimated to be the years 1995 (95% HPD; 1987-1997) and 2000 (95% HPD; 1998-2001), respectively. The nucleotide substitution rate of the BA genotype [(4.58×10-3 (95% HPD; 3.89-5.29×10-3) substitution/site/year] was slightly faster than the BA9 lineage [4.03×10-3 (95% HPD; 4.65-5.2492×10-3)]. The BA9 lineage was categorized into 3 sub lineages (I, II and III) based on the Bayesian and Network analyses. The local transmission pattern suggested that BA9 is the predominant lineage of BA viruses that has been circulating in India since 2002 though showing fluctuations in its effective population size. The BA9 lineage established its global distribution with report from 23 different countries over the past 16 years. The present study augments our understanding of RSV infection, its epidemiological dynamics warranting steps towards its overall global surveillance.

Global distribution of NA1 genotype of respiratory syncytial virus and its evolutionary dynamics assessed from the past 11 years.

Respiratory syncytial virus (RSV) is a potent pathogen having global distribution. The main purpose of this study was to gain an insight into distribution pattern of the NA1 genotype of group A RSV across the globe together with its evolutionary dynamics. We focused on the second hypervariable region of the G protein gene and used the same for Phylogenetic, Bayesian and Network analyses. Eighteen percent of the samples collected from 500 symptomatic pediatric patients with acute respiratory tract infection (ARI) were found to be positive for RSV during 2011-15 from New Delhi, India. Of these, group B RSV was predominant and clustered into two different genotypes (BA and SAB4). Similarly, group A viruses clustered into two genotypes (NA1 and ON1). The data set from the group A viruses included 543 sequences from 23 different countries including 67 strains from India. The local evolutionary dynamics suggested consistent virus population of NA1 genotype in India during 2009 to 2014. The molecular clock analysis suggested that most recent common ancestor of group A and NA1 genotype have emerged in during the years 1953 and 2000, respectively. The global evolutionary rates of group A viruses and NA1 genotype were estimated to be 3.49 × 10-3 (95% HPD, 2.90-4.17 × 10-3) and 3.56 × 10-3 (95% HPD, 2.91 × 10-3-4.18 × 10-3) substitution/site/year, respectively. Analysis of the NA1 genotype of group A RSV reported during 11 years i.e. from 2004 to 2014 showed its dominance in 21 different countries across the globe reflecting its evolutionary dynamics. The Network analysis showed highly intricate but an inconsistent pattern of haplotypes of NA1 genotype circulating in the world. Present study seems to be first comprehensive attempt on global distribution and evolution of NA1 genotype augmenting the optimism towards the vaccine development.

Unveiling the stimulatory effects of tartrazine on human and bovine serum albumin fibrillogenesis: Spectroscopic and microscopic study.

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.

Negatively charged food additive dye "Allura Red" rapidly induces SDS-soluble amyloid fibril in beta-lactoglobulin protein.

Recent studies have led to an increased interest to categorize small molecular inhibitors of protein fibrillation. In this study, we used spectroscopy, microscopy and gel electrophoresis techniques that provides an elaborated description of the Allura Red-induced amyloid fibrillation in the ß-LG protein at two pHs (7.4 and 3.5). The spectroscopy results show that ß-LG protein form aggregates in the presence of Allura Red (0.04-15.0mM) at pH 3.5 due to electrostatic and hydrophobic interactions. However, at pH 7.4, the ß-LG does not interact electrostatically with Allura Red and therefore no aggregation occurred. The Allura Red-induced aggregates have an amyloid-like structure that was confirmed by far-UV CD, Congo Red and transmission electron microscopy (TEM). The CD spectrum of ß-LG contains single minima at ∼218nm, which shifts towards higher wavelength minima at ∼225nm in the presence of Allura Red, characteristics of the cross ß-sheet structure. The TEM results suggest that ß-LG form long straight fibril when exposed to Allura Red at pH 3.5. The Allura Red-induced amyloid fibril is SDS-soluble confirmed by SDS-PAGE techniques. A far UV CD result shows the conversion of Allura Red induced cross ß-sheet structure into alpha-helical structure in the presence of increasing concentration of SDS. The results of this study suggest that the electrostatic, as well as hydrophobic interactions play an important role during Allura Red-induced ß-LG fibrillation.

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis.

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA-ANS complex. Binding of ANS to BSA showed strong binding (Ka = 4.4 µM) with native conformation in comparison to glycated state (Ka = 8.4 µM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (Ka = 23 µM) for glycated albumin compared with native state (Ka = 16 µM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow's site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.

Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis.

Human mortalin is a Hsp70 mitochondrial protein that plays an essential role in the biogenesis of mitochondria. The deregulation of mortalin expression and its functions could lead to several age-associated disorders and some types of cancers. In the present study, we optimized the expression and purification of recombinant human mortalin by the use of two-step chromatography. Low temperature (18°C) and 0.5mM (IPTG) was required for optimum mortalin expression. Chaperone activity of mortalin was assessed by the citrate synthase and insulin protection assay, which suggested their protective role in mitochondria. Folding and unfolding assessments of mortalin were carried out in the presence of guanidine hydrochloride (GdnHCl) by intrinsic fluorescence measurement, ANS (8-analino 1-nephthlene sulfonic acid) binding and CD (circular dichroism) analysis. Under denaturing conditions, mortalin showed decrease in tryptophan fluorescence intensity along with a red shift of 11nm. Moreover, ANS binding studies illustrated decrease in hydrophobicity. CD measurement of mortalin showed a predominant helical structure. However, the secondary structure was lost at low concentration of GdnHCl (1M). We present a simple and robust method to produce soluble mortalin and warranted that chaperones are also susceptible to unfolding and futile to maintain protein homeostasis.

Spectral and thermal properties of novel eye lens ζ-crystallin.

Eye lenses are exposed to thermal, solar radiations, dryness that enhances cataractogenesis. Some animal lenses contain novel proteins in bulk quantities. ζ-crystallin occurred in three ecologically divergent species, but it's physiological role not known. The truncated variant of ζ-crystallin causes hereditary cataract. Guinea pig ζ-crystallin is temperature-sensitive and rapidly aggregates at 41°C. Camels adopted to survive above 50°C, which raises an interesting question about how it retains lens proteins in the soluble state? Here, we have optimized expression and purification of recombinant camel ζ-crystallin. We have studied thermodynamic and spectroscopic properties using orthogonal techniques. Dynamic multimode spectroscopy results showed that camel ζ-crystallin unfolds via single transition with Tm value of 60.8±0.1°C and van't Hoff enthalpy of 714.7±7.1kJ/mol. Thermal-shift assay calculates Tm value of 62°C at pH 7. Additionally, the conformational stability of ζ-crystallin increases with ionic-strength. The influence of pH on ζ-crystallin was evaluated where the protein was found to be stable in the pH range of 6-9, but its stability drastically decreases below pH 6. Our results also showed that quaternary structure of ζ-crystallin drastically changed as a result of lowering pH. This study provides significant understandings onto the conformational, thermodynamic and unfolding pathway of camel ζ-crystallin.

Camelus dromedarius glucose transporter 4: in silico analysis, cloning, expression, purification and characterisation in E. coli.

Camels have exceptional carbohydrate metabolism as their plasma glucose level is high and have low whole body insulin sensitivity, similar to that observed in type 2 diabetes patients. We aimed at studing an important component of insulin signalling pathway, the GLUT4, in camel. Camelus dromedarius GLUT4 (CdGLUT4) CDS is 1530 nucleotide in length that encodes for a 55KDa protein. CdGLUT4 has 23 amino acid substitutions and 3N-glycosylation sites, compared to 2 in Human GLUT4. 3 D structures of CdGLUT4 and HsGLUT4 generated by homology modelling revealed conservation of characteristic signature motifs. CdGLUT4 was cloned and expressed optimally in C43(DE3)pLysS strain and maximum detergent solubility was observed in n-Dodecyl-ß-d-maltopyranoside. These preliminary data provide information on residual differences between CdGLUT4 and HsGLUT4 that may be responsible for camel's unique glucose metabolism. These differences are postulated to assist in designing and development of efficacious GLUT4 that might help in management of diabetic patients.

Co-Circulation of 72bp Duplication Group A and 60bp Duplication Group B Respiratory Syncytial Virus (RSV) Strains in Riyadh, Saudi Arabia during 2014.

Respiratory syncytial virus (RSV) is an important viral pathogen of acute respiratory tract infection (ARI). Limited data are available on molecular epidemiology of RSV from Saudi Arabia. A total of 130 nasopharyngeal aspirates were collected from children less than 5 years of age with ARI symptoms attending the Emergency Department at King Khalid University Hospital and King Fahad Medical City, Riyadh, Saudi Arabia between October and December, 2014. RSV was identified in the 26% of the hospitalized children by reverse transcriptase PCR. Group A RSV (77%) predominated during the study as compared to group B RSV (23%). The phylogenetic analysis of 28 study strains clustered group A RSV in NA1 and ON1 genotypes and group B viruses in BA (BA9) genotype. Interestingly, 26% of the positive samples clustered in genotypes with duplication in the G protein gene (ON1 for group A and BA for group B). Both the genotypes showed enhanced O-linked glycosylation in the duplicated region, with 10 and 2 additional sites in ON1 and BA respectively. Selection pressure analysis revealed purifying selection in both the ON1 and BA genotypes. One codon each in the ON1 (position 274) and BA genotypes (position 219) were positively selected and had high entropy values indicating variations at these amino acid positions. This is the first report describing the presence of ON1 genotype and the first report on co-circulation of two different genotypes of RSV with duplication in the G protein gene from Saudi Arabia. The clinical implications of the simultaneous occurrence of genotypes with duplication in G protein gene in a given population especially in the concurrent infections should be investigated in future. Further, the ongoing surveillance of RSV in this region will reveal the evolutionary trajectory of these two genotypes with duplication in G protein gene from largest country in the Middle East.

Glycation Induced Generation of Amyloid Fibril Structures by Glucose Metabolites.

The non-enzymatic reaction (glycation) of reducing sugars with proteins has received increased interest in dietary and therapeutic research lately. In the present work, the impact of glycation on structural alterations of camel serum albumin (CSA) by different glucose metabolites was studied. Glycation of CSA was evaluated by specific fluorescence of advanced glycation end-products (AGEs) and determination of available amino groups. Further, conformational changes in CSA during glycation were also studied using 8-analino 1-nephthlene sulfonic acid (ANS) binding assay, circular dichroism (CD) and thermal analysis. Intrinsic fluorescence measurement of CSA showed a 22 nm red shift after methylglyoxal treatment, suggesting glycation induced denaturation of CSA. Rayleigh scattering analysis showed glycation induced turbidity and aggregation in CSA. Furthermore, ANS binding to native and glycated-CSA reflected perturbation in the environment of hydrophobic residues. However, CD spectra did not reveal any significant modifications in the secondary structure of the glycated-CSA. Thioflavin T (ThT) fluorescence of CSA increased after glycation, illustrated cross ß-structure and amyloid formation. Transmission electron microscopy (TEM) analysis further reaffirms the formation of aggregate and amyloid. In summary, glucose metabolites induced conformational changes in CSA and produced aggregate and amyloid structures.

Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli.

The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 µM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.

Spectroscopic and thermodynamic properties of recombinant heat shock protein A6 from Camelus dromedarius.

Heat shock protein A6, also known as HSP70B', is a member of the Hsp70 family of molecular chaperones. Under stressed conditions, the level of HSPA6 increases substantially, and the protein has been targeted as a biomarker of cellular stress in several studies. We report the spectroscopic and thermodynamic properties of Arabian camel species cHSPA6, determined by measurement of intrinsic and extrinsic fluorescence emission, and use of far-UV circular dichroism and dynamic multimode spectroscopy. Our results showed that cHSPA6 has similar binding affinity for both ATP and ADP (K D = ~50 nM). Binding of ATP and ADP reduced the surface hydrophobicity of the protein, and slightly altered its secondary structure, suggesting localized conformational rearrangement after ATP or ADP binding. Dynamic multimode spectroscopy revealed that cHSPA6 unfolds through three transitions with melting points (T m) of 42.3 ± 0.2, 61.3 ± 0.1, and 81.2 ± 0.2 °C. To the best of the author's knowledge, and literature search, this is the first report of the spectroscopic and thermodynamic properties of the Arabian camel heat shock protein.

Benzo(a)pyrene induced structural and functional modifications in lung cystatin.

Cystatins are thiol proteinase inhibitors ubiquitously present in the mammalian body. They serve a protective function to regulate the activities of endogenous proteinases, which may cause uncontrolled proteolysis and damage. In the present study, the effect of benzo(a)pyrene [BaP] on lung cystatin was studied to explore the hazardous effects of environmental pollutant on structural and functional integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR, and fluorescence spectroscopy. The enhancement of total protein fluorescence with a red shift of 5 nm suggests structural scratch of lung cystatin by benzo(a)pyrene. Further, ANS binding studies reaffirm the unfolding of the thiol protease inhibitor (GLC-I) after treating with benzo(a)pyrene. The results of FT-IR spectroscopy reflect perturbation of the secondary conformation (alpha-helix to ß-sheet) in goat lung cystatin on interaction with BaP. Finally, functional inactivation of cystatin on association with BaP was checked by its papain inhibitory activity. Benzo(a)pyrene (10 µM) caused complete inactivation of goat lung cystatin. Benzo(a)pyrene-induced loss of structure and function in the thiol protease inhibitor could provide a caution for lung injury caused by the pollutants and smokers.