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.
Introduction: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. Methods and Results: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2-and Th1type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. Conclusion: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.
The insulin−protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn−insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin− protamine complex remains unknown. In this study, it was observed that Zn−insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive ΔC m value (+880 cal mol −1 ), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry (N) value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn−insulin−protamine complex. In fluorescence, Zn−insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.
The real motivation in the present work is to tune the synthesis variables that can result in a highly fluorescent and stable DNA copper nanocluster (CuNC) and also to understand the intricate mechanism behind this process. Here, carefully optimized concentrations of various reactants enabled the creation of a DNA-encapsulated CuNC for AT-DNA, displaying a size of <1.0 nm as confirmed by transmission electron microscopy and dynamic light scattering. The extremely small size of the AT-DNACuNC supports the discrete electronic transitions, also characterized by an exceptionally strong negative circular dichroism (CD) band around 350 nm, whose intensity is well correlated with the observed strong fluorescence emission intensity. This remarkably strong CD can open new applications in the detection and quantification of a specific DNACuNC. Further, timedependent fluorescence analysis suggested stronger photostabilization of these DNACuNCs. The simulation study, based on Cu ion distribution, explained how AT-DNA is a better candidate for NC formation than GC-DNA. In conclusion, the better-tuned synthesis procedure has resulted in a highly compact, well-defined three-dimensional conformation that promotes a more favorable microenvironment to sequester a DNA-based CuNC with high brightness and outstanding photostability.
There is a successful use of protamine-insulin formulation (Zn+insulin+protamine) to treat diabetes in which protamine is added to the stable form of hexameric insulin (Zn-insulin). The biophysical characterization of Zn-insulin, which can dissociate to form biologically active monomers, is well reported. However, its interaction with protamine, which is at the core of the mode of action in many pharmaceutical formulations, is unresolved. Through biophysical characterization, we have tried to dissect the interactions driving the Zn-insulin-protamine complexation. Based on the thermal melting study, it was found that protamine indulges in the destabilization of Zn-insulin. Fluorescence results revealed that Zn-insulin experiences Tyr quenching in the presence of protamine, undergoing a significant conformational change. As shown by the molecular docking study, protamine disturbs the H-bonding network at the dimer interface by binding to the amino acid residues involved in the dimer stabilization. It may result in the freeying of B-chain, introducing conformational fluctuations in the insulin. This is well supported by the loss of helical content seen in circular dichroism. Further, the insulin-protamine complex formation was strongly dominated by hydrogen bonding and a few hydrophobic contacts. The endothermic heat and positive entropy observed in isothermal titration calorimetry in the dissociation of Zn-insulin-protamine is a reflection of that. Finally, the ANS binding study proposed the adaption of a flexible conformation by the Zn-insulin-protamine complex containing exposed hydrophobic residues, a potential arrangement for successful receptor binding.
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