Non-enzymatic glycation of human angiogenin: Effects on enzymatic activity and binding to hRI and DNA.

The effects of non-enzymatic glycation on the structural and functional properties of human angiogenin (hAng) have been investigated with respect to the formation of advanced glycated end products (AGEs), on prolonged treatment with d-Glucose, d-Fructose and d-Ribose at 37 °C. Fluorescence studies show the formation of fluorescent AGEs which exhibit emission maxima at 406 nm and 435 nm. Glycation of hAng with ribose leads to the maximum loss of its functional characteristic properties, as compared to fructose and glucose, along with the formation of higher oligomers. An increase in the incubation time results in the formation of higher oligomers with a concomitant decrease in the ribonucleolytic activity. The increase in the hydrodynamic radii of the glycated samples compared to native hAng is indicative of structural perturbations. The ribonucleolytic activity and the DNA binding ability of glycated hAng has been investigated by an agarose gel-based assay. Glycated hAng was unable to bind with human placental ribonuclease inhibitor (hRI), otherwise known to form one of the strongest protein-protein interaction systems with an affinity in the femtomolar range.

Thermodynamics of the Association of Aminoglycoside Antibiotics with Human Angiogenin.

BACKGROUND: The body needs to maintain a firm balance between the inducers and inhibitors of angiogenesis, the process of proliferation of blood vessels from pre-existing ones. Human angiogenin (hAng), being a potent inducer of angiogenesis, is a cause of tumor cell proliferation, therefore its inhibition becomes a vital area of research. Aminoglycosides are linked ring systems consisting of amino sugars and an aminocyclitol ring and are in use in clinical practices for a long time. These compounds have found clinical uses as antibacterial agents that inhibit bacterial protein synthesis. OBJECTIVE: Gentamycin C1, Kanamycin A, Neomycin B, Paromomycin I, and Streptomycin A are commonly used aminoglycoside antibiotics that have been used for the present study. Among these, Neomycin has reported inhibitory activity against angiogenin-induced angiogenesis on the chicken chorioallantoic membrane. This study focuses on the thermodynamic parameters involved in the interactions of these antibiotics with hAng. METHODS: Agarose gel-based assay, Fluorescence quenching studies and Docking studies. RESULTS: Anti-ribonucleolytic effect of the antibiotics was observed qualitatively using an agarose gelbased assay, which shows that Neomycin exhibits the most efficient inhibition of hAng. Fluorescence quenching studies at different temperatures, using Stern-Volmer and van't Hoff equations provide information about the thermodynamics of binding, which furthermore highlights the higher binding constant of Neomycin. Docking studies showed that the antibiotics preferably interact with the nuclear translocation site, except Streptomycin, which shows affinity towards the ribonucleolytic site of the protein with very less affinity value. CONCLUSION: The study has shown the highly spontaneous formation of Neomycin-hAng complex, giving an exothermic reaction with increase in the degree of freedom of the protein-ligand complex.

ß-cyclodextrin encapsulation of curcumin elicits an altered mode of angiogenin inhibition: In vitro and in vivo studies.

The interaction of curcumin (Cur) with human angiogenin (hAng), a potent blood vessel inducer responsible for angiogenesis is found to change following encapsulation within the ß-cyclodextrin (ßCD) cavity. The enhanced bioavailability and increase in the binding stoichiometry of hAng:Cur-ßCD (1:2) leads to increased affinity, hence an increase in the association constant. The altered mode of hAng inhibition of Cur from a non-competitive (KI = 23.7 ± 2.2 µM) to a mixed type (KI = 19.8 ± 1.4 µM), after encapsulation provides an insight into interaction patterns. Isothermal titration calorimetry (ITC) experiments indicate the formation of multiple favorable non-covalent interactions (also confirmed by docking studies), which implies negative enthalpy changes (-ΔHo) and restriction in the dynamic mobility of the free protein molecule resulting in a very less positive entropy change (TΔSo). This leads to a medium magnitude for the spontaneous free energy change associated with the interaction/binding process. The spontaneity of binding indicates a more favorable value for the Cur-ßCD (ΔGo = -7.75 kcal/mol) compared to Cur (ΔGo = -7.49 kcal/mol). In vivo studies also demonstrate the anti-angiogenic effect of Cur/Cur-ßCD confirmed by the significant decrease in blood vessel density and branching index.

Enhancement of angiogenin inhibition by polyphenol-capped gold nanoparticles.

Angiogenin (Ang), is a ribonucleolytic protein that is associated with angiogenesis, the formation of blood vessels. The involvement of Ang in vascularisation makes it a potential target for the identification of compounds that have the potential to inhibit the process. The compounds may be assessed for their ability to inhibit the ribonucleolytic activity of the protein and subsequently blood vessel formation, a crucial requirement for tumor formation. We report an inhibition of the ribonucleolytic activity of Ang with the gallate containing green tea polyphenols, ECG and EGCG that exhibits an increased efficacy upon forming polyphenol-capped gold nanoparticles (ECG-AuNPs and EGCG-AuNPs). The extent of inhibition was confirmed using an agarose gel-based assay followed by fluorescence titration studies that indicated a hundred fold stronger binding of polyphenol-capped gold nanoparticles (GTP-AuNPs) compared to the bare polyphenols. Interestingly, we found a change in the mode of inhibition from a noncompetitive type to a competitive mode of inhibition in case of the GTP-AuNPs, which is in agreement with the 'n' values obtained from the fluorescence quenching studies. The effect on angiogenesis has also been assessed by the chorioallantoic membrane (CAM) assay. We find an increase in the inhibition potency of GTP-AuNPs that could find applications in the development of anti-angiogenic compounds.

Positional preferences in flavonoids for inhibition of ribonuclease A: Where "OH" where?

Flavonoids are a class of polyphenols that possess diverse properties. The structure-activity relationship of certain flavonoids and resveratrol with ribonuclease A (RNase A) has been investigated. The selected flavonoids have a similar skeleton and the positional preferences of the phenolic moieties toward inhibition of the catalytic activity of RNase A have been studied. The results obtained for RNase A inhibition by flavonoids suggest that the planarity of the molecules is necessary for effective inhibitory potency. Agarose gel electrophoresis and precipitation assay experiments along with kinetic studies reveal Ki values for the various flavonoids in the micromolar range. Minor secondary structural changes of RNase A were observed after interaction with the flavonoids. An insight into the specific amino acid involvement in the binding of the substrate using docking studies is also presented. The dipole moment of the flavonoids that depends on the orientation of the hydroxyl groups in the molecule bears direct correlation with the inhibitory potency against RNase A. The direct association of this molecular property with enzyme inhibition can be exploited for the design and development of inhibitors of proteins.

pH-Dependent Nitrotyrosine Formation in Ribonuclease A is Enhanced in the Presence of Polyethylene Glycol (PEG).

Protein nitration can occur as a result of peroxynitrite-mediated oxidative stress. Excess production of peroxynitrite (PN) within the cellular medium can cause oxidative damage to biomolecules. The in vitro nitration of Ribonuclease A (RNase A) results in nitrotyrosine (NT) formation with a strong dependence on the pH of the medium. In order to mimic the cellular environment in this study, PN-mediated RNase A nitration has been carried out in a crowded medium. The degree of nitration is higher at pH 7.4 (physiological pH) compared to pH 6.0 (tumor cell pH). The extent of nitration increases significantly when PN is added to RNase A in the presence of crowding agents PEG 400 and PEG 6000. PEG has been found to stabilize PN over a prolonged period, thereby increasing the degree of nitration. NT formation in RNase A also results in a significant loss in enzymatic activity.