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Core Histones Are Glutaminyl Substrates for Tissue Transglutaminase

Chicken erythrocyte core histones are glutaminyl substrates in the transglutaminase (TGase) reaction with monodansylcadaverine (DNC) as donor amine. The modification is very fast when compared with that of many native substrates of TGase. Out of the 18 glutamines of the four histones, nine (namely glutamine 95 of H2B; glutamines 5, 19, and 125 of H3; glutamines 27 and 93 of H4; and glutamines 24, 104, and 112 of H2A) are the amine acceptors in free histones. The use of Gln112 of H2A requires a temperature-dependent partial unfolding of the histone, showing that structural determinants are decisive for the glutamine specificity. The structures of H2A and H2B do not appreciably change upon modification with DNC as determined by circular dichroism, and core particles reconstituted from these DNC-modified histones are indistinguishable from native nucleosome cores. When the reaction is carried out with native nucleosomes, only glutamines 5 and 19 of H3, which are located in the N-terminal tail, and glutamine 22 of H2B, which is not labeled in free histone, are modified. Methylamine and putrescine also are incorporated into nucleosomes by the TGase reaction. Our results reveal several possibilities for the application of the TGase reaction in the chromatin field, and taking into account that histones are easily cross-linked or modified by polyamines in vitro, the possibility that they may be TGase substrates in vivo is discussed.

Bond-Based 2D Quadratic Fingerprints in QSAR Studies. Virtual and In Vitro Tyrosinase Inhibitory Activity Elucidation

In this report, we show the results of quantitative structure–activity relationship (QSAR) studies of tyrosinase inhibitory activity, by using the bond‐based quadratic indices as molecular descriptors (MDs) and linear discriminant analysis (LDA), to generate discriminant functions to predict the anti‐tyrosinase activity. The best two models [Eqs (6) and (12)] out of the total 12 QSAR models developed here show accuracies of 93.51% and 91.21%, as well as high Matthews correlation coefficients (C) of 0.86 and 0.82, respectively, in the training set. The validation external series depicts values of 90.00% and 89.44% for these best two equations (6) and (12), respectively. Afterwards, a second external prediction data are used to perform a virtual screening of compounds reported in the literature as active (tyrosinase inhibitors). In a final step, a series of lignans is analysed using the in silico‐developed models, and in vitro corroboration of the activity is carried out. An issue of great importance to remark here is that all compounds present greater inhibition values than Kojic Acid (standard tyrosinase inhibitor: IC50 = 16.67 μm). The current obtained results could be used as a framework to increase the speed, in the biosilico discovery of leads for the treatment of skin disorders.

Addressing substrate glutamine requirements for tissue transglutaminase using substance P analogues

We have investigated the effect on the substrate requirements for guinea pig liver (tissue) transglutaminase of a set of 11 synthetic glutamine substitution analogues making up the full sequence of the naturally occurring tissue transglutaminase substrate substance P. While a number of peptide sequences derived from proteins that are well‐recognized as tissue transglutaminase substrates have been studied, the enzyme activity using substitution analogues of full‐length natural substrates has not been investigated as thoroughly. Thus, our set of substance P analogues only differs from one to other by one amino acid mutation while the length (of the peptide) is maintained as in the natural parent peptide. Our results indicate that a glutamine residue is not recognized as substrate by the enzyme whether it is placed at the N‐ or C‐terminal or between two positively charged residues or between two proline residues. To further address the effect on enzyme activity of charged amino acids in the vicinity of the reactive glutamine residue, a new set of synthetic charge replacement analogues of substance P has been also studied. Together, the results have identified new minimal requirements for modification of a particular glutamine residue in a polypeptide chain. It would be of interest to set up a full set of such requirements in order to highlight potential glutamine residues as enzyme targets in the growing list of proteins that are being described as transglutaminase substrates.

Structural characterisation of the natural membrane-bound state of melittin: a fluorescence study of a dansylated analogue.

The binding of a dansylated analogue of melittin (DNC-melittin) to natural membranes is described. The cytolytic peptide from honey bee venom melittin was enzymatically labelled in its glutamine-25 with the fluorescent probe monodansylcadaverine using guinea pig liver transglutaminase. The labelled peptide was characterised functionally in cytolytic assays, and spectroscopically by circular dichroism and fluorescence. The behaviour of DNC-melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. We used resonance energy transfer to measure the state of aggregation of melittin on the membrane plane in synthetic and natural lipid bilayers. When bound to erythrocyte ghost membranes, the extent of energy transfer was found to be equivalent to when bound to small unilamellar vesicles of phosphatidylcholine. Our results correlate best with a proposed model in which the initial interaction between melittin and the red blood cells could be merely electrostatic and the peptide remains in a low alpha-helical conformation. The next step would be a peptide stabilisation in the membrane in a monomeric alpha-helical conformation that would imply the collapse of the membrane structure and liberation of the cell contents.

HPLC study on the ‘history’ dependence of gramicidin A conformation in phospholipid model membranes

A novel HPLC methodology for the study of gramicidin A reconstituted in model membranes has been tested in comparison with circular dichroism data. It is shown that this chromatographic technique not only corroborates most of the recent spectroscopic results but allows one to explain them in terms of mass fractions of different actual conformational species of GA in the phospholipid assemblies. In particular, the dependence of the inserted peptide configuration on the organic solvent and other parameters involved in the ‘history’ of the sample preparation and handling has been analyzed by HPLC in two phospholipid model systems: small unilamellar vesicles and micelles. Moreover, a slow conformational transition of GA towards a β6.3‐helical configuration, accelerated by heat incubation, has been also chromatographically visualized and quantitatively interpreted.

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimerisation motif that mediates transmembrane (TM) helix association, one of the best‐suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Further, we have used this minimised GpA motif to assess the influence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix–helix association and/or dissociation between TM fragments.

Time-dependent monomerization of gramicidin A, enhanced by phosphatidylcholine in non-polar solvents : A high-performance liquid chromatographic and spectrofluorometric study

Abstract The usefulness of size-exclusion high-performance liquid chromatography for the study of gramicidin A dimer—monomer conformational equilibrium in non

New high-performance liquid chromatography-based methodology for monitoring the conformational transitions of self-associating hydrophobic peptides, incorporated into liposomes

A new high-performance size-exclusion chromatographic strategy is reported for the analysis of the hydrophobic self-associating peptide gramicidin A, incorporated into artificial phospholipid vesicles (liposomes). The method is based on the direct injection of a few microlitres of the gramicidin A-containing liposome suspension into the column, which is eluted with a non-polar solvent, such as tetrahydrofuran. The type and amount of information which can be derived from this methodology have been evaluated. Using this chromatographic approach, a correlation has been unambiguously shown to exist between the organization of the peptide in the vesicles and a number of variables involved in the method of preparation of liposomes. Finally, a gramicidin A conformational transition has been monitored in the phospholipid vesicles which proved to be dependent on the class of phospholipid present in the liposome.

Tyrosinase Enzyme: 1. An Overview on a Pharmacological Target

The tyrosinase enzyme (EC 1.14.18.1) is an oxidoreductase inside the general enzyme classification and is involved in the oxidation and reduction process in the epidermis. These chemical reactions that the enzyme catalyzes are of principal importance in the melanogenesis process. This process of melanogenesis is related to the melanin formation, a heteropolymer of indolic nature that provides the different tonalities in the skin and helps to the protection from the ultraviolet radiation. However, a pigment overproduction, come up by the action of the tyrosinase, can cause different disorders in the skin related to the hyperpigmentation. Several studies mainly focused on the characteristics of the enzyme have been reported. In this work, an approximation to general aspects related to this enzyme is made. Besides, it is treated the researches that have been published in the part of the biochemical anatomy dealing with diseases associated with this protein (melanogenesis), its active place and its physiological states, the molecular mechanism, the methods carried out to detect the inhibitory activity, and the used substrates.

Mark–Houwink Parameters of Biosynthetic Poly(γ-glutamic acid) in Aqueous Solution

A combined viscosity–light scattering–gel permeation chromatography (GPC) study was carried out on bacterially produced poly(γ-glutamic acid) (PGGA). PGGA samples with weight-average molecular weights ranging from 8×104 up to 8×105 g·mol–1 dissolved in phosphate buffer at 0.13 M ionic strength were used. It was found that the Mark–Houwink relation is acceptably obeyed, giving K and a values of 1.84×10–6 dL·g–1 and 1.16, respectively. As expected, GPC analysis showed that PGGA does not follow the universal calibration plot and that deviations can not be avoided by modifying the ionic strength.