A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana.

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines.

Non-peptide molecules in the pedicellariae of Toxopneustes roseus.

Toxopneustes roseus is a species of sea urchin with a wide distribution along the eastern Pacific coast. It belongs to the Toxopneustidae family and, like its members, has well-developed globiferous pedicellariae that exert a variety of pharmacological actions. We identified six volatile non-peptide molecules from its globiferous pedicellariae by using GC-MS and RP-HPLC-MS/MS, including: benzoic acid; 2-aminoethanol (MEA); 2-(dimethylamine) ethanol (DMAE); 1- (4-bromophenyl)-1-phenylethanol (BPPE); 2-[1-(4-bromophenyl)-1- phenylethoxy]-N,N-dimethylethanamine (EMB); and 2-[1-(4-chlorphenyl)-1- phenylethoxy]-N,N-dimethylethanamine (CLX). The construction of a pharmacophore model and the in silico molecular docking of EMB and CLX into the human voltage-gated sodium channel hNaV1.7 allowed establishing that these molecules are structurally similar to local anesthetics and other NaV channel blockers and can bind to the same site receptor in NaV channels; suggesting that both molecules are active components in T. roseus venom. Furthermore, a viable endogenous biopathway is proposed in which T. roseus can synthesize EMB and CLX from benzoic acid, MEA, DMAE, and BPPE as their precursors, which would emphasize the importance of these molecules in the metabolism of this sea urchin.

Detection and characterization by circular dichroism of a stable intermediate state formed in the thermal unfolding of papain.

The thermal unfolding of papain was studied at pH 2.6 by means of circular dichroism and difference spectroscopy. The transition curves obtained from ellipticity changes at 208 and 220 nm were biphasic, i.e., they showed two distinct successive steps, demonstrating the existence of an intermediate state of stable secondary conformation in the denaturation process. Difference-spectroscopy studies indicated that considerable exposure of aromatic side-chains is involved in both steps of the transition. Since papain has two domains in its molecular structure, our results suggest that they unfold in a successive way and rather independently. Furthermore, the structural characteristics of the intermediate state, obtained from its circular dichroism spectrum in the far-ultraviolet region, seem to point out that the second domain (residues 111-212) is the most stable part of the molecule.

Stability of the C-terminal peptide of CETP mediated through an (i, i + 4) array.

Based on circular dichroism (CD), we have found an essential (i, i + 4) alpha-helix stabilizing array in the C-terminus region for the cholesteryl ester transfer protein (CETP) between histidine 466 and aspartic acid 470. This region apparently corresponds to an amphipathic alpha-helix. The behavior of this peptide in solution in comparison with a mutant peptide (D470N) was also analyzed by dynamic light scattering (DLS). The results showed that alpha-helix stabilization is not due to peptide aggregation. The thermodynamic estimation of stability supports the idea that the phenomenon is carried out through an (i, i + 4) array. The representation of the C-terminal region as an amphipathic alpha-helical peptide shows that lipid-binding activity might be in part due to both the asymmetric polar/non-polar residue distribution and to the presence of an (i, i + 4) array important for helix stability.

Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes.

The aligned amino acid sequences of TIM from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a positional identity of 68%. The two enzymes have markedly similar catalytic properties. Agents that interact with their interface Cys inhibit TcTIM and TbTIM; and those TIMs that lack this Cys (such as human TIM) are largely or completely insensitive to these agents. The susceptibility of TcTIM to the agents is approximately 100 times higher than that of TbTIM. To ascertain the cause of this large difference, the crystal structure of TcTIM was solved at 1.83 A resolution. The two enzymes are very similar homodimers. In TcTIM and TbTIM their respective Cys, 15 or 14, forms part of the dimer interface. In both, the contacts of the Cys with residues of the other subunit are almost identical. Nevertheless, there are noteworthy differences between the two; the existence of glutamine 18 in TbTIM instead of glutamic acid in TcTIM at the beginning of helix 1 decreases the contacts between this portion of the protein and helix 3 of the other subunit. In addition, TcTIM has proline at position 24 in the first helix of the TIM barrel; this is absent in the other TIM. Pro24 disrupts the regular helix arrangement, making the pitch of this helix 1.2 A longer than in TbTIM. When Pro24 of TcTIM was substituted for Glu, the sensitivity of TcTIM to sulfhydryl reagents increased about fivefold, possibly as a consequence of an increase in the space between the first portion of helix 1 and helix 3 of the other subunit. Therefore, it may be concluded that the geometry of the latter region is central in the accessibility to agents that perturb the interface Cys. In human TIM this region is more compact.

Aspirin blocks binding of photosensitizer SnET2 into human serum albumin: implications for photodynamic therapy.

Tin etiopurpurin dichloride (SnET2) is one of the photosensitizers under investigation to be used in photodynamic therapy of prostate cancer. The drug is delivered intravenously, transported in vivo by liposomes and plasma proteins and localized within the prostate. SnET2 exists in two tautomeric forms (I - closed ring, II - open ring) with I converting spontaneously into the more energetically stable form II at physiological pH. Up to approximately 50% of the drug can be carried by serum albumin, although this association can increase photo-bleaching and diminish the drug efficiency. Molecular modeling and force field calculations indicate that Sudlow Site I in human serum albumin (HSA) is the most probable binding site for both forms of SnET2, with the porphyrin moiety nestling between domains IIA and IB, and the esterolytic side group oriented toward domain IIIA of HSA. Other drugs, including aspirin, bind to the same part of HSA. SnET2 does not bind to HSA when pre-incubated with aspirin, which confirms that its place of binding to this protein must be located near Lys199. This observation could be exploited to improve photo-efficiency of SnET2 by finding drugs that could compete with the photosensitizer for binding into Sudlow Site I of HSA.

Crystal structure of hevein at 2.8 A resolution.

The three-dimensional structure of hevein, a low molecular weight protein isolated from the latex of Hevea brasiliensis, has been determined by X-ray diffraction at 2.8 A resolution. The protein crystallizes in space group P2(1)2(1)2(1), with lattice constants a = 21.78, b = 31.86, c = 51.12 A. The structure was solved by molecular replacement methods using the domain C of wheat germ agglutinin (WGA) as search model. The positions and individual isotropic temperature factors of the 324 atoms have been refined by the Hendrickson and Konnert restrained refinement procedure. While tight restraints have been maintained on the bonded distances and angles, the R-factor has dropped to 24.1% and an averaged B value of 9.5 A2, using 78% (802) of the total possible number of reflections in the resolution range 5-2.8 A. The tertiary structure is very similar to that of domain C of WGA from residues 3-31.

Isolation and characterization of a protease from the marine sponge Spheciospongia vesparia.

A protein that showed activity against proteic (casein and hide powder azure) and synthetic (BAEE and HLPA) substrates was isolated from the marine sponge Spheciospongia vesparia. The protease was purified from an aqueous extract by ammonium sulfate precipitation, gel filtration, hydrophobic and HPLC-anion exchange chromatographies. The purified protease showed a single band in SDS-PAGE minigels and had a molecular weight of 29,600, but when submitted to isoelectric focusing it showed 2 bands with isoelectric points of 4.56 and 4.43. Its catalytic action was inhibited by EDTA and 1,10-phenanthroline, so it seemed to be a metalloprotease.

Organoselenium compounds as potential therapeutic and chemopreventive agents: a review.

Selenium is an essential trace element. It is, however toxic at concentration little above which is required for health. Selenium is incorporated into proteins as selenocysteine, the 21(st) amino acid. Selenoproteins are found in bacteria, archaea and eukaryotes. Biochemical and physicochemical properties of selenium result in the unique redox characteristics of selenocysteine and its use in antioxidant enzymes. In this context of a redox reaction is the reduction of reactive oxygen metabolites by glutathione peroxidases, helping to maintain membrane integrity, reduces the oxidative damage to lipids, lipoproteins, and DNA. Selenium has structural and enzymatic roles. Selenium influences a number of endocrine processes, most notably, those involved in thyroid hormone synthesis and metabolism. Se is needed for the proper functioning of the immune system, a role in viral suppression, AIDS, and also is implicated in delaying the aging process. Its deficiency has been linked to a number of disorders such as heart disease, diabetes, and diseases of the liver, and it is required for sperm motility and may reduce the risk of miscarriage. Se supplementation has recently moved from the realm of correcting nutritional deficiencies to one of pharmacological intervention, especially in the clinical domain of cancer chemoprevention. During the last few years, a tremendous effort has been directed toward the synthesis of stable organoselenium compounds that could be used as antioxidants, enzyme modulators, antitumor, antimicrobials, antihypertensive agents, antivirals and cytokine inducers. The biochemistry and pharmacology of selenium-based compounds are subjects of intense current interest, especially from the point of view of public heath. The purpose of this review is to discuss the recent pharmacological applications of organoselenium compounds as therapeutic agents in the treatment of several diseases.

Synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine; an amino-estrogen with prolonged anticoagulant and brief estrogenic effects.

The synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, is described. It was characterized by ir, nmr, mass spectrometry and chemical analysis. The crystal structure of this compound was determined by single-crystal x-ray diffraction. Prolame belongs to space group P212121. Cell dimensions are: a = 8.356(2), b = 13.343(4) and c = 16.119(4) A. Z = 4; R = 4.1%.

Synthesis, structure, spectra and redox activities of model compounds for methanogenic bacteria.

Synthetically accessible benzimidazoles have been synthesized and the benzimidazole ligands were complexed with nickel(II) nitrate. A nickel(II) complex of N,N'-bis(benzimidazole-2-ylethyl)ethylenediamine was crystallized in single-crystal form and the structure was investigated by X-ray crystallography. The structure of the complex is bicapped axial coordinated octahedral. Ni(bbes)(2+)(2)[bbes=bis(benzimidazole-2-ylethyl)sulfide] exhibits broad low energy bands in electronic spectra and high redox potential in cyclic voltammetry (CV) rather than Ni(enbzim)(2+) [enbzim =N,N'-bis(benzimidazol-2-ylethyl)ethylenediamine], where high energy well separated bands were observed in the visible region and a more negative redox potential was detected in CV. Experimental studies show that an increasing amount of pi-orbital interaction with the Ni(2+)ion, irrespective of chelate ring may favour the higher redox potential. The higher redox potential of methanogenic bacterial [Ni(II)/Ni(I)] than nickel compounds is one of the main factors for the degradation of organic biodegradable compounds and the further transformation to methane.

Purification, crystallization, and preliminary X-ray characterization of a 36 kDa amaranth globulin.

The purpose of this study was to purify, crystallize, and characterize by X-ray diffraction an amaranth globulin for its subsequent structure elucidation. A 36-kDa amaranth globulin was extracted by sequential precipitation and purified by gel filtration and cationic exchange columns. It was crystallized at 18 degrees C from 4 M sodium formate. Suitable crystals for X-ray analysis were found to belong to the tetragonal crystal system with cell dimensions of a = b = 195.5 A and c = 164.14 A. Two possible tetragonal space groups P4(1)2(1)2 or P4(3)2(1)2 were determined. The crystals diffracted up to 2.5 A.

Structure of the mexicain-E-64 complex and comparison with other cysteine proteases of the papain family.

Mexicain is a 23.8 kDa cysteine protease from the tropical plant Jacaratia mexicana. It is isolated as the most abundant product after cation-exchange chromatography of the mix of proteases extracted from the latex of the fruit. The purified enzyme inhibited with E-64 [N-(3-carboxyoxirane-2-carbonyl)-leucyl-amino(4-guanido)butane] was crystallized by sitting-drop vapour diffusion and the structure was solved by molecular replacement at 2.1 A resolution and refined to an R factor of 17.7% (R(free) = 23.8%). The enzyme belongs to the alpha+beta class of proteins and the structure shows the typical papain-like fold composed of two domains, the alpha-helix-rich (L) domain and the beta-barrel-like (R) domain, separated by a groove containing the active site formed by residues Cys25 and His159, one from each domain. The four monomers in the asymmetric unit show one E-64 molecule covalently bound to Cys25 in the active site and differences have been found in the placement of E-64 in each monomer.