RESUMO
New porphyrin-Schiff base conjugates bearing one (6) and two (7) basic amino groups were synthesized by condensation between tetrapyrrolic macrocycle-containing amine functions and 4-(3-(N,N-dimethylamino)propoxy)benzaldehyde. This approach allowed us to easily obtain porphyrins substituted by positive charge precursor groups in aqueous media. These compounds showed the typical Soret and four Q absorption bands with red fluorescence emission (ΦF ~ 0.12) in N,N-dimethylformamide. Porphyrins 6 and 7 photosensitized the generation of O2(1Δg) (ΦΔ ~ 0.44) and the photo-oxidation of L-tryptophan. The decomposition of this amino acid was mainly mediated by a type II photoprocess. Moreover, the addition of KI strongly quenched the photodynamic action through a reaction with O2(1Δg) to produce iodine. The photodynamic inactivation capacity induced by porphyrins 6 and 7 was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the photoinactivation of these microorganisms was improved using potentiation with iodide anions. These porphyrins containing basic aliphatic amino groups can be protonated in biological systems, which provides an amphiphilic character to the tetrapyrrolic macrocycle. This effect allows one to increase the interaction with the cell wall, thus improving photocytotoxic activity against microorganisms.
Assuntos
Aminoácidos Básicos/química , Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Porfirinas/química , Bases de Schiff/farmacologia , Anti-Infecciosos/química , Antifúngicos/química , Bases de Schiff/químicaRESUMO
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
Assuntos
Calicreínas/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Furina/química , Furina/metabolismo , Humanos , Hidrólise , Calicreínas/química , Calicreínas/genética , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/química , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3 , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeos/metabolismo , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.
Assuntos
Aminoácidos Básicos/química , Corantes Fluorescentes/química , Serina Endopeptidases/química , Proteínas não Estruturais Virais/química , Sítios de Ligação , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Furina/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Oligopeptídeos/química , Proteínas Recombinantes/química , Sais/química , Especificidade por SubstratoRESUMO
Ribosomes, ribosomal proteins (r-proteins), and messenger and transfer RNAs catalyze the synthesis of proteins in organisms. To understand and define the components involved in this event in Taenia solium, we isolated and characterized a T. solium cDNA encoding the basic ribosomal protein S15a (TsS15a). The TsS15a cDNA produces a protein with M(r) (relative molecular mass) 14,988, which contains 22.3% of basic amino acids. Analysis comparing TsS15a protein with other S15a r-proteins indicates that this protein is highly conserved. A recombinant TsS15a protein with similar M(r) was produced in bacteria. Antibodies against recombinant TsS15a react with a 15-kDa protein in extracts from all life stages of T. solium and from all helminths tested. Hybridization studies showed the presence of two genes encoding a mRNA of 0.5 kb. Moreover, the gene presents an intron of 30 bp. Our phylogenetic analysis using S15a r-proteins reproduced the topologies reported for 16/18S rRNA.
Assuntos
Clonagem Molecular , Genes de Helmintos , Proteínas de Helminto/genética , Proteínas Ribossômicas/genética , Taenia solium/genética , Sequência de Aminoácidos , Aminoácidos Básicos/química , Animais , Sequência de Bases , Western Blotting , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/isolamento & purificação , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Proteínas de Helminto/química , Íntrons/genética , Dados de Sequência Molecular , Peso Molecular , Hibridização de Ácido Nucleico , Filogenia , RNA Mensageiro/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Ribossômicas/química , Proteínas Ribossômicas/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Taenia solium/classificaçãoRESUMO
We explored the unique substrate specificity of the primary S, subsite of human urinary kallikrein (hK1), which accepts both Phe or Arg synthesizing and assaying peptides derived from Phenylacetyl-Phe-Ser-Arg-EDDnp, a previously described inhibitor with analgesic and anti-inflammatory activities [Emim et al., Br. J. Pharmacol. 130 (2000), 1099-1107]. Phe was substituted by amino acids containing larger aliphatic or aromatic side chains as well as by non-natural basic amino acids, which were designed to combine a large hydrophobic and/or aromatic group with a positively-charged group at their side chains. In general, all peptides with basic amino acids represented better inhibitors than those with hydrophobic amino acids. Furthermore, the S1 subsite specificity proved to be much more selective than the mere distinction between Phe and Arg, for minor differences in the side chains of the non-natural amino acids resulted in major differences in the Ki values. Finally, we present a series of peptides that were assayed as competitive inhibitors for human tissue kallikrein that may lead to the development of novel peptides, which are both more potent and selective.