RESUMEN
The cysteine proteinase B of Leishmania parasites is an important virulence factor. In this study we have expressed, isolated and characterized for the first time a recombinant CPB from Leishmania braziliensis, the causative agent of mucocutaneous leishmaniosis. The mature region of the recombinant CPB shares a high percentage identity with its Leishmania mexicana CPB2.8 (rCPB2.8DeltaCTE) counterpart (76.36%) and has identical amino acid residues at the S(1), catalytic triad and S'(1) subsites. Nevertheless, when the kinetics of substrate hydrolysis was measured using a combinatorial library of internally quenched fluorescent peptides based upon the lead sequence Abz-KLRSSKQ-EDDnp, significant differences were obtained. These results suggest that the differences in substrate utilization observed between the L. mexicana and L. braziliensis CPs must be related to amino acid modifications outside the core of the active site cleft. Moreover, a potent inhibitor with Pro at P1 and high affinity for L. braziliensis recombinant CPB showed less affinity to L. mexicana CPB 2.8, which preferred Phe, Leu, and Asn at the same position.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Leishmania braziliensis/enzimología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Cinética , Leishmania braziliensis/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P2-P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.
Asunto(s)
Catepsinas/metabolismo , Virus del Dengue/enzimología , Transferencia Resonante de Energía de Fluorescencia , Biblioteca de Péptidos , Péptidos/química , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Reactivos de Enlaces Cruzados/química , Humanos , Cinética , Péptidos/metabolismo , Polietilenglicoles/química , Soluciones/química , Especificidad por SustratoRESUMEN
The CPB genes of the protozoan parasite Leishmania mexicana encode stage-regulated cathepsin L-like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1-S3 and S1'-S3' subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3' by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz-FRAK(Dnp)-OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3' subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2' position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.
Asunto(s)
Sustitución de Aminoácidos/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Leishmania mexicana/enzimología , Animales , Sitios de Unión , Western Blotting , Línea Celular , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A library consisting of about half of 800 000 possible peptidotriazoles on 450 000 beads was prepared by solid-phase peptide synthesis combined with a regiospecific copper(I)-catalyzed 1,3-dipolar cycloaddition between a resin-bound alkyne and a protected amino azide. The central [1,2,3]-triazole was flanked on each side by two randomized amino acids introduced in a combinatorial approach. Importantly, the formation of the triazole could be performed quantitatively in a randomized fashion. The library was screened on solid phase for inhibitory effect against a recombinant cysteine protease, Leishmania mexicana CPB2.8DeltaCTE and sorted by a high-throughput instrument, COPAS beadsorter (up to 200 000 beads/h). Forty-eight hits were analyzed by MALDI-TOF MS providing structural information about the protease specificity, and 23 peptidotriazoles were resynthesized and evaluated in solution, with the best inhibitor displaying a K(i) value of 76 nM. A one-pot procedure was used to convert Fmoc-amino azides into their corresponding Boc derivatives. The crucial influence of weak interactions with a spacer used for detection by MALDI-TOF MS on screening results was observed.
Asunto(s)
Proteasas de Cisteína , Leishmania mexicana , Caspasas , Técnicas Químicas Combinatorias , Biblioteca de Genes , Modelos Moleculares , Estructura Molecular , TriazolesRESUMEN
A one-bead-two-compound inhibitor library was synthesized by the split-mix method for the identification of inhibitors of a recombinant cysteine protease from Leishmania mexicana, CPB2.8DeltaCTE. The inhibitor library was composed of octapeptides with a centrally located reduced bond introduced by reductive amination of the resin-bound amines with Fmoc amino aldehydes. The library was screened on solid phase, and less than 1% of the library contained active compounds. The inhibitors displayed great specificity in the subsites flanking the enzyme catalytic triad with Cha and Ile/Leu preferred in P(2), Phe in P(1), Cha and Ile/Leu in P(1)', and Ile/Leu in P(2)'. Some of the inhibitors were resynthesized, and the kinetics of inhibition were determined in solution-phase assays. Most of the inhibitors had micromolar K(i) values, and a few inhibited the enzyme at nanomolar concentrations. One inhibitor, DKHF(CH(2)NH)LLVK (K(i) = 1 microm), was tested for antiparasite efficacy and shown to affect parasite survival with an IC(50) of approximately 50 microm.