Navegando por Palavras-chave "cysteine proteases"
Agora exibindo 1 - 3 de 3
Resultados por página
Opções de Ordenação
- ItemSomente MetadadadosComparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L(Elsevier B.V., 2004-10-15) Puzer, L.; Cotrin, S. S.; Alves, MFM; Egborge, T.; Araujo, M. S.; Juliano, M. A.; Juliano, L.; Bromme, D.; Carmona, A. K.; Universidade Federal de São Paulo (UNIFESP); Mt Sinai Sch MedCathepsins V and L have high identity and few structural differences. in this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S-3 to S'(2) subsites. the cathepsin V subsites S-1 and S-3 present a broad specificity while cathepsin L has preference for positively charged residues. the S-2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. the S-1' and S-2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens. (C) 2004 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosDefining the substrate specificity of mouse cathepsin P(Elsevier B.V., 2005-03-01) Puzer, L.; Barros, NMT; Oliveira, V; Juliano, M. A.; Lu, G. Z.; Hassanein, M.; Juliano, L.; Mason, R. W.; Carmona, A. K.; Universidade Federal de São Paulo (UNIFESP); Univ Cidade São Paulo; Alfred I DuPont Hosp Children; Univ DelawareCathepsin P is a recently discovered placental cysteine protease that is structurally related to the more ubiquitously expressed, broad-specificity enzyme, cathepsin L. We studied the substrate specificity requirements of recombinant mouse cathepsin P using fluorescence resonance energy transfer (FRET) peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp (Abz, ortho-aminobenzoic acid and EDDnp, N-[2,4-dinitroplieiiyl]ethyleiiediamine). Systematic modifications were introduced resulting in five series of peptides to map the S-3 to S-2' subsites of the enzyme. the results indicate that the subsites S-1, S-2, S-1' and S-2', present a clear preference for hydrophobic residues. the specificity requirements of the S, subsite were found to be more restricted, preferring hydrophobic aliphatic amino acids. the S-3 subsite of the enzyme presents a broad specificity, accepting negatively charged (Glu), positively charged (Lys, Arg), and hydrophobic aliphatic or aromatic residues (Val, Phe). for several substrates, the activity of cathepsin P was markedly regulated by kosmotropic salts, particularly Na2SO4. No significant effect on secondary or tertiary structure could be detected by either circular dichroism or size exclusion chromatography, indicating that the salts most probably disrupt unfavorable ionic interactions between the substrate and enzyme active site. A substrate based upon the preferred P-3 to P-2' defined by the screening study, ortho-aminobenzoic-Glu-Ile-Phe-Val-Phe-Lys-Gln-N-(2,4-dinitrophenyl)ethylenediamine (cleaved at the Phe-Val bond) was efficiently hydrolyzed in the absence of high salt. the k(cat)/K-m for this substrate was almost two orders of magnitude higher than that of the original parent compound. These results show that cathepsin P, in contrast to other mammalian cathepsins, has a restricted catalytic specificity. (C) 2004 Elsevier Inc. All rights reserved.
- ItemSomente MetadadadosInterplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex(Elsevier B.V., 2006-01-01) Svensjo, E.; Batista, P. R.; Brodskyn, C. I.; Silva, R.; Lima, APCA; Schmitz, V; Saraiva, E.; Pesquero, J. B.; Mori, MAS; Muller-Esterl, W.; Scharfstein, J.; Universidade Federal do Rio de Janeiro (UFRJ); Ctr Pesquisa Goncalo Moniz; Universidade Federal de São Paulo (UNIFESP); Univ FrankfurtKinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules. Peaking at 15 min, the parasite-induced leakage was drastically enhanced by captopril (Cap), an inhibitor of angiotensin-converting enzyme (ACE), a kinin-degrading metallopeptidase. the enhanced microvascular responses were cancelled by HOE-140, an antagonist of the B, bradykinin receptor (13,R), or by pre-treatment of promastigotes with the irreversible cysteine proteinase inhibitor N-methylpiperazine-urea-Phe-homoPhe-vinylsulfone-benzene (N-Pip-hF-VSPh). in agreement with the above-mentioned data, the promastigotes vigorously induced edema in the paw of Cap-treated J129 mice, but not Cap-B2R-/(-) mice. Analysis of parasite-induced breakdown of high molecular weight kininogens (HK), combined with active site-affinity-labeling with biotin-N-Pip-hF-VSPh, identified 35-40 kDa proteins as kinin-releasing cysteine peptidases. We then checked if macrophage infectivity was influenced by interplay between these kinin-releasing parasite proteases, kininogens, and kinin-degrading peptidases (i.e. ACE). Our studies revealed that full-fledged B2R engagement resulted in vigorous increase of L. chagasi uptake by resident macrophages. Evidence that inflammatory macrophages treated with HOE-140 became highly susceptible to amastigote outgrowth, assessed 72 h after initial macrophage interaction, further suggests that the kinin/B2R activation pathway may critically modulate inflammation and innate immunity in visceral leishmaniasis. (c) 2005 Elsevier SAS. All rights reserved.