Navegando por Palavras-chave "Proteolytic enzymes"

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    Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?
    (Elsevier Science Bv, 2017) Silva, Roberta N. [UNIFESP]; Oliveira, Lilian C. G. [UNIFESP]; Parise, Carolina B. [UNIFESP]; Oliveira, Juliana R. [UNIFESP]; Severino, Beatrice; Corvino, Angela; di Vaio, Paola; Temussi, Piero A.; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Luiz [UNIFESP]; Juliano, Maria A. [UNIFESP]
    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-(2,4dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R down arrow R-ACC and Z-R down arrow 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. (C) 2017 Elsevier B.V. All rights reserved.
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    Specificity studies on Kallikrein-related peptidase 7 (KLK7) and effects of osmolytes and glycosaminoglycans on its peptidase activity
    (Elsevier B.V., 2015-01-01) Oliveira, Juliana R. [UNIFESP]; Bertolin, Thiago C. [UNIFESP]; Andrade, Douglas [UNIFESP]; Oliveira, Lilian C. G. [UNIFESP]; Kondo, Marcia Y. [UNIFESP]; Santos, Jorge A. N. [UNIFESP]; Blaber, Michael; Juliano, Luiz [UNIFESP]; Severino, Beatrice; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Maria A. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Florida State Univ; Univ Naples Federico II
    KLK7 substrate specificity was evaluated by families of fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLFSSK-Q-EDDnp (Abz = ortho-aminobenzoic acid and Q-EDDnp = glutaminyl-N-[2,4-dinitrophenyl] ethylenediamine), by one bead-one peptide FRET peptide library in PEGA resin, and by the FRET peptide libraries Abz-GXX-Z-XX-Q-EDDnp (Z and X are fixed and random natural amino acids, respectively). KLK7 hydrolyzed preferentially F,Y or M, and its S-1' and S-2' subsites showed selectivity for hydrophilic amino acids, particularly Rand K. This set of specificities was confirmed by the efficient kininogenase activity of KLK7 on Abz-MISLM(down arrow)KRPPGFSPF(down arrow)RSSRI-NH2 ((down arrow)indicates cleavage), hydrolysis of somatostatin and substance P and inhibition by kallistatin. the peptide Abz-(NLYRVE)-R-down arrow-Q-EDDnp is the best synthetic substrate so far described for KLK7 [k(cat)/K-m, = 455 (mM s)(-1)] that was designed from the KLK7 substrate specificity analysis. It is noteworthy that the NLYRVE sequence is present in human semaphorin 6B. KLK7 is activated by GAGs, inhibited by neutral salts, and activated by high concentration of kosmotropic salt. Pyroglutamic acid inhibited KLK7 (K-i = 33 mM) and is present in skin moisturizing factor (124 mM). the KLK7 specificity described here and elsewhere reflects its participation in patho-physiological events in skin, the gastrointestinal tract and central nervous system, where KLK7 is significantly expressed. (C) 2014 Elsevier B.V. All rights reserved.