Navegando por Palavras-chave "substrate recognition"

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    Investigating the substrate specificity and oligomerisation of the leader protease of foot and mouth disease virus using NMR
    (Elsevier B.V., 2007-11-02) Cencic, Regina; Mayer, Christina; Juliano, Maria A.; Juliano, Luiz; Konrat, Robert; Kontaxis, Georg; Skern, Tim; Med Univ Vienna; Universidade Federal de São Paulo (UNIFESP); Univ Vienna
    The leader protease (Lb(pro)) of foot-and-mouth disease virus frees itself during translation from the viral polyprotein by cleavage between its own C terminus and the N terminus of the subsequent protein, VP4. Lb(pro) also specifically cleaves the host proteins eukaryofic initiation factor (eIF) 4GI and 4GII, thus disabling host cell protein synthesis. We used NMR to study full-length Lb(pro) as well as a shortened species lacking six C-terminal amino acid residues (sLb(pro)) to examine the mechanism of self-processing, the quaternary structure and the substrate specificity. Both Lb(pro) forms have the same structure in solution as in the crystal. in the solution structure of sLb(pro), the 12 residue C-terminal extension was flexible and disordered. in contrast, the 18 residue C-terminal extension of full-length Lb(pro) was bound by the substrate-binding site of a neighbouring molecule, resulting in the formation of a stable dimer in solution. the Lbpro dimer could not be dissociated by increasing the ionic strength or by dilution. Furthermore, titration with model peptides mimicking the substrates destabilised the dimer interface without dissociating the dimer. the peptides were, however, bound by sLb(pro) in the canonical substrate binding site. Peptide binding gave rise to chemical shifts of residues around the sLb(pro) substrate binding site. Shifts of Asn146 and Glu147 indicated that these residues might form the enzyme's S1' site and interact with the P1' arginine residue of the eIF4GI cleavage site. Furthermore, differences in substrate specificity between sLb(pro) and Lb(pro) observed with an in vitro translated protein indicate some involvement of the C terminus in substrate recognition. (C) 2007 Elsevier B.V. All rights reserved.
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    New insights into the substrate specificity of macrophage elastase MMP-12
    (Walter De Gruyter Gmbh, 2016) Lamort, Anne-Sophie; Gravier, Rodolphe; Laffitte, Anni; Juliano, Luiz [UNIFESP]; Zani, Marie-Louise; Moreau, Thierry
    Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence ProX-X down arrow Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.