RESUMO
Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.
Assuntos
Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Haplorrinos , Humanos , Processamento de Imagem Assistida por Computador , Metaloproteinases da Matriz Associadas à Membrana , Membranas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Conformação Proteica , Propriedades de Superfície , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Células Tumorais Cultivadas , Vaccinia virusRESUMO
Beta-lactamases are resistance enzymes for beta-lactam antibiotics. These enzymes hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. Of the four classes of known beta-lactamases, the enzymes of class D are the least understood. We report herein the high-resolution (1.9 A) crystal structure of the class D OXA-10 beta-lactamase inhibited by a penicillanate derivative. The structure provides evidence that the carboxylated Lys-70 (a carbamate) is intimately involved in the mechanism of the enzyme.
Assuntos
beta-Lactamases/química , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Inibidores de beta-Lactamases , beta-Lactamases/metabolismoRESUMO
Classical trajectory simulations are performed to study energy transfer in collisions of protonated triglycine (Gly)(3) and pentaglycine (Gly)(5) ions with n-hexyl thiolate self-assembled monolayer (SAM) and diamond [111] surfaces, for a collision energy E(i) in the range of 10-110 eV and a collision angle of 45 degrees. Energy transfer to the peptide ions' internal degrees of freedom is more efficient for collision with the diamond surface; i.e., 20% transfer to peptide vibration/rotation at E(i) = 30 eV. For collision with diamond, the majority of E(i) remains in peptide translation, while the majority of the energy transfer is to surface vibrations for collision with the softer SAM surface. The energy-transfer efficiencies are very similar for (Gly)(3) and (Gly)(5). Constraining various modes of (Gly)(3) shows that the peptide torsional modes absorb approximately 80% of the energy transfer to the peptide's internal modes. The energy-transfer efficiencies depend on E(i). These simulations are compared with recent experiments of peptide SID and simulations of energy transfer in Cr(CO)(6)(+) collisions with the SAM and diamond surfaces.