title: Mechanism of heparin acceleration of tissue inhibitor of metalloproteases-1 (TIMP-1) degradation by the human neutrophil elastase.
abstract: Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k₂ = 21±1 s⁻¹) was much higher than the HNE deacylation step (k₃ = 0.57±0.05 s⁻¹). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k₁ 2.4-fold and reducing k₋₁ 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k₂ value, whereas the k₃ value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.
authors: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Mogi das Cruzes, Brazil.
authors: Gabriel L C
authors: GL
authors: Nunes
authors: Alyne
authors: A
authors: Simões
authors: Fábio H
authors: FH
authors: Dyszy
authors: Claudio S
authors: CS
authors: Shida
authors: Maria A
authors: MA
authors: Juliano
authors: Luiz
authors: L
authors: Juliano
authors: Tarsis F
authors: TF
authors: Gesteira
authors: Helena B
authors: HB
authors: Nader
authors: Gillian
authors: G
authors: Murphy
authors: Alain F
authors: AF
authors: Chaffotte
authors: Michel E
authors: ME
authors: Goldberg
authors: Ivarne L S
authors: IL
authors: Tersariol
authors: Paulo C
authors: PC
authors: Almeida
mesh_terms: D020134:Catalytic Domain; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D019272:Leukocyte Elastase / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D013379:Substrate Specificity / Q000187:drug effects; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D019715:Tissue Inhibitor of Metalloproteinase-1; D006493:Heparin; D019272:Leukocyte Elastase