Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

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

Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli.

International audienceThe C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein

Circular dichroism of F19ec,ri, F19bac,r and F19bac.

<p>Superimposition of the circular dichroism far-UV spectra of F19ec,ri (red), F19bac,r (blue) and F19bac (green).</p

SDS-PAGE of purified F19 fragments.

<p>Samples were denatured in 2% SDS in the presence of 5% 2-mercaptoethanol before being subjected to 12% SDS-PAGE. Lanes 1 and 3: molecular weight markers; lane 2: F19bac; lane 4: F19ec.</p

The "pre-molten globule," a new intermediate in protein folding.

In vitro folding studies of several proteins revealed the formation, within 2-4 msec, of transient intermediates with a large far-UV ellipticity but no amide proton protection. To solve the contradiction between the secondary structure contents estimated by these two methods, we characterized the isolated C-terminal fragment F2 of the tryptophan synthase beta 2 subunit. In beta 2, F2 forms its tertiary interactions with the F1 N-terminal region. Hence, in the absence of F1, isolated F2 should remain at an early folding stage with no long-range interactions. We shall show that isolated F2 folds into, and remains in, a "state" called the pre-molten globule, that indeed corresponds to a 2- to 4-msec intermediate. This condensed, but not compact, "state" corresponds to an array of conformations in rapid equilibrium comprising native as well as nonnative secondary structures. It fits the "new view" on the folding process

Circular dichroism spectra of F19ec,ri and F19bac.

<p>Superimposition of the circular dichroism spectra of <i>in vitro</i> renatured F19ec,ri (red) and of F19bac (green) used as a reference for F19 native conformation.</p

NOESY spectra of F19ec,rf and F19ec, ri.

<p>Two regions of NOESY spectra of F19ec,rf, which was isolated from renatured MBP-F19 (blue) and F19ec,ri (green), which was denatured and refolded <i>in vitro</i> in its isolated form. <b>A</b> Low field region and <b>B</b> amide/aromatic <i>versus</i> aliphatic regions.</p

NOESY spectra of F19ec and F19ec, rf.

<p>Two regions of NOESY spectra of F19ec (blue) and F19ec,rf isolated from renatured MBP-F19 (in green). <b>A</b> Low field region and <b>B</b> amide/aromatic <i>versus</i> aliphatic regions.</p

Solution equilibrium association constants of F19-antibody interactions.^a

a<p>Association constants (K_A) were determined at 25°C in PBS-Tween buffer pH 7.4 supplemented with 0.02% bovine serum albumin. K_A values are expressed in M⁻¹. The experimental error represents 20–25% of the corresponding K_A value.</p>b<p>MBP-F19 fusion protein natively produced in <i>E. coli</i> periplasm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057086#pone.0057086-Planson1" target="_blank">[16]</a>,</p>c<p>denatured and reduced fusion protein,</p>d<p>fusion protein renatured <i>in vitro</i>,</p>e<p>ratio of the K_A values obtained for the native and denatured fusion protein.</p