Modulation of Plasminogen Activator Inhibitor 1 by Triton X-100 – Identification of Two Consecutive Conformational Transitions

Gils, Ann; Declerck, Paul

doi:10.1055/s-0037-1615189

Cited by 74 publications

(43 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…PAI-1 exhibiting substrate behavior will be recovered as the large N-terminal fragment produced by reactive center cleavage, whereas the only 33 amino acid long C-terminal fragment will not be recovered by the gel system used here. Inert PAI-1, for instance the latent form, will be recovered in the position of native full-length PAI-1 (45). Control experiments (not shown) ensured that no further changes in the reaction between PAI-1 and LMW-uPA occurred by prolonging the incubation time beyond the routinely used 2 min.…”

Section: Analysis Of Functional Behavior Of Pai-1 By Reaction With Lmmentioning

confidence: 97%

A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

Egelund¹,

Einholm²,

Pedersen³

et al. 2001

Journal of Biological Chemistry

104

View full text Add to dashboard Cite

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environmentsensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizing compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4-dianilino-1,1-bisnaphthyl-5,5-disulfonic acid. The fluorescence increase could be competed by all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA. With some neutralizers, the second step was associated with PAI-1 polymerization. Vitronectin reduced the susceptibility to the neutralizers. Changes in sensitivity to activity neutralization by point mutations were compatible with the various neutralizers having overlapping, but not identical, binding sites in the region around ␣-helices D and E and ␤-strand 1A, known to act as a flexible joint when ␤-sheet A opens and the reactive center loop inserts as ␤-strand 4A during reaction with target proteinases. The defined binding area may be a target for development of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases.Plasminogen activator inhibitor-1 (PAI-1) 1 is a fast and specific inhibitor of the serine proteinases urokinase-type (uPA) and tissuetype plasminogen activator (tPA) and, as such, an important regulator of extracellular proteolysis in turn over of extracellular matrix and in fibrinolysis (for reviews see Refs. 1 and 2). PAI-1 binds with high affinity to vitronectin (for reviews see Refs. 3 and 4) and may regulate cell migration and adhesion by inhibition of vitronectin binding of integrins and the uPA receptor (5-10). The PAI-1 level in malignant tumors is one of the most informative biochemical markers of a poor prognosis (for reviews see Refs. 11 and 12), and PAI-1 seems to be causally involved in tumor invasion and angiogenesis (13). A high PAI-1 level in blood plasma is a risk factor for ischemic cardiovascular disease and venous thromboembolism (for review see Ref. 14). PAI-1 is therefore a potential target for both anti-cancer and anti-thrombotic therapy.PAI-1 belongs to the serpin superfamily. Serpins are composed of 3 ␤-sheets and 9 ␣-helices. Serpins and their target proteinases form stable complexes by interaction of the active site of the proteinases with the reactive center peptide bond (P 1 -P 1 Ј) in the solvent-exposed, ϳ20-amino acid long peptide loop, the reactive center loop (RCL) (for reviews see Refs. 2 and 15-17). There is both structural and biochemical evidence that complex formation is associated with the P 1 -P 1 Ј bond being cle...

show abstract

Section: Analysis Of Functional Behavior Of Pai-1 By Reaction With Lmmentioning

confidence: 97%

A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

Egelund¹,

Einholm²,

Pedersen³

et al. 2001

Journal of Biological Chemistry

104

View full text Add to dashboard Cite

show abstract

“…Under physiological conditions, the latency transition is irreversible; however, denaturant-induced refolding can reconstitute PAI-1 in the native, active form (26). Several latency-inducing agents have been described, including amphipathic nonionic detergents (27,28) and monoclonal antibodies or peptides (29)(30)(31). These latter agents are thought to stabilize a prelatent intermediate and thereby shift the structural equilibrium toward the latent conformation.…”

mentioning

confidence: 99%

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Reinke

Sanders

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration-approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family.fibrinolysis | thrombolysis | fibrosis | cancer P lasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, chronic fibrotic and inflammatory diseases, and tumor invasion and metastasis (1-6). These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7-16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.PAI-1 is the most potent physiologic inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively) (17). Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19). However, before the protease can dissociate from the serpin, a dramatic conformational change occurs [termed the stressed to relaxed (S to R) transition], in which the cleaved RCL inserts into the central β-sheet A, translocating the covalently-bound protease 70 Å to the base of β-sheet A (20). This conformational change results in distortion of the protease active site (21,22) and thereby prevents the deacylation reaction, inactivating the protease. Should this conformational change be interrupted, the protease can complete its cleavage of the serpin RCL, remaining active and leaving the serpin in a cleaved, inactive, loop-inserted state (23).PAI-1 is unique among serpins in that it readily autoinactivates into a so-called latent form, where the PAI-1 ...

show abstract

“…Depending on the mobility of the RCL, the presence of co-factors (e.g. heparin) and the proteinase used, PAI-1 can also be cleaved in a substrate-type reaction, resulting in an inactivated inhibitor (12)(13)(14)(15). Some serpins require secondary interactions for efficient inhibition of the target proteinase.…”

mentioning

confidence: 99%

Immobilization of the Distal Hinge in the Labile Serpin Plasminogen Activator Inhibitor 1

Taeye¹,

Compernolle

Dewilde

et al. 2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsinsusceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of ␣hF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of ␣hF, subsequently resulting in substrate behavior. Plasminogen activator inhibitor-1 (PAI-1)1 is the most important physiological inhibitor of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) (1). It plays an important role in the regulation of the fibrinolytic activity in human blood (2). In addition to its role in fibrinolytic processes, PAI-1 has been demonstrated to be involved in a variety of other (patho)physiological processes.PAI-1 is a member of the superfamily of the serpins. The three-dimensional structure of these glycoproteins consists of three ␤-sheets, nine ␣-helices, and a reactive center loop (RCL, P16 -P10Ј). Typically their active site comprises the P1-P1Ј (bait) residues, located on the RCL, thereby serving as a pseudo-substrate for the proteinase (3). N-terminally, the RCL is connected via the so-called proximal hinge (P14 -P18) to ␤-strand 5 of the large central ␤-sheet A (s5A). C-terminally, the RCL is connected to s1C (i.e. the distal hinge region).PAI-1 is unique among the serpins because of its functional and conformational flexibility. The active conformation of PAI-1 inhibits its target proteinases by the formation of a stable, inactive complex. After the formation of an initial, reversible Michaelis-like complex, the proteinase cleaves the active site of PAI-1 and ...

show abstract

Modulation of Plasminogen Activator Inhibitor 1 by Triton X-100 – Identification of Two Consecutive Conformational Transitions

Cited by 74 publications

References 30 publications

A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

A Regulatory Hydrophobic Area in the Flexible Joint Region of Plasminogen Activator Inhibitor-1, Defined with Fluorescent Activity-neutralizing Ligands

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Immobilization of the Distal Hinge in the Labile Serpin Plasminogen Activator Inhibitor 1

Contact Info

Product

Resources

About