title: Plasminogen activator inhibitor-1 is locked in active conformation and polymerizes upon binding ligands neutralizing its activity.
abstract: Plasminogen activator inhibitor-1 (PAI-1), a member of the serpin super-family, forms a covalent complex with its target proteinases, such as tissue and urokinase plasminogen activators. Thus, PAI-1 controls the physiological and pathological proteolysis. An abnormal expression of PAI-1 has been observed in different diseases, which can be treated by returning the proteolysis back to normal physiological levels. It has been reported that some PAI-1 inhibitors neutralize its activity by accelerating the conversion of PAI-1 into a latent form. We have found small organic chemicals that also neutralize PAI-1 activity, but by a different mechanism. Using the NBD fluorescent probe [N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] incorporated into the reactive center loop (RCL) of PAI-1, we measured the kinetics of conversion from an active to a latent form. Unexpectedly, we found that some inhibitors of PAI-1 arrest this serpin in its active form instead of increasing the speed of conversion. Using docking calculations, we located two possible binding sites for these chemicals. The sites are in proximity of the P1/P1' amino acids of the RCL of PAI-1. Binding in this area can inactivate PAI-1 and additionally create a steric obstacle on the RCL making insertion of this loop between the A3 and A5 strands more difficult; hence abolishing a necessary step in the conversion of this protein into the latent form. Additionally, PAI-1 inhibitors link the RCL of one PAI-1 molecule with the strand 3C and strand 4C or helix A and strand 1B regions of the other PAI-1 molecule aiding polymerization or stabilizing the junction of the two. The polymerization of PAI-1 reduces PAI-1 activity by encapsulating the critical RCL fragment inside the formed PAI-1/PAI-1 polymers.
authors.affiliation: Urology Research Center, Medical University of Ohio, Toledo, 43614, USA. jjankun@meduohio.edu
authors.forename: Jerzy
authors.initials: J
authors.lastname: Jankun
authors.forename: Zofia
authors.initials: Z
authors.lastname: Specht
authors.forename: Maria
authors.initials: M
authors.lastname: Szkudlarek
authors.forename: Robert
authors.initials: R
authors.lastname: Greenfield
authors.forename: Bhavna
authors.initials: B
authors.lastname: Gaikwad
authors.forename: Latchezar
authors.initials: L
authors.lastname: Trifonov
authors.forename: Jean
authors.initials: J
authors.lastname: Vaugeois
authors.forename: Ansari M
authors.initials: AM
authors.lastname: Aleem
authors.forename: Venkatesha
authors.initials: V
authors.lastname: Basrur
authors.forename: Steven H
authors.initials: SH
authors.lastname: Selman
authors.forename: Maria I
authors.initials: MI
authors.lastname: Zavodszky
authors.forename: Ewa
authors.initials: E
authors.lastname: Skrzypczak-Jankun
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D001704:Biopolymers / Q000378:metabolism*; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence
chemical_list: D001704:Biopolymers; D005456:Fluorescent Dyes; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1; D015849:Plasminogen Inactivators