A peptide accelerating the conversion of plasminogen activator inhibitor-1 to an inactive latent state.
The serpin plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and a potential therapeutic target in cancer and cardiovascular diseases. Accordingly, formation of a basis for development of specific PAI-1-inactivating agents is of great interest. One possible inactivation mode for PAI-1 is conversion to the inactive, so-called latent state. We have now screened a phage-displayed peptide library with PAI-1 as bait and isolated a 31-residue cysteine-rich peptide that will be referred to as paionin-4. A recombinant protein consisting of paionin-4 fused to domains 1 and 2 of the phage coat protein g3p caused a 2- to 3-fold increase in the rate of spontaneous inactivation of PAI-1. Paionin-4-D1D2 bound PAI-1 with a K(D) in the high nanomolar range. Using several biochemical and biophysical methods, we demonstrate that paionin-4-D1D2-stimulated inactivation consists of an acceleration of conversion to the latent state. As demonstrated by site-directed mutagenesis and competition with other PAI-1 ligands, the binding site for paionin-4 was localized in the loop between alpha-helix D and beta-strand 2A. We also demonstrate that a latency-inducing monoclonal antibody has an overlapping, but not identical binding site, and accelerates latency transition by another mechanism. Our results show that paionin-4 inactivates PAI-1 by a mechanism clearly different from other peptides, small organochemical compounds, or antibodies, whether they cause inactivation by stimulating latency transition or by other mechanisms, and that the loop between alpha-helix D and beta-strand 2A can be a target for PAI-1 inactivation by different types of compounds.
Department of Molecular Biology, Aarhus University, 8000 Aarhus C, Denmark.
Lisa
L
Mathiasen
Daniel M
DM
Dupont
Anni
A
Christensen
Grant E
GE
Blouse
Jan K
JK
Jensen
Ann
A
Gils
Paul J
PJ
Declerck
Troels
T
Wind
Peter A
PA
Andreasen
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D000939:Epitopes; D006493:Heparin / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D006493:Heparin