title: Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library.
abstract: The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA-PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA-PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Anders
authors.initials: A
authors.lastname: Malmendal
authors.forename: Birgit
authors.initials: B
authors.lastname: Schiøtt
authors.forename: Sune
authors.initials: S
authors.lastname: Skeldal
authors.forename: Katrine E
authors.initials: KE
authors.lastname: Pedersen
authors.forename: Leyla
authors.initials: L
authors.lastname: Celik
authors.forename: Niels Chr
authors.initials: NC
authors.lastname: Nielsen
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.forename: Troels
authors.initials: T
authors.lastname: Wind
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D019921:Biotinylation; D002460:Cell Line; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D003553:Cystine / Q000737:chemistry; D004705:Endocytosis / Q000187:drug effects*; D005354:Fibrosarcoma / Q000473:pathology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism; D008823:Micelles; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000302:isolation & purification / Q000494:pharmacology*; D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: C514519:LDLR-related protein 1A, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008823:Micelles; D009363:Neoplasm Proteins; D009842:Oligopeptides; D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1; D011973:Receptors, LDL; C051321:SERPINE1 protein, human; C036053:VLDL receptor; C514518:paionin-1; D003553:Cystine; D014568:Urokinase-Type Plasminogen Activator