title: RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1.
abstract: The hallmark of serpins is the ability to undergo the so-called "stressed-to-relaxed" switch during which the surface-exposed reactive center loop (RCL) becomes incorporated as strand 4 in central beta-sheet A. RCL insertion drives not only the inhibitory reaction of serpins with their target serine proteases but also the conversion to the inactive latent state. RCL insertion is coupled to conformational changes in the flexible joint region flanking beta-sheet A. One interesting serpin is plasminogen activator inhibitor-1 (PAI-1), a fast and specific inhibitor of the serine proteases tissue-type and urokinase-type plasminogen activator. Via its flexible joints' region, native PAI-1 binds vitronectin and relaxed, protease-complexed PAI-1 certain endocytosis receptors. From a library of 35-nucleotides long 2'-fluoropyrimidine-containing RNA oligonucleotides, we have isolated two aptamers binding PAI-1 by the flexible joint region with low nanomolar K(D) values. One of the aptamers exhibited measurable binding to native PAI-1 only, while the other also bound relaxed PAI-1. While none of the aptamers inhibited the antiproteolytic effect of PAI-1, both aptamers inhibited vitronectin binding and the relaxed PAI-1-binding aptamer also endocytosis receptor binding. The aptamer binding exclusively to native PAI-1 increased the half-life for the latency transition to more than 6 h, manyfold more than vitronectin. Contact with Lys124 in the flexible joint region was critical for strong inhibition of the latency transition and the lack of binding to relaxed PAI-1. We conclude that aptamers yield important information about the serpin conformational switch and, because they can compete with high-affinity protein-protein interactions, may provide leads for pharmacological intervention.
authors.affiliation: Danish-Chinese Center for Proteases and Cancer, Aarhus University,10C Gustav Wieds Vej, 8000 Aarhus C, Denmark.
authors.forename: Jeppe B
authors.initials: JB
authors.lastname: Madsen
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Dupont
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Andersen
authors.forename: Anne F
authors.initials: AF
authors.lastname: Nielsen
authors.forename: Lu
authors.initials: L
authors.lastname: Sang
authors.forename: Ditte M
authors.initials: DM
authors.lastname: Brix
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Thomas
authors.initials: T
authors.lastname: Broos
authors.forename: Maarten L V
authors.initials: ML
authors.lastname: Hendrickx
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D052157:Aptamers, Nucleotide; D017395:Plasminogen Activator Inhibitor 1