scholarly journals 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

2006 ◽  
Vol 399 (3) ◽  
pp. 387-396 ◽  
Author(s):  
Jan K. Jensen ◽  
Anders Malmendal ◽  
Birgit Schiøtt ◽  
Sune Skeldal ◽  
Katrine E. Pedersen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Pai 1

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.

scholarly journals Binding of urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex to the endocytosis receptors α2-macroglobulin receptor/low-density lipoprotein receptor-related protein and very-low-density lipoprotein receptor involves basic residues in the inhibitor

1998 ◽  
Vol 329 (1) ◽  
pp. 55-63 ◽  
Author(s):  
W. Kees RODENBURG ◽  
Lars KJØLLER ◽  
H. Helle PETERSEN ◽  
A. Peter ANDREASEN
Keyword(s):  
Plasminogen Activator ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Plasminogen Activator Inhibitor ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Low Density Lipoprotein Receptor ◽  
Α2 Macroglobulin ◽  
Density Lipoprotein Receptor ◽  
Pai 1

The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors α2-macroglobulin receptor/low-density lipoprotein receptor-related protein (α2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA·PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified α2MR/LRP and VLDLR respectively. α2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA·PAI-1 complex to both α2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by α-helix D and β-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease·serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.

Three Complement-Type Repeats of the Low-Density Lipoprotein Receptor-Related Protein Define a Common Binding Site for RAP, PAI-1, and Lactoferrin

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3277-3285 ◽  
Author(s):  
Brian Vash ◽  
Neil Phung ◽  
Sima Zein ◽  
Dianne DeCamp
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Dependent Manner ◽  
Related Protein ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein ◽  
Pai 1

Abstract The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding. © 1998 by The American Society of Hematology.

scholarly journals A proximal pair of positive charges provides the dominant ligand-binding contribution to complement-like domains from the LRP (low-density lipoprotein receptor-related protein)

2012 ◽  
Vol 443 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Peter G. W. Gettins ◽  
Klavs Dolmer
Keyword(s):  
Binding Energy ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Related Protein ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein ◽  
Pai 1

The LRP (low-density lipoprotein receptor-related protein) can bind a wide range of structurally diverse ligands to regions composed of clusters of ~40 residue Ca2+-dependent, disulfide-rich, CRs (complement-like repeats). Whereas lysine residues from the ligands have been implicated in binding, there has been no quantification of the energetic contributions of such interactions and hence of their relative importance in overall affinity, or of the ability of arginine or histidine residues to bind. We have used four representative CR domains from the principal ligand-binding cluster of LRP to determine the energetics of interaction with well-defined small ligands that include methyl esters of lysine, arginine, histidine and aspartate, as well as N-terminally blocked lysine methyl ester. We found that not only lysine but also arginine and histidine bound well, and when present with an additional proximal positive charge, accounted for about half of the total binding energy of a protein ligand such as PAI-1 (plasminogen activator inhibitor-1). Two such sets of interactions, one to each of two CR domains could thus account for almost all of the necessary binding energy of a real ligand such as PAI-1. For the CR domains, a central aspartate residue in the sequence DxDxD tightens the Kd by ~20-fold, whereas DxDDD is no more effective. Together these findings establish the rules for determining the binding specificity of protein ligands to LRP and to other LDLR (low-density lipoprotein receptor) family members.

scholarly journals Differential endocytosis of tissue plasminogen activator by serpins PAI-1 and PAI-2 on human peripheral blood monocytes

2010 ◽  
Vol 104 (12) ◽  
pp. 1133-1142 ◽  
Author(s):  
Jodi A. Lee ◽  
David R. Croucher ◽  
Marie Ranson
Keyword(s):  
Peripheral Blood ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Blood Monocytes ◽  
Low Density Lipoprotein Receptor ◽  
Peripheral Blood Monocytes ◽  
Density Lipoprotein Receptor ◽  
Pai 1

SummaryGeneration of the broad spectrum protease plasmin is facilitated by the tissue (t-PA) and urokinase (u-PA) plasminogen activators, within multiple physiological and disease states. Finely tuned control of this proteolytic cascade is exerted by the plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and 2 (PAI-2/SERPINB2). Expression of this network of activators and inhibitors by cells of myeloid lineage appears to be highly interchangeable between physiological environments, and whilst the role of PAI-1 and PAI-2 in regulating u-PA-dependent functions is well established, the interaction between t-PA and PAI-2 on these cell types is poorly characterised. To this end, we used freshly isolated peripheral blood monocytes (PBM) as a model of a t-PA-dependent cellular environment. We demonstrate that while both PAI-1 and PAI-2 could inhibit surface-bound t-PA and are internalised predominately via low-density-lipoprotein receptor family members, PAI-1 enhanced the endocytosis of t-PA, whereas PAI-2 did not. Surface plasmon resonance analyses revealed differential binding affinities between the very-low-density-lipoprotein receptor and t-PA and t-PA:PAI-1 complexes in addition to those previously described with low-density-lipoprotein receptor-related protein. Moreover, t-PA:PAI-2 bound to both endocytosis receptors with similar kinetics to t-PA. These differential biochemical interactions between t-PA and the t-PA:PAI complexes may underlie the observed differences in endocytosis mechanisms on the PBMs. This suggests that while PAI-1 and PAI-2 function similarly in the control of cellular plasmin generation by t-PA, they may have disparate effects on the alternative functions of t-PA via modulation of its engagement with endocytosis receptors.

Three Complement-Type Repeats of the Low-Density Lipoprotein Receptor-Related Protein Define a Common Binding Site for RAP, PAI-1, and Lactoferrin

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3277-3285 ◽  
Author(s):  
Brian Vash ◽  
Neil Phung ◽  
Sima Zein ◽  
Dianne DeCamp
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Dependent Manner ◽  
Related Protein ◽  
Low Density Lipoprotein Receptor ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein ◽  
Pai 1

The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding. © 1998 by The American Society of Hematology.

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