High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor–related protein 1 requires lysines 80, 88, and 207

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Abstract

It is well-established that complexes of plasminogen-activator inhibitor 1 (PAI-1) with its target enzymes bind tightly to low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), but the molecular details of this interaction are not well-defined. Furthermore, considerable controversy exists in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In this study, we examined the binding of free PAI-1 and complexes of PAI-1 with low-molecular-weight urokinase-type plasminogen activator to LRP1. Our results confirmed that uPA:PAI-1 complexes bind LRP1 with ∼100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirmed an essential requirement of lysine residues in PAI-1 for the interactions of both PAI-1 and uPA:PAI-1 complexes with LRP1. Results of surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and uPA:PAI-1 complexes interact with complementary sites on LRP1. An ionic-strength dependence of binding suggested the critical involvement of two charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of uPA:PAI-1 complexes with LRP1. An enhanced affinity resulting from the interaction of three regions of the uPA:PAI-1 complex with LDLa repeats on LRP1 provided an explanation for the increased affinity of uPA:PAI-1 complexes for LRP1. Mutational analysis revealed an overlap between LRP1 binding and binding of a small-molecule inhibitor of PAI-1, CDE-096, confirming an important role for Lys-207 in the interaction of PAI-1 with LRP1 and of the orientations of Lys-207, -88, and -80 for the interaction of uPA:PAI-1 complexes with LRP1.

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VADAR: a web server for quantitative evaluation of protein structure quality.

Leigh Willard, Anuj Ranjan, Haiyan Zhang … (2003)

VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and/or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible surface area, backbone and side chain dihedral angles, secondary structure, hydrogen bonding partners, hydrogen bond energies, steric quality, solvation free energy as well as local and overall fold quality. These derived parameters can be used to rapidly identify both general and residue-specific problems within newly determined protein structures. The VADAR web server is freely accessible at http://redpoll.pharmacy.ualberta.ca/vadar.

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Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnants.

Rachel R. Hammer, J. Herz, M. Gotthardt … (1998)

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) has been postulated to participate in a number of diverse physiological and pathological processes ranging from the homeostasis of plasma lipoproteins, atherosclerosis, and fibrinolysis to neuronal regeneration and survival. It has not been possible to demonstrate in vivo the physiological significance of LRP for each of these complex processes by a conventional gene knockout approach because LRP is essential for embryonic development. Here we have used the Cre/loxP recombination system to achieve inducible, tissue-specific and quantitative disruption of the LRP gene in adult mice. Inactivation of LRP in the livers of LDL receptor-deficient mice resulted in the accumulation of cholesterol-rich remnant lipoproteins in the circulation. In normal animals, this caused a compensatory upregulation of the LDL receptor in the liver. Conditional gene targeting has thus allowed us to isolate a specific physiological function of LRP for in vivo analysis and has provided unequivocal evidence for another LDL receptor-independent cholesterol clearance pathway in liver.

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The Role of Plasminogen Activator Inhibitor Type-1 in Fibrosis.

Panagiotis Flevaris, Douglas S. Vaughan (2017)

Extracellular matrix (ECM) deposition during wound healing is a physiological response to an insult. Wound healing becomes deregulated in the setting of chronic injury or long-standing metabolic disease, leading to the accumulation of ECM components and fibrosis. Matrix protein turnover is determined by the rate of synthesis as well as the rate of proteolytic degradation and clearance by matrix metalloproteinases (MMPs). The persistent activation of interstitial myofibroblasts, coupled with defects in matrix proteolysis, ultimately disrupts tissue architecture and leads to biochemical and mechanical organ dysfunction with eventual organ failure. Plasminogen activator inhibitor type-1 (PAI-1) regulates tissue homeostasis and wound healing by inhibiting plasmin-mediated MMP activation. Multiple reports using models of liver, lung, and kidney fibrosis suggest that PAI-1 deficiency or inhibition of PAI-1 activity attenuates fibrosis. The disinhibition of plasmin-mediated MMP activation leads to collagen degradation and its diminished accumulation, resulting in the reduction of fibrotic matrix deposition in these organs. Paradoxically, homozygous deficiency of PAI-1 promotes age-dependent spontaneous cardiac fibrosis, suggesting a protective role for PAI-1 in the heart. It remains unclear whether PAI-1-deficient cardiac fibroblasts have increased proliferative, migratory, or differentiation capabilities, that allow them to overcome increased plasmin and MMP activity and matrix clearance. In this review, we examine the specific roles of PAI-1 in fibrosis of different organs including the lung, liver, kidney, and cardiovascular system.