Helle H. Petersen

The plasminogen activation system in tumor growth, invasion, and metastasis

Abstract. Generation of the serine proteinase plasmin from the extracellular zymogen plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type plasminogen activators (uPA and tPA). The plasminogen activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the plasminogen activation system may allow utilization of its components as targets for anti-invasive therapy.

A Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptide with an Unusual P2 Residue and an Extended Protease Binding Surface Demonstrates New Modalities for Enzyme Inhibition

To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent Ki, which at 25 °C and pH 7.4 was ∼500 nm. At the same conditions, the equilibrium dissociation constant KD, monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ∼400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg4 of upain-1 as the P1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P1, P2, P3′, P4′, and the P5′ residues of upain-1. Substitution with alanine of the P2 residue, Trp3, converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.

Binding areas of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis.

Some endocytosis receptors related to the low‐density lipoprotein receptor, including low‐density lipoprotein receptor‐related protein‐1A, very‐low‐density lipoprotein receptor, and sorting protein‐related receptor, bind protease‐inhibitor complexes, including urokinase‐type plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), and the uPA–PAI‐1 complex. The unique capacity of these receptors for high‐affinity binding of many structurally unrelated ligands renders mapping of receptor‐binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor‐binding area of the uPA–PAI‐1 complex by site‐directed mutagenesis. Substitution of a cluster of basic residues near the 37‐loop and 60‐loop of uPA reduced the receptor‐binding affinity of the uPA–PAI‐1 complex approximately twofold. Deletion of the N‐terminal growth factor domain of uPA reduced the affinity 2–4‐fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA–PAI‐1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in α‐helix D and α‐helix E of PAI‐1. The localization of the implicated residues in the 3D structures of uPA and PAI‐1 shows that they form a continuous receptor‐binding area spanning the serpin as well as the A‐chain and the serine protease domain of uPA. Our results suggest that the 10–100‐fold higher affinity of the uPA–PAI‐1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI‐1 together on the same binding entity.

150 Breast carcinoma epithelial cells express a very low density lipoprotein receptor variant lacking the olinked glycosylation domain encoded by exon 16, but with normal binding activity for serine proteinase/serpin complexes and Mr 40,000 receptor associated protein

Very-low density lipoprotein receptor (VLDLR) belongs to the low-density lipoprotein receptor family of endocytosis receptors. It binds a variety of different ligands, including apolipoprotein E, Mr-40,000 receptor-associated-protein (RAP), and some serine proteinase/serpin complexes. We previously demonstrated the occurrence of two forms of VLDLR in SDS/PAGE, migrating with Mr 105,000 and Mr 130,000, respectively [Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjøller, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. & Andreasen, P. A. (1995) J. Biol. Chem. 270, 20,855-20,869]. We now demonstrate that these two forms correspond to forms with the absence (type-II) and presence (type-I) of the O-linked glycosylation domain encoded by exon 16, respectively. We show that the two forms have the same binding affinity to RAP and serine proteinase/serpin complexes. Using reverse transcription and PCR, we demonstrate that the splice variation giving rise to the two forms is highly cell specific. In particular, we demonstrate that human breast carcinomas express predominantly or exclusively the variant lacking exon 16. By immunohistochemistry, we demonstrate that VLDLR is mainly expressed by the epithelial cancer cells in these carcinomas. The VLDLR variant expressed by epithelial cancer cells could function in the clearance of cell-surface-associated serine proteinase/serpin complexes in breast carcinomas.