Analysis of cortical cell polarity by imaging flow cytometry.

Metastasis is the main cause of cancer-related death and therapies specifically targeting metastasis are highly needed. Cortical cell polarity (CCP) is a prometastatic property of circulating tumor cells affecting their ability to exit blood vessels and form new metastases that constitute a promising point of attack to prevent metastasis. However, conventional fluorescence microscopy on single cells and manual quantification of CCP are time-consuming and unsuitable for screening regulators. In this study, we developed an imaging flow cytometry-based method for high-throughput screening of factors affecting CCP in melanoma cells. The artificial intelligence-supported analysis method we developed is highly reproducible, accurate, and orders of magnitude faster than manual quantification. Additionally, this method is flexible and can be adapted to include additional cellular parameters. In a small-scale pilot experiment using polarity-, cytoskeleton-, or membrane-affecting drugs, we demonstrate that our workflow provides a straightforward and efficient approach for screening factors affecting CCP in cells in suspension and provide insights into the specific function of these drugs in this cellular system. The method and workflow presented here will facilitate large-scale studies to reveal novel cell-intrinsic as well as systemic factors controlling CCP during metastasis.

Human IFT52 mutations uncover a novel role for the protein in microtubule dynamics and centrosome cohesion.

Mutations in genes encoding components of the intraflagellar transport (IFT) complexes have previously been associated with a spectrum of diseases collectively termed ciliopathies. Ciliopathies relate to defects in the formation or function of the cilium, a sensory or motile organelle present on the surface of most cell types. IFT52 is a key component of the IFT-B complex and ensures the interaction of the two subcomplexes, IFT-B1 and IFT-B2. Here, we report novel IFT52 biallelic mutations in cases with a short-rib thoracic dysplasia (SRTD) or a congenital anomaly of kidney and urinary tract (CAKUT). Combining in vitro and in vivo studies in zebrafish, we showed that SRTD-associated missense mutation impairs IFT-B complex assembly and IFT-B2 ciliary localization, resulting in decreased cilia length. In comparison, CAKUT-associated missense mutation has a mild pathogenicity, thus explaining the lack of skeletal defects in CAKUT case. In parallel, we demonstrated that the previously reported homozygous nonsense IFT52 mutation associated with Sensenbrenner syndrome [Girisha et al. (2016) A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin. Genet., 90, 536-539] leads to exon skipping and results in a partially functional protein. Finally, our work uncovered a novel role for IFT52 in microtubule network regulation. We showed that IFT52 interacts and partially co-localized with centrin at the distal end of centrioles where it is involved in its recruitment and/or maintenance. Alteration of this function likely contributes to centriole splitting observed in Ift52-/- cells. Altogether, our findings allow a better comprehensive genotype-phenotype correlation among IFT52-related cases and revealed a novel, extra-ciliary role for IFT52, i.e. disruption may contribute to pathophysiological mechanisms.

Biochemical and structural analyses suggest that plasminogen activators coevolved with their cognate protein substrates and inhibitors.

Protein sequences of members of the plasminogen activation system are present throughout the entire vertebrate phylum. This important and well-described proteolytic cascade is governed by numerous protease-substrate and protease-inhibitor interactions whose conservation is crucial to maintaining unchanged protein function throughout evolution. The pressure to preserve protein-protein interactions may lead to either co-conservation or covariation of binding interfaces. Here, we combined covariation analysis and structure-based prediction to analyze the binding interfaces of urokinase (uPA):plasminogen activator inhibitor-1 (PAI-1) and uPA:plasminogen complexes. We detected correlated variation between the S3-pocket-lining residues of uPA and the P3 residue of both PAI-1 and plasminogen. These residues are known to form numerous polar interactions in the human uPA:PAI-1 Michaelis complex. To test the effect of mutations that correlate with each other and have occurred during mammalian diversification on protein-protein interactions, we produced uPA, PAI-1, and plasminogen from human and zebrafish to represent mammalian and nonmammalian orthologs. Using single amino acid point substitutions in these proteins, we found that the binding interfaces of uPA:plasminogen and uPA:PAI-1 may have coevolved to maintain tight interactions. Moreover, we conclude that although the interaction areas between protease-substrate and protease-inhibitor are shared, the two interactions are mechanistically different. Compared with a protease cleaving its natural substrate, the interaction between a protease and its inhibitor is more complex and involves a more fine-tuned mechanism. Understanding the effects of evolution on specific protein interactions may help further pharmacological interventions of the plasminogen activation system and other proteolytic systems.

Crystal structure of tetrameric human Rabin8 GEF domain.

Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF-domain for in vitro activity assays.

Latency transition of plasminogen activator inhibitor type 1 is evolutionarily conserved.

Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central ß-sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI-1 variants undergo latency transition with a similar rate. Since the functional stability of PAI-1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.

Distinctive binding modes and inhibitory mechanisms of two peptidic inhibitors of urokinase-type plasminogen activator with isomeric P1 residues.

Two isomeric piperidine derivatives (meta and para isomers) were used as arginine mimics in the P1 position of a cyclic peptidic inhibitor (CPAYSRYLDC) of urokinase-type plasminogen activator. The two resulting cyclic peptides showed vastly different affinities (∼70 fold) to the target enzyme. X-ray crystal structure analysis showed that the two P1 residues were inserted into the S1 specificity pocket in indistinguishable manners. However, the rest of the peptides bound in entirely different ways on the surface of the enzyme, and the two peptides have different conformations, despite the highly similar sequence. These results demonstrate how the subtle difference in P1 residue can dictate the exosite interactions and the potencies of peptidic inhibitors, and highlight the importance of the P1 residue for protease inhibition. This study provides important information for the development of peptidic agents for pharmacological intervention.

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Bicyclic peptide inhibitor of urokinase-type plasminogen activator: mode of action.

The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide-protease interactions will aid future designs of bicyclic peptide protease inhibitors.

Urokinase-type plasminogen activator-like proteases in teleosts lack genuine receptor-binding epidermal growth factor-like domains.

Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed to be central to the functions of uPA, as uPA-catalyzed plasminogen activation activity appeared to be confined to cell surfaces through the binding of uPA to uPAR. However, a functional uPAR has so far only been identified in mammals. We have now cloned, recombinantly produced, and characterized two zebrafish proteases, zfuPA-a and zfuPA-b, which by several criteria are the fish orthologs of mammalian uPA. Thus, both proteases catalyze the activation of fish plasminogen efficiently and both proteases are inhibited rapidly by plasminogen activator inhibitor-1 (PAI-1). But zfuPA-a differs from mammalian uPA by lacking the exon encoding the uPAR-binding epidermal growth factor-like domain; zfuPA-b differs from mammalian uPA by lacking two cysteines of the epidermal growth factor-like domain and a uPAR-binding sequence comparable with that found in mammalian uPA. Accordingly, no zfuPA-b binding activity could be found in fish white blood cells or fish cell lines. We therefore propose that the current consensus of uPA-catalyzed plasminogen activation taking place on cell surfaces, derived from observations with mammals, is too narrow. Fish uPAs appear incapable of receptor binding in the manner known from mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation.

The binding mechanism of a peptidic cyclic serine protease inhibitor.

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.

Elucidation of the contribution of active site and exosite interactions to affinity and specificity of peptidylic serine protease inhibitors using non-natural arginine analogs.

There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.

RNA aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor-1.

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.

Nonproteolytic induction of catalytic activity into the single-chain form of urokinase-type plasminogen activator by dipeptides.

Serine proteases are initially synthesized as single-chain proenzymes with activities that are many orders of magnitude lower than those of the mature enzyme. Proteolytic cleavage of an exposed loop liberates a new amino terminus that inserts into a hydrophobic pocket and forms a stabilizing salt bridge with a ubiquitously conserved aspartate residue, resulting in a conformational change organizing the mature oxyanion hole. In a decisive 1976 work, Huber and Bode [Bode, W., and Huber, R. (1976) FEBS Lett. 68, 231-236] demonstrated that peptides sequentially similar to the new amino terminus in combination with a catalytic site inhibitor could specifically induce a trypsin-like conformation in trypsinogen. We now demonstrate that an Ile-Ile or Ile-Val dipeptide can induce limited enzyme activity in the single-chain zymogen form of urokinase-type plasminogen activator (uPA) or its K158A variant, which cannot be activated proteolytically. Furthermore, the slow formation of a covalent serpin-protease complex between single-chain uPA and PAI-1 is significantly accelerated in the presence of specific dipeptide sequences. The technique of using a dipeptide mimic as a surrogate for the liberated amino terminus further provides a novel means by which to covalently label the immature active site of single-chain uPA with a fluorescent probe, permitting fluorescence approaches for direct observations of conformational changes within the protease domain during zymogen activation. These data demonstrate the structural plasticity of the protease domain, reinforce the notion of "molecular sexuality", and provide a novel way of studying conformational changes of zymogens during proteolytic activation.

Characterization of a site on PAI-1 that binds to vitronectin outside of the somatomedin B domain.

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between alpha-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a K(D) value approximately 100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.

A peptide accelerating the conversion of plasminogen activator inhibitor-1 to an inactive latent state.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and a potential therapeutic target in cancer and cardiovascular diseases. Accordingly, formation of a basis for development of specific PAI-1-inactivating agents is of great interest. One possible inactivation mode for PAI-1 is conversion to the inactive, so-called latent state. We have now screened a phage-displayed peptide library with PAI-1 as bait and isolated a 31-residue cysteine-rich peptide that will be referred to as paionin-4. A recombinant protein consisting of paionin-4 fused to domains 1 and 2 of the phage coat protein g3p caused a 2- to 3-fold increase in the rate of spontaneous inactivation of PAI-1. Paionin-4-D1D2 bound PAI-1 with a K(D) in the high nanomolar range. Using several biochemical and biophysical methods, we demonstrate that paionin-4-D1D2-stimulated inactivation consists of an acceleration of conversion to the latent state. As demonstrated by site-directed mutagenesis and competition with other PAI-1 ligands, the binding site for paionin-4 was localized in the loop between alpha-helix D and beta-strand 2A. We also demonstrate that a latency-inducing monoclonal antibody has an overlapping, but not identical binding site, and accelerates latency transition by another mechanism. Our results show that paionin-4 inactivates PAI-1 by a mechanism clearly different from other peptides, small organochemical compounds, or antibodies, whether they cause inactivation by stimulating latency transition or by other mechanisms, and that the loop between alpha-helix D and beta-strand 2A can be a target for PAI-1 inactivation by different types of compounds.

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 alpha-helix D and alpha-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.

Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C.

Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule.

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 K(i), which at 25 degrees C and pH 7.4 was approximately 500 nm. At the same conditions, the equilibrium dissociation constant K(D), monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was approximately 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 Arg(4) of upain-1 as the P(1) residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P(1), P(2), P(3)', P(4)', and the P(5)' residues of upain-1. Substitution with alanine of the P(2) residue, Trp(3), 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 P(2) 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.

Plasminogen activator inhibitor-I and tumour growth, invasion, and metastasis.

In recent decades, evidence has been accumulating showing the important role of urokinase-type plasminogen activator (uPA) in growth, invasion, and metastasis of malignant tumours. The evidence comes from results with animal tumour models and from the observation that a high level of uPA in human tumours is associated with a poor patient prognosis. It therefore initially came as a surprise that a high tumour level of the uPA inhibitor plasminogen activator inhibitor-I (PAI-I) is also associated with a poor prognosis, the PAI-I level in fact being one of the most informative biochemical prognostic markers. We review here recent investigations into the possible tumour biological role of PAI-I, performed by animal tumour models, histological examination of human tumours, and new knowledge about the molecular interactions of PAI-I possibly underlying its tumour biological functions. The exact tumour biological functions of PAI-I remain uncertain but PAI-I seems to be multifunctional as PAI-I is expressed by multiple cell types and has multiple molecular interactions. The potential utilisation of PAI-I as a target for anti-cancer therapy depends on further mapping of these functions.

Biochemical importance of glycosylation of plasminogen activator inhibitor-1.

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC(50)-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosylated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended specifically on glycosylation of either one or the other of the utilised sites. The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites. Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1-inactivating compounds of potential clinical importance.