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Factor VII-activating protease (FSAP) is a novel plasma-derived serine protease structurally homologous to tissue-type and urokinase-type plasminogen activators. We demonstrate that plasminogen activator inhibitor-1 (PAI-1), the predominant inhibitor of tissue-type and urokinase-type plasminogen activators in plasma and tissues, is an inhibitor of FSAP as well. We detected PAI-1.FSAP complexes in addition to high levels of extracellular RNA, an important FSAP cofactor, in bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome. Hydrolytic activity of FSAP was inhibited by PAI-1 with a second-order inhibition rate constant (K(a)) of 3.38 +/- 1.12 x 10(5) m(-1).s(-1). Residue Arg(346) was a critical recognition element on PAI-1 for interaction with FSAP. RNA, but not DNA, fragments (>400 nucleotides in length) dramatically enhanced the reactivity of PAI-1 with FSAP, and 4 microg.ml(-1) RNA increased the K(a) to 1.61 +/- 0.94 x 10(6) m(-1).s(-1). RNA also stabilized the active conformation of PAI-1, increasing the half-life for spontaneous conversion of active to latent PAI-1 from 48.4 +/- 8 min to 114.6 +/- 5 min. In contrast, little effect of DNA on PAI-1 stability was apparent. Residues Arg(76) and Lys(80) in PAI-1 were key elements mediating binding of nucleic acids to PAI-1. FSAP-driven inhibition of vascular smooth muscle cell proliferation was antagonized by PAI-1, suggesting functional consequences for the FSAP-PAI-1 interaction. These data indicate that extracellular RNA and PAI-1 can regulate FSAP activity, thereby playing a potentially important role in hemostasis and cell functions under various pathophysiological conditions, such as acute respiratory distress syndrome.

Disseminated intravascular coagulation (DIC), an acute syndrome of systemic thrombus formation in microvasculatures throughout the body, can be induced by severe infections, e.g. sepsis. Anticoagulants are clinically used to alleviate the intensities of DIC. However, anticoagulants only reduce the thrombus formation but have negligible effects on the inflammatory conditions. We previously reported embelin, a natural product, as an inhibitor of plasminogen activator inhibitor-1 (PAI-1), suggesting the potent antithrombotic property. In this study, we used three thrombotic mice models to confirm the antithrombotic property of embelin. By combining the anti-inflammatory and the antithrombotic properties, we proposed embelin as a potent therapeutic agent for sepsis-induced DIC, which involves both inflammation and thrombosis. In a lipopolysaccharides-induced septic mice model, embelin not only significantly ameliorated the inflammation levels, but also effectively reduced the pulmonary hemorrhages and the micro-thrombi formations in lung. In contrast, low-molecular-weight-heparin, an anticoagulant, only moderately ameliorated the pulmonary hemorrhages and thrombotic obstructions, but had non-measurable effect on the inflammatory conditions. In addition, embelin alleviated the dysregulation of the global coagulation in septic mice, but did not affect the global coagulation in normal mice. Our current study demonstrates the antithrombotic property of embelin and the potency of the treatment or prevention of syndromes combining inflammation and thrombosis, e.g. sepsis-induced DIC.

Some peptide sequences can behave as either substrates or inhibitors of serine proteases. Working with a cyclic peptidic inhibitor of the serine protease urokinase-type plasminogen activator (uPA), we have now demonstrated a new mechanism for an inhibitor-to-substrate switch. The peptide, CSWRGLENHAAC (upain-2), is a competitive inhibitor of human uPA, but is also slowly converted to a substrate in which the bond between Arg4 and Gly5 (the P1-P1′ bond) is cleaved. Substituting the P2 residue Trp3 to an Ala or substituting the P1 Arg4 residue with 4-guanidino-phenylalanine strongly increased the substrate cleavage rate. We studied the structural basis for the inhibitor-to-substrate switch by determining the crystal structures of the various peptide variants in complex with the catalytic domain of uPA. While the slowly cleaved peptides bound clearly in inhibitory mode, with the oxyanion hole blocked by the side chain of the P3′ residue Glu7, peptides behaving essentially as substrates with a much accelerated rate of cleavage was observed to be bound to the enzyme in substrate mode. Our analysis reveals that the inhibitor-to-substrate switch was associated with a 7 A translocation of the P2 residue, and we conclude that the inhibitor-to-substrate switch of upain-2 is a result of a major conformational change in the enzyme-bound state of the peptide. This conclusion is in contrast to findings with so-called standard mechanism inhibitors in which the inhibitor-to-substrate switch is linked to minor conformational changes in the backbone of the inhibitory peptide stretch.

Serine proteases play important roles in numerous physiological and pathophysiological processes. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms of enzymes. Here, we determined the crystal structures of a serine protease, murine plasma kallikrein (mPK), and its complex with a peptidic inhibitor. Although mPK in the complex adopts a canonical protease structure, the apo-mPK exhibits a previously unobserved structural feature: the entrance of the intact S1 pocket is blocked by Glu217. In addition, molecular dynamics simulations and functional assays support the flexibility of Glu217 and suggest that this flexibility plays a role in regulating the activity of serine proteases. Enzymes: EC: 3.4.21.34.

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

It is a wide-held assumption that professional development and change within purchasing and supply management (PSM) organisations can be explained and guided by a maturity model. In this paper the guidance which the maturity model concept offers to understand a PSM organisation's performance is assessed. The methodology is based on the outcomes of a literature review of PSM maturity models, development of an organisational change framework and the learning from three qualitative case studies. An alternative understanding of the development of the PSM organisation is offered through an organisational change framework, composing 1) movement transitions, 2) scalability of change, 3) acceptability of change, and 4) the substantive element of change. The research found that extant PSM maturity models are too rigid for PSM managers to apply, and although maturity models are commonly accepted in PSM literature, in practice, they may produce the opposite effect of what is promised. The PSM maturity models suggest that their application will lead to increased status and influence of PSM within the organisation; expectations that may not be met. PSM organisations’ change processes are subjected to a range of situational and contextual power relations which must be considered in order to advance the specific PSM organisation roles and responsibilities.

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases.

Urokinase-type plasminogen activator (uPA) is a diagnostic marker for breast and prostate cancers recommended by American Society for Clinical Oncology and German Breast Cancer Society. Inhibition of uPA was proposed as an efficient strategy for cancer treatments. In this study, we report peptide-based uPA inhibitors with high potency and specificity comparable to monoclonal antibodies. We revealed the binding and inhibitory mechanisms by combining crystallography, molecular dynamic simulation, and other biophysical and biochemical approaches. Besides, we showed that our peptides efficiently inhibited the invasion of cancer cells via intervening with the processes of the degradation of extracellular matrices. Furthermore, our peptides significantly suppressed the tumor growth and the cancer metastases in tumor-bearing mice. This study demonstrates that these uPA peptides are highly potent anticancer agents and reveals the mechanistic insights of these uPA inhibitors, which can be useful for developing other serine protease inhibitors.

Background The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce. Results Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)—and three-LU domain containing genes in general—occurred later in evolution and was first detectable after coelacanths. Conclusions This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members—hitherto unknown in mammals—provide new perspectives on the evolution of this important enzyme system. Electronic supplementary material The online version of this article (10.1186/s12862-019-1353-z) contains supplementary material, which is available to authorized users.

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. ispartof: Journal of Biological Chemistry vol:291 issue:29 pages:15156-15168 ispartof: location:United States status: published

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain).

Most serpins are fast and specific inhibitors of extracellular serine proteases controlling biological processes such as blood coagulation, fibrinolysis, tissue remodeling, and inflammation. The inhibitory activity of serpins is based on a conserved metastable structure and their conversion to a more stable state during reaction with the target protease. However, the metastable state also makes serpins vulnerable to mutations, resulting in disease caused by inactive and misfolded monomeric or polymeric forms ("serpinopathy"). Misfolding can occur either intracellularly (type-I serpinopathies) or extracellularly (type-II serpinopathies). We have isolated a 2'-fluoropyrimidine-modified RNA aptamer, which inhibits a mutation-induced inactivating misfolding of the serpin α1-antichymotrypsin. It is the first agent able to stabilize a type-II mutation of a serpin without interfering with the inhibitory mechanism, thereby presenting a solution for the long-standing challenge of preventing pathogenic misfolding without compromising the inhibitory function.

Fibrinolysis is a process responsible for the dissolution of formed thrombi to re‐establish blood flow after thrombus formation. Plasminogen activator inhibitor‐1 (PAI‐1) inhibits urokinase‐type and tissue‐type plasminogen activator (uPA and tPA) and is the major negative regulator of fibrinolysis. Inhibition of PAI‐1 activity prevents thrombosis and accelerates fibrinolysis. However, a specific antagonist of PAI‐1 is currently unavailable for therapeutic use. We screened a panel of uPA variants with mutations at and near the active site to maximize their binding to PAI‐1 and identified a potent PAI‐1 antagonist, PAItrap. PAItrap is the serine protease domain of urokinase containing active‐site mutation (S195A) and four additional mutations (G37bR–R217L–C122A–N145Q). PAItrap inhibits human recombinant PAI‐1 with high potency (K d = 0.15 nM) and high specificity. In vitro using human plasma, PAItrap showed significant thrombolytic activity by inhibiting endogenous PAI‐1. In addition, PAItrap inhibits both human and murine PAI‐1, allowing the evaluation in murine models. In vivo, using a laser‐induced thrombosis mouse model in which thrombus formation and fibrinolysis are monitored by intravital microscopy, PAItrap reduced fibrin generation and inhibited platelet accumulation following vascular injury. Therefore, this work demonstrates the feasibility to generate PAI‐1 inhibitors using inactivated urokinase.

Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7–18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19,20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19]. Keywords: Tpa, Serine protease, PAI-1, Serpin, Michaëlis complex, Crystal structure, Fibrinolysis, Thrombolytic agents, Structural biology

The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinasetype plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.

All serine proteases hydrolyze peptide bonds by the same basic mechanism and have very similar active sites, in spite of the fact that individual proteases have different physiological functions. We here report a strategy for designing high-affinity and high-specificity serine protease inhibitors using a versatile peptide scaffold, a 10-mer peptide, mupain-1 (CPAYSRYLDC). Mupain-1 was previously reported as a specific inhibitor of murine urokinase-type plasminogen activator (K(i) = 0.55 µM) without measurable affinity to plasma kallikrein (K(i) > 1000 µM). On the basis of a structure-based rational design, we substituted five residues of mupain-1 and converted it to a potent plasma kallikrein inhibitor (K(i) = 0.014 µM). X-ray crystal structure analysis showed that the new peptide was able to adapt a new set of enzyme surface interactions by a slightly changed backbone conformation. Thus, with an appropriate re-engineering, mupain-1 can be redesigned to specific inhibitors of other serine proteases.

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy.

SummaryPlasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin super-family 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 β–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.Supplementary Material to this article is available online at www.thrombosis-online.com.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.

Scientific reports 7(1), 3385(2017). doi:10.1038/s41598-017-03457-7, Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general., Published by Nature Publishing Group, London

RNA aptamers, selected from large synthetic libraries, are attracting increasing interest as protein ligands, with potential uses as prototype pharmaceuticals, conformational probes, and reagents for specific quantification of protein levels in biological samples. Very little is known, however, about their effects on protein conformation and dynamics. We have employed hydrogen/deuterium exchange (HDX) mass spectrometry to study the effect of RNA aptamers on the structural flexibility of the serpin plasminogen activator inhibitor-1 (PAI-1). The aptamers have characteristic effects on the biochemical properties of PAI-1. In particular, they are potent inhibitors of the structural transition of PAI-1 from the active state to the inactive, so-called latent state. This transition is one of the largest conformational changes of a folded protein domain without covalent modification. Binding of the aptamers to PAI-1 is associated with substantial and widespread protection against deuterium uptake in PAI-1. The aptamers induce protection against exchange with the solvent both in the protein-aptamer interface as well as in other specific areas. Interestingly, the aptamers induce substantial protection against exchange in α-helices B, C and I. This observation substantiates the relevance of structural instability in this region for transition to the latent state and argues for involvement of flexibility in regions not commonly associated with regulation of latency transition in serpins.

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.

Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities for elucidating fundamental allosteric mechanisms. The monoclonal antibody mU1 has previously been shown to be able to inhibit the function of murine urokinase-type plasminogen activator in vivo. We have now mapped the epitope of mU1 to the catalytic domain's 37- and 70-loops, situated about 20 Å from the S1 specificity pocket of the active site. Our data suggest that binding of mU1 destabilizes the catalytic domain and results in conformational transition into a state, in which the N-terminal amino group of Ile16 is less efficiently stabilizing the oxyanion hole and in which the active site has a reduced affinity for substrates and inhibitors. Furthermore, we found evidence for functional interactions between residues in uPA's C-terminal catalytic domain and its N-terminal A-chain, as deletion of the A-chain facilitates the mU1-induced conformational distortion. The inactive, distorted state is by several criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI‐1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI‐1 counteracts the negative feedback and behaves as a proteolysis‐triggered stabilizer of uPAR‐mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N‐terminal vitronectin fragments that can be detected in vivo , underscoring the potential physiological relevance of the process. ![][1] This study identifies a novel function for the plasminogen activation cascade in the regulation of cell adhesion. Urokinase and plasmin cleave the RGD motif of vitronectin in a process that is accelerated by the urokinase receptor and counteracted by the plasminogen activator inhibitor‐1. [1]: /embed/graphic-1.gif

RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 1016 different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. WIREs RNA 2016, 7:744-757. doi: 10.1002/wrna.1360 For further resources related to this article, please visit the WIREs website.

In drug development, molecular intervention strategies are usually based on interference with a single protein function, such as enzyme activity or receptor binding. However, in many cases, protein drug targets are multifunctional, with several molecular functions contributing to their pathophysiological actions. Aptamers and peptides are interesting synthetic building blocks for the design of multivalent molecules capable of modulating multiple functions of a target protein. Here, we report a molecular trap with the ability to interfere with the activation, catalytic activity, receptor binding, etc. of the serine protease urokinase-type plasminogen activator (uPA) by a rational combination of two RNA aptamers and a peptide with different inhibitory properties. The assembly of these artificial inhibitors into one molecule enhanced the inhibitory activity between 10- and 10,000-fold toward several functions of uPA. The study highlights the potential of multivalent designs and illustrates how they can easily be constructed from aptamers and peptides using nucleic acid engineering, chemical synthesis, and bioconjugation chemistry. By aptamer to aptamer and aptamer to peptide conjugation, we created, to the best of our knowledge, the first trivalent molecule which combines three artificial inhibitors binding to three different sites in a protein target. We hypothesize that by simultaneously preventing all of the functional interactions and activities of the target protein, this approach may represent an alternative to siRNA technology for a functional knockout.

The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases.

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.

Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. Typically, aptamers are identified from libraries containing up to 10(16) different RNA or DNA sequences by 5-10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.

To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1(PN-1) encoded by the SERPINE2 gene in different cell types.Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR.A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2'-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect.We have carefully mapped the CpG methylation pattern in two CpG islands in the 5' part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2′-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA–PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2′-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

To analyze the process of tumor cell intravasation, we used the human tumor-chick embryo spontaneous metastasis model to select in vivo high (PC-hi/diss) and low (PC-lo/diss) disseminating variants from the human PC-3 prostate carcinoma cell line. These variants dramatically differed in their intravasation and dissemination capacities in both chick embryo and mouse spontaneous metastasis models. Concomitant with enhanced intravasation, PC-hi/diss exhibited increased angiogenic potential in avian and murine models. PC-hi/diss angiogenesis and intravasation were dependent on increased secretion of vascular endothelial growth factor (VEGF), since treating developing tumors with a function-blocking anti-VEGF antibody simultaneously inhibited both processes without affecting primary tumor growth. PC-hi/diss cells were also more migratory and invasive, suggestive of heightened ability to escape from primary tumors due to matrix-degrading activity. Consistent with this suggestion, PC-hi/diss cells produced more of the serine protease urokinase-type plasminogen activator (uPA) as compared with PC-lo/diss. The functional role of uPA in PC-hi/diss dissemination was confirmed by inhibition of invasion, angiogenesis, and intravasation with specific function-blocking antibodies that prevented uPA activation and blocked uPA activity. These processes were similarly sensitive to aprotinin, a potent inhibitor of serine proteases, including uPA-generated plasmin. Thus, our comparison of the PC-3 intravasation variants points to key roles for the uPA-plasmin system in PC-hi/diss intravasation, possibly via (1) promoting tumor cell matrix invasion and (2) facilitating development of VEGF-dependent angiogenic blood vessels.

Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.

Background Activated protein C (APC) reduces mortality in severe sepsis. Protecting APC in the circulatory system from inactivation by serine protease inhibitors (serpins) could improve its therapeutic efficiency. Significantly elevated levels of a serpin plasminogen activator inhibitor 1 (PAI-1) correlate with a lethal outcome in severe sepsis and disseminated intravascular coagulation. Intermolecular mechanisms were employed to redirect the reaction between APC and PAI-1 from the inhibitory to the substrate pathway, which results in the catalytic neutralization of the serpin. Methods The effects of anti-PAI-1 monoclonal antibodies (mAbs) and vitronectin, as well as their fragments, on the kinetics and stoichiometry of the reaction between PAI-1 and APC were studied using SDS PAGE and fluorescence spectroscopy. Results MAbs with epitopes at α-helix F redirected 70–80% of the reaction between PAI-1 and APC, to the substrate pathway. Vitronectin and its SMB domain did not affect the stoichiometry of acyl-enzyme formation, but enhanced the effect of mAbs. While vitronectin induced a more than two-fold increase in the rate of the reaction between PAI-1 and APC, neither mAbs (mAb fragments), nor SMB domain of vitronectin affected it. Conclusions Ligands interacting with α-helix F of PAI-1 demonstrated a potential for the protection of APC from inactivation by PAI-1. Since the mechanism of proteinase/serpin interaction is universal, a similar design and approach could be employed for enhancing the inactivation of other serpins in order to preserve APC activity in the circulation. Rational pharmacological targeting of the inhibitors of APC could have therapeutic utility.

Plasminogen activator inhibitor-1 (PAI-1): A molecule at the crossroads to cell survival or cell death

Beginning in the early 90 es, evidence has been accumulating that a high level of plasminogen activator inhibitor-1 (PAI-1) protein in extracts of human primary malignant tumours is one of the most informative biochemical markers of a poor prognosis in several human cancer types. This observation has given the impetus to numerous studies of the role of PAI-1 in tumour growth, invasion, and metastasis. Recent mapping of cell types expressing PAI-1 in human tumours and studies with tumours growing on mice with targeted disruption of the PAI-1 gene have given results consistent with the idea that PAI-1 expressed by stromal fibroblasts and endothelial cells promotes tumour growth and spread. PAI-1 expressed by these cells therefore seems to be a potential therapeutic target in cancer. Confusingly, however, PAI-1 is also expressed by other cell types in tumours, and in some cancer types, the predominant PAI-1-expressing cells are the malignant epithelial cells themselves. Adding to the complexity is the fact that PAI-1 is not only a plasminogen activator inhibitor, but also engages in other molecular interactions, i.e., binds the extracellular matrix protein vitronectin and endocytosis receptors of the low density lipoprotein receptor family. Further progress towards the utilisation of PAI-1 as a therapeutic target in cancer will depend on understanding the role of PAI-1 expressed by different cell types in tumours and on development of compounds inhibiting separately each molecular interaction of PAI-1. The eventual use of PAI-1 as a therapeutic target will depend on mapping PAI-1 levels and PAI-1 expressing cell types in tumours of individual patients.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain).

Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·;PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·;PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1β pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation.