Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.73 (urokinase-type plasminogen activator)
 10,685 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and beta-trypsin by PAI-1. Alanine substitutions at the distal P4' (Glu-350) and P5' (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex formation (k(a)) and overall inhibition (k(app)) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation (k(lim)) increased by 3.3-fold. The effects of double mutations on k(a), k(lim), and k(app) were small with uPA and nonexistent with beta-trypsin. We provide the first kinetic evidence that the removal of exosite interactions significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into beta-sheet A. Moreover, mutational analysis indicates that the P5' residue contributes more to the mechanism of tPA inhibition, notably by promoting the formation of a final Michaelis complex.
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PMID:The contribution of the exosite residues of plasminogen activator inhibitor-1 to proteinase inhibition. 1459 4

In this letter we report the synthesis and biochemical evaluation of selective, irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator (uPA). A diphenyl phosphonate group was introduced on the substratelike peptide Z-d-Ser-Ala-Arg, and modification of the guanidine side chain was investigated. A guanylated benzyl group appeared the most promising side chain modification. A k(app) value in the 10(3) M(-1) s(-1) range for uPA was obtained, together with a selectivity index higher than 240 toward other trypsin-like proteases such as tPA, thrombin, plasmin, and FXa.
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PMID:Development of irreversible diphenyl phosphonate inhibitors for urokinase plasminogen activator. 1511 82

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.
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PMID:Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays. 1574 19

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alphavbeta3. Vitronectin (VN) induces cell migration by binding to alphavbeta3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alphavbeta3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alphavbeta3 and activates alphavbeta3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alphavbeta3 and uPAR-alpha5beta1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alphavbeta3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.
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PMID:Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity: generation of a new integrin inhibitor. 1586 11

The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.
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PMID:Cross-talk between fMLP and vitronectin receptors triggered by urokinase receptor-derived SRSRY peptide. 1586 65

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.
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PMID: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. 1614 Dec 8

[reaction: see text] A hydroxyethylene isostere of the tripeptide Arg-Gly-Leu, representing an important fragment of a novel cyclic-peptide-based uPA inhibitor, was synthesized in few steps employing as the key step a samarium diiodide promoted coupling of either the 4-thiopyridyl ester of N(alpha)-Fmoc- or N(alpha)-Cbz-protected L-ornithine with the N-acryloyl derivative of L-leucine methyl ester. Epimerization under the coupling conditions at the chiral center in the alpha-position to the ketone was demonstrated not to take place. A stereoselective reduction of the Cbz-protected aminoketone obtained from this radical reaction was promoted by the same single-electron reducing agent in the presence of methanol providing the syn-amino alcohol with a diastereoselectivity of 85:15. With the use of lithium tri-tert-butoxyaluminum hydride in methanol, the corresponding anti-isomer was obtained almost exclusively. Subsequent elaboration of the ornithine moiety in the anti-isomer by introduction of the guanidine group followed by hydrolysis of the C-terminal ester bond and protection of the alcohol as its tert-butyldimethylsilyl ether provided the desired tripeptide mimic. The long reaction times required for the radical addition reactions with N(delta)-Boc-L-ornithine (up to 5 days) led to a short study where a series of 4-thiopyridyl esters of Cbz-protected amino acids were reacted with two acrylates. Whereas N(delta)-Boc-L-ornithine, alanine, phenylalanine, proline, and leucine all provided the aminoketone in 43-79% yield, valine only afforded traces of the coupling product.
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PMID:Synthesis of a hydroxyethylene isostere of the tripeptide Arg-Gly-Leu via a convergent acyl-like radical addition strategy. 1614 78

The charge of Lys300(c143) located within a flexible loop(297-313) of sc-uPA has been identified as an important determinant for its high intrinsic activity. Mutations affecting the flexibility of the loop also modulate the intrinsic activity. Glu-plasminogen activation by sc-uPA is strongly promoted by fibrin fragment E but not fibrin fragment D-dimer, whereas plasminogen activation by t-PA is strongly promoted by fragment D-dimer but not fragment E. To further investigate the effect of conformation changes in the flexible loop on catalytic properties of sc-uPA, cassette mutations at Pro309(c152) were made and characterized. It was found that the activation of Pro309(c152) mutants by Lys-plasmin was only moderately affected. In contrast, the intrinsic and two-chain activities of Pro309(c152) mutants against S2444 were both significantly decreased. The two-chain activities of these mutants against Glu-plasminogen were also reduced in a range of 1.1- to 127-fold. The mutations of Pro309(c152) to Trp/Phe and Arg/Asp more significantly affected both intrinsic and two-chain activities, while only a moderate decrease in activity was found with mutations to Ala/Ser/Thr. In contrast to wild-type sc-uPA, plasminogen activation by Pro309(c152) mutants was found to be promoted by both fibrin fragment E and D-dimer. In the presence of 2.0 microM D-dimer, plasminogen activation by mutant Pro309(c152) --> His was promoted by 22-fold, while only 2.0-fold promotion was found with mutant Pro309(c152) --> Gly. In conclusion, these findings demonstrated that conformation changes in the flexible loop of sc-uPA not only affect its intrinsic and two-chain activity, but also extend its promotion of plasminogen activation by fragment E to D-dimer.
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PMID:Mutagenesis at Pro309 of single-chain urokinase-type plasminogen activator alters its catalytic properties. 1623 30

The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1' Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1' Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1' Thr increased thrombosis. Substitution of Serp-1 P2-P7 with Ala6 allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.
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PMID:Identification of myxomaviral serpin reactive site loop sequences that regulate innate immune responses. 1640 26

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55 degrees C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), beta-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bbeta-chains and Aalpha-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.
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PMID:Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. 1657 57


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