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)

Plasminogen activator (PA) is a key enzyme in control of the cascade of extracellular proteolytic activities, proteases that degrade the extracellular components. Mammalian cells produce two molecular forms of PA, the urokinase type (u-PA) and the tissue type (t-PA); the u-PA type enzyme regulates cell migration/invasion and related tissue plasticity events. Thus, these plasticity properties of cells are defined by their PAs' biochemical profiles. The capacity of the differentiating glial cells of the central nervous system (CNS) to express and regulate the two types of PA activities has been examined as a function of cell age in culture. Results of the study suggest that only the immature astrocyte is endowed with these plasticity properties. Differentiating heterogeneous rat glial cells in culture express PA activity. Astroglia were identified as the primary source for the glial PA activity, as no PA activity was detected in the purified oligodendroglia. Cellular PA activity levels of differentiating rat and mouse astroglia are developmentally regulated. The specific activity of PA reached its highest level in rat astroglia at a cell age corresponding to 20-32 postnatal days (P20-P32) and in mouse astroglia at P8-P14; thereafter, this declined (three- to fourfold decrease) within 2 weeks to a low value. At comparable ages (P0-P35), the magnitudes of the PA specific activities of the differentiating rat astroglia and of the developing cerebrum, the tissue from which these cells were purified, were similar. Differentiating rat astroglia produce u-PA and t-PA, the cellular content of both is developmentally regulated, and the u-PA form is only found in the immature cells. u-PA is the predominant form in the immature astrocyte until age P13. Both forms are found in cells at ages P14-P30, and at later stages u-PA disappears while the t-PA type persists as the sole form. After 3 more weeks neither of the PA types was detected. Astroglia express also PA inhibitory activity; the rat astroglial PA inhibitor (PAI) seemed to be identical to PAI-1, one of the known types of PAIs. Stimulation of astroglial proliferation by their subculturing in contrast to Schwann cells did not lead to an increase; rather, beyond a certain cell age (P13) it resulted in a threefold irreversible decline in the PA specific activity of the daughter cells. It has been established that various biochemical properties of CNS mature glia appear on schedule with cell age in culture, thus defining "mature"glia in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Developmental transition in plasticity properties of differentiating astrocytes: age-related biochemical profile of plasminogen activators in astroglial cultures. 214 27

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
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PMID:Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. 796 84

Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function.
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PMID:Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins. 912 38

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator.PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).
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PMID:Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2. 955 75

Plasminogen activator inhibitor-1 (PAI-1), an inhibitor of urokinase plasminogen activator, is paradoxically associated with a poor prognosis in breast cancer. PAI-1 is linked to several processes in the metastatic cascade. However, the role of PAI-1 in metastatic processes, which may be independent of protease inhibitory activity, is not fully understood. We report herein that PAI-1, when added exogenously to or stably transfected in human MDA-MB-435 breast carcinoma cells, had disparate effects on adhesion to extracellular matrix proteins and motility in vitro. Specifically, exogenously added PAI-1 inhibited cell adhesion to vitronectin but not fibronectin, in agreement with the literature. By contrast, stably transfected PAI-1 stimulated adhesion to both proteins. Wild-type PAI-1 was required for this stimulation, because expression of a non-protease inhibitory P14 (T333R) PAI-1 mutant failed to enhance adhesion. Compared with non-inhibitory PAI-1, wild-type PAI-1 also increased cell motility in chemotaxic assays. Furthermore, stable transfection of a related serine protease inhibitor, plasminogen activator inhibitor-3 (PAI-3, or protein C inhibitor) gave results similar to wild-type PAI-1. The stimulatory activity of PAI-3 was not seen with a non-protease inhibitory P14 PAI-3 mutant (T341R). We show that a downstream effect of endogenous wild-type PAI-1 and PAI-3 overexpression, but not their non-inhibitory counterparts, was the altered expression of alpha(2), alpha(3), alpha(4), alpha(5), and beta(1) integrin subunits. Additionally, blocking antibodies to beta(1) integrin inhibited PAI-1-induced adhesion. Our data provide experimental support for the stimulatory and inhibitory effects of PAI-1 in metastasis and introduce PAI-3 as another serpin potentially important in malignant disease.
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PMID:Plasminogen activator inhibitor-1 and -3 increase cell adhesion and motility of MDA-MB-435 breast cancer cells. 1217 77

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.
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PMID:A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition. 1235

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.
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PMID:Expression of active plasminogen activator inhibitor-1 reduces cell migration and invasion in breast and gynecological cancer cells. 1514 46

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.
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PMID:Characterization of recombinant human protein C inhibitor expressed in Escherichia coli. 1575 93

Plasminogen activator inhibitor-1 (PAI-1) regulates the proteolytic activity of urokinase-type plasminogen activator (uPA) and there is increasing evidence for a PAI-1 role in regulating cell migration/invasion by other mechanisms like vitronectin (VN) binding and lipoprotein-receptor related protein (LRP) binding. We examined the role of PAI-1 to interact with uPA, VN, and LRP in MDA-MB-435 breast cancer and SKOV-3 ovarian cancer cells in a wound-induced cell migration assay. By different experimental conditions with PAI-1, expressed either by stable transfection or by adenoviral infection (wild-type PAI-1 and an inactive reactive site P14 mutant, T333R) or by addition of recombinant PAI-1 and various mutants (stable wild-type, P14 T333R, reactive center P1 R346A, reduced VN-binding Q123K, and reduced LRP-binding R76E), we found that the PAI-1-VN interaction was foremost in suppressing wound-induced cell migration. Likewise, a VN-antibody that blocks cell adhesion to VN mimicked the effect of PAI-1 to reduce wound-induced SKOV-3 cell migration. However, manipulation of the endogenous plasminogen activator system (using blocking antibodies to PAI-1 and to uPA) in wound-induced SKOV-3 cell migration demonstrated an important role for uPA inhibition by PAI-1 that was dependent on plasminogen. By contrast, smooth muscle cell wound-induced migration was promoted by exogenously added PAI-1, but this enhancement was dependent on PAI-1 binding to LRP, not binding to VN. These findings contribute to the overall complexity of the role of PAI-1 in regulating cell migration; especially since both VN binding and uPA inhibition are involved in suppressing wound-induced breast and ovarian cancer cell migration.
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PMID:Wound-induced migration of MDA-MB-435 and SKOV-3 cancer cells is regulated by plasminogen activator inhibitor-1. 1607 25

Wild-type plasminogen activator inhibitor type 1 (PAI-1) is a fast-acting uPA and tPA inhibitor with half-life of 1-2 h. Recombinant PAI-1 with two mutations, Q197C and G355C, shows a very long half-life (VLHL). An introduced disulfide bridge holds together two central, parallel strands of beta-sheet A, preventing their separation to incorporate residues P4-P14 during the serpin's transition into latency. An active PAI-1 is usually described as a single structure with the reactive center loop (RCL) with P1-P1' (R369-M370) extended far from the bulk of the serpin's body. We have found that VLHL PAI-1 exists in several active forms that travel with different electrophoretic mobilities. Under aerobic conditions, two distinct active forms are observed. Upon reduction of cysteines, the VLHL mutant converts into the latent form, which spontaneously reactivates into a fully or partially active serpin, with yet another mobility. Utilizing electrophoresis, zymography (to check PAI-1 activity toward uPA) and theoretical calculations for molecular modeling, we have characterized active 1, 2, 3 and latent conformers of VLHL PAI-1 and their behaviors at normal and elevated temperatures, and in normal or reducing environments. VLHL PAI-1 activity is not affected, and the molecules do not polymerize unless reduced and/or heated. VLHL PAI-1 associates into dimers and bigger oligomers when -SH groups become available for oxidation and formation of intra- or intermolecular -S-S- bridges between conformers of different shapes and activities. We postulate that the active structures differ in RCL conformation and their position in relation to the gate region and the rest of the molecule.
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PMID:VLHL plasminogen activator inhibitor spontaneously reactivates from the latent to active form. 1908 7


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