Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 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

The serpin plasminogen activator inhibitor-1 (PAI-1) spontaneously adopts an inactive or latent conformation by inserting the N-terminal part of the reactive center loop as strand 4 into the major beta-sheet (sheet A). To examine factors that may regulate reactive loop insertion in PAI-1, we determined the inactivation rate of the inhibitor in the pH range 4.5-13. Below pH 9, inactivation led primarily to latent PAI-1, and one predominant effect of pH on the corresponding rate constant could be observed. Protonation of a group exhibiting a pKa of 7.6 (25 degrees C, ionic strength = 0.15 M) reduced the rate of formation of latent PAI-1 by a factor of 35, from 0.17 h-1 at pH 9 to about 0.005 h-1 below pH 6. The ionization with a pKa 7.6 was found to have no effect on the rate by which PAI-1 inhibits trypsin and is therefore unlikely to change the flexibility of the loop or the orientation of the reactive center. The peptides Ac-TEASSSTA and Ac-TVASSSTA (cf. P14-P7 in the reactive loop of PAI-1) formed stable complexes with PAI-1 and converted the inhibitor to a substrate for tissue type plasminogen activator. We found that peptide binding and formation of latent PAI-1 are mutually exclusive events, similarly affected by the pKa 7.6 ionization. This is direct evidence that external peptides can substitute for strand 4 in beta-sheet A of PAI-1 and that the pKa 7.6 ionization regulates insertion of complementary, internal or external, strands into this position. A model that accounts for the observed pH effects is presented, and the identity of the ionizing group is discussed based on the structure of latent PAI-1. The group is tentatively identified as His-143 in helix F, located on top of sheet A.
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PMID:The acid stabilization of plasminogen activator inhibitor-1 depends on protonation of a single group that affects loop insertion into beta-sheet A. 749 70

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

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.
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PMID:Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells. 892 13

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

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

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