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)

We have studied conformational changes of type-1 plasminogen-activator inhibitor (PAI-1) during a temperature-dependent inhibitor-substrate transition by measuring susceptibility of the molecule to non-target proteinases. When incubated at 0 degree C instead of the normally used 37 degrees C, a tenfold decrease in the specific inhibitory activity of active PAI-1 was observed. Accordingly, PAI-1 was recovered in a reactive-centre-cleaved form from incubations with urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) at 0 degree C, but not at 37 degrees C. It thus behaved as a substrate for the target proteinases at the lower temperature. Active PAI-1 was exposed to a variety of non-target proteinases, including elastase, papain, thermolysin, trypsin, and V8 proteinase. It was found that specific peptide bonds in the reactive centre loop (RCL) and strand 5 in beta-sheet A (s5A) had a temperature-dependent proteolytic susceptibility, while the P17-P16 (E332-S333) bond, forming the hinge between s5A and the RCL, showed indistinguishable susceptibility to proteolysis by V8 proteinase at 0 degree and 37 degrees C. In latent and reactive-centre-cleaved PAI-1, all the bonds were resistant to proteolysis at the higher as well as the lower temperature. An anti-PAI-1 monoclonal antibody maintained the inhibitory activity of PAI-1 and prevented reactive centre cleavage at 0 degree C, and thus prevented substrate behaviour. Concomitantly, it caused specific changes in proteolytic susceptibility of s5A and the RCL, but it did not affect cleavage of the P17-P16 bond by V8 proteinase. Our observations suggest that temperature-dependent conformational changes of beta-sheet A and the RCL determine whether the serpin act as an inhibitor or a substrate. Furthermore they suggest that the RCL of PAI-1 is fully extracted from beta-sheet A in the inhibitory as well as in the substrate form, favoring a so-called induced conformational state model to explain why inhibitory activity requires partial insertion of the RCL into beta-sheet A.
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PMID:Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1. 889 86

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

PAI-1, the physiological inhibitor of tissue-type and urokinase-type plasminogen activator, is a unique member of the serpins as it exists in three distinct conformations: an active inhibitory conformation, a non-inhibitory substrate conformation, and a non-reactive latent conformation. Proline substitution of single residues in the P16-P20 region (situated at the proximal hinge of the reactive site loop) of wild-type PAI-1 (wtPAI-1) and a stabilized PAI-1-variant (PAI-1-stab; N150H, K154T, Q301P, Q319L, and M354I, t(1/2)=150), respectively, resulted in two series of PAI-1-variants with different properties. In wtPAI-1 only substitution at P18 resulted in a pronounced u-PA specificity and substrate behaviour towards t-PA. In contrast, in PAI-1-stab substitution at either P18, P19 or P20 resulted in a u-PA specificity and a significantly increased substrate behaviour towards t-PA and u-PA. Importantly, analysis of the kinetics of inhibition did not reveal any differences in the second-order rate constants of inhibition (k approximately 10(7)M(-1)s(-1)). The pronounced differences observed for identical mutations in wtPAI-1 vs PAI-1-stab demonstrate that not merely the sequence of the reactive loop but also intramolecular interactions between the hF/s3A-loop and the main part of the molecule govern the functional and conformational behaviour of PAI-1.
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PMID:Comparative analysis of the proteinase specificity in wild-type and stabilized plasminogen activator inhibitor-1: evidence for contribution of intramolecular flexibility. 1535 69