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1.
Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.  相似文献   

2.
The lysosomal cysteine proteinases cathepsin L and cathepsin B were examined for their effect on the neutrophil elastase inhibitory activity of human alpha 1-proteinase inhibitor (alpha 1PI). Human cathepsin L catalytically inactivated human alpha 1PI by cleavage of the bonds Glu354-Ala355 and Met358-Ser359 (the serine proteinase inhibitory site). Cathepsin B did not inactivate alpha 1PI, even when equimolar amounts of enzyme were employed. Cathepsin L is the first human proteinase shown to catalytically inactivate alpha 1PI. These findings, in conjunction with other reports, suggest that alpha 1PI contains a proteolytically sensitive region encompassing residues 350-358. Taken together with the discovery of the elastinolytic activity of cathepsin L (Mason, R. W., Johnson, D. A., Barrett, A. J., and Chapman, H. A. (1986) Biochem. J. 233, 925-927), the present findings emphasize the possible importance of cathepsin L in the pathological proteolysis of elastin and diminish the role that can be attributed to cathepsin B in such processes.  相似文献   

3.
Hsieh MC  Cooperman BS 《Biochemistry》2002,41(9):2990-2997
Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.  相似文献   

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It has been found that two active in neutral medium thiol proteinases from bovine spleen, cathepsin L and cathepsin H, bring about rapid and irreversible inactivation of alpha 1-proteinase inhibitor (alpha 1PI)--one of the major plasma inhibitors of serine proteinases. The activity of the enzymes studied did not change upon the interaction with alpha 1PI. With stoichiometric proteinase/inhibitor ratio, the inactivation of alpha 1PI under the effect of cathepsin L was instantaneous, while under the effect of cathepsin H it occurred within 30-60 min. The products of alpha 1PI inactivation had an inhibitory effect on the rate of its reaction with cathepsin L. alpha 1PI inactivation under the action of cathepsin L and cathepsin H was accompanied by the decrease in the molecular mass of the inhibitor from 54 kDA to 46 kDa. This was, probably, caused by the hydrolysis of the peptide bond formed by NH2 group of threonine. The 46 kDa fragment did not undergo further degradation. It did not bind to immobilized trypsin but retained antigenic properties. The results obtained show that the limited proteolysis is a mechanism of the inhibitor inactivation. It is suggested that under some conditions thiol proteinases, upon their release from the cell, participate in the control of effective alpha 1PI concentration.  相似文献   

8.
alpha(1)-Protease inhibitor (alpha(1)PI), the most abundant serine protease inhibitor found in human plasma (at 30-60 microM), is a glycoprotein (53 kDa) having a single cysteine residue at position 232 (Cys(232)). We have found that Cys(232) of human alpha(1)PI was readily S-nitrosylated by nitric oxide (NO) without affecting inhibitory activity to trypsin or elastase. S-nitrosylated alpha(1)PI (S-NO-alpha(1)PI) not only retained inhibitory activity against these serine proteases, but also gained thiol protease inhibitory activity against a Streptococcus pyogenes protease; the parental alpha(1)PI did not have this activity. Furthermore, S-NO-alpha(1)PI exhibited bacteriostatic activity against Salmonella typhimurium at concentrations of 0.1-10 microM, which were 20- to 3000-fold stronger than those of the other NO-generating compounds or S-nitroso compounds such as S-nitrosoalbumin and S-nitrosoglutathione. NO appears to be transferred into the bacterial cells from S-NO-alpha(1)PI via transnitrosylation, as evidenced by electron spin resonance spectroscopy with an NO spin trap. Thus, we conclude that S-NO-alpha(1)PI may be generated from the reaction between alpha(1)PI and NO under inflammatory conditions, in which production of both is known to increase. As a result, new functions, i.e., antibacterial and thiol protease inhibitory activities of alpha(1)PI, were generated.  相似文献   

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Alpha(1)-protease inhibitor (alpha(1)PI) is an acute phase plasma protein, and possesses a single cysteine residue at position 232. A single cysteinyl sulfhydryl of human alpha(1)PI is found to be readily S-nitrosylated by nitric oxide (NO) in vitro without affecting the inhibitory capacity against bovine trypsin or elastase, a major target protease of alpha(1)PI in vivo. S-nitroso-alpha(1)PI (S-NO-alpha(1)PI) was also formed by the reaction of alpha(1)PI with NO produced excessively by a murine macrophage cell line (RAW264 cells) upon infection with Salmonella typhimurium and in an ex vivo perfusion system of the liver obtained from lipopolysaccharide-treated rats. S-NO-alpha(1)PI (10(-9)-10(-6) M) induces a dose-dependent relaxation of the ring preparation of rabbit aorta. Also, S-NO-alpha(1)PI but not alpha(1)PI shows a potent inhibitory effect on platelet aggregation. Unprecedented observation is that S-NO-alpha(1)PI showed a potent bacteriostatic effect against a wide range of bacteria at the concentration of 1-10 microM, which was 10-1000-fold stronger than that of NO and other S-nitrosylated compounds including S-nitrosylated albumin and S-nitrosylated glutathione. These results suggest that S-NO-alpha(1)PI is produced as an NO sink under inflammatory conditions, where production of both alpha(1)PI and NO is highly up-regulated, and it may function as a soluble factor which consists of an innate defense system through not only the protease inhibitory activity but also its antibacterial activity and facilitating the peripheral blood flow. Therefore, S-nitrosylation of alpha(1)PI occurring under physiological conditions in vivo should diversify the biological functions contributing to cytoprotective effects of alpha(1)PI.  相似文献   

11.
Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.  相似文献   

12.
IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the beta-adrenergic receptor kinase or the Deltap85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the alpha(1B)-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced alpha(1B)-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation. The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gbetagamma subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in alpha(1B)-AR phosphorylation and desensitization.  相似文献   

13.
Coagulation and complement proteinases are activated in sepsis, and one approach to therapy is to develop proteinase inhibitors that will specifically inhibit these proteinases without inhibiting activated protein C, a proteinase that is beneficial to survival. In this study, we made mutants of the serpin alpha(1)-PI, designed to mimic the specificity of C1-inhibitor. The P3-P2-P1 residues of alpha1-PI were changed from IPM to LGR and PFR, sequences preferred by C1s and kallikrein, respectively. Inhibition of C1s, kallikrein, factor XIIa, and activated protein C was assessed by SDS-PAGE, and by determination of the k(app) and SI. alpha(1)-PI-LGR inhibited C1s with a rate of 7790 M(-1)s(-1), but only minimal inhibition of C1 in a hemolytic assay was observed. Kallikrein, factor XIIa, and activated protein C were inhibited with rates of 382,180 M(-1)s(-1), 10,400 M(-1)s(-1), and 3500 M(-1)s(-1), respectively. alpha(1)-PI-PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4' residue of alpha(1)-PI-LGR Pro to Glu reduced the activity with C1s, consistent with the idea that C1s requires hydrophobic residues in this region of the serpin for optimal interaction. The data provide insight into the requirements for kallikrein and C1s inhibition necessary for designing inhibitors with appropriate properties for further investigation as therapeutic agents.  相似文献   

14.
Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.  相似文献   

15.
Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.  相似文献   

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The crystal structure of proteolytically modified human alpha 1-antichymotrypsin (ACT), a member of the serpin superfamily, has been solved by Paterson search techniques and refined to an R-factor of 18.0% at 2.7 A resolution with mean deviations from standard bond lengths and angles of 0.013 A and 3.1 degrees, respectively. The final model consists of 374 amino acid residues, 126 solvent molecules and five sugar residues. Asn70 could be identified unambiguously as a glycosylation site and Asn104 is probably also glycosylated. The structure of cleaved ACT is compared with cleaved alpha 1-antitrypsin (alpha 1 PI) and with plakalbumin, which are prototypical models for cleaved and intact serpins, respectively. Cleaved ACT is very similar to cleaved alpha 1 PI; in particular, it has strand s4A, which is liberated by proteolysis, inserted as the middle strand in beta-sheet A. ACT and alpha 1 PI differ locally only at sites of insertions, except at the segment s3C-turn-s4C, which is displaced by several angstr?m units. This region of ACT is involved in DNA binding.  相似文献   

19.
Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G(1) to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT(1A)). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000) J. Biol. Chem. 275, 26349-26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) -136 and -96 and between bp -29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the -136 to -96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21(ras), Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the -29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the -136 to -96 bp region of the cyclin D1 promoter and by activation of the -29 to +139 bp region, both in a p21(ras)/Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.  相似文献   

20.
Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G(0)/G(1) transition) and again in late G(1). The two ubiquitous PI3K isoforms (p110alpha and p110beta) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110alpha was activated first in G(0)/G(1), followed by a minor p110beta activity peak. In late G(1), p110alpha activation preceded that of p110beta, which showed the maximum activity at this time. p110beta activation required Ras activity, whereas p110alpha was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110alpha and -beta activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110alpha in blocking early G(1) events. We show that inhibition of either p110alpha or p110beta reduced cell cycle entry. These results reveal that PI3Kalpha and -beta present distinct activation requirements and kinetics in G(1) phase, with a selective action of PI3Kalpha at the G(0)/G(1) phase transition. Nevertheless, PI3Kalpha and -beta both regulate S-phase entry.  相似文献   

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