Transforming growth factor-beta1 as a regulator of the serpins/t-PA axis in cerebral ischemia.
(33/1699)
The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-beta1 (TGF-beta1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT-PCR and Western blotting, we observed that only the expression of PAI-1 was modulated by TGF-beta1 treatment via a TGF-beta-inducible element contained in the PAI-1 promoter (CAGA box). Finally, we compared the specificity of TGF-beta1 action with other members of the TGF-beta family by using luciferase reporter genes. These data show that TGF-beta and activin were able to induce the overexpression of PAI-1 in astrocytes, but that bone morphogenetic proteins, glial cell line-derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t-PA axis and the mechanism by which TGF-beta may be neuroprotective. (+info)
The zinc finger protein A20 interacts with a novel anti-apoptotic protein which is cleaved by specific caspases.
(34/1699)
A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases. (+info)
A novel serpin expressed by blood-borne microfilariae of the parasitic nematode Brugia malayi inhibits human neutrophil serine proteinases.
(35/1699)
Serine proteinase inhibitors (serpins) play a vital regulatory role in a wide range of biological processes, and serpins from viruses have been implicated in pathogen evasion of the host defence system. For the first time, we report a functional serpin gene from nematodes that may function in this manner. This gene, named Bm-spn-2, has been isolated from the filarial nematode Brugia malayi, a causative agent of human lymphatic filariasis. Polymerase chain reaction (PCR) and Western blot experiments indicate that Bm-spn-2 is expressed only by microfilariae (Mf), which are the long-lived blood-dwelling larval stage. A survey of the greater than 14,000 expressed sequence tags (ESTs) from B malayi deposited in dbEST shows that greater than 2% of the ESTs sequenced from Mf cDNA libraries correspond to Bm-spn-2. Despite its abundance in the microfilarial stage, Bm-spn-2 has not been found in any other point in the life cycle. The predicted protein encoded by Bm-spn-2 contains 428 amino acids with a putative signal peptide. Antibodies to recombinant Bm-SPN-2 protein react specifically with a 47.5-kD native protein in Mf extract. Bm-SPN-2 is one of the largest of the 93 known serpins, due to a 22 amino acid carboxy-terminal extension, and contains the conserved serpin signature sequence. Outside these regions, levels of homology are low, and only a distant relationship can been seen to a Caenorhabditis elegans serpin. The Bm-spn-2 gene contains 6 introns, 2 of which appear to be shared by both nematode species. The B malayi introns have an extended and conserved 3' splice site and are relatively large compared with C elegans. A panel of mammalian serine proteinases were screened and Bm-SPN-2 protein was found to specifically inhibit enzymatic activity of human neutrophil cathepsin G and human neutrophil elastase, but not a range of other serine proteinases. It is possible that Bm-SPN-2 could function as a stage-specific serpin in the blood environment of the microfilarial parasite in protection from human immunity and thus may be a good candidate for protective vaccine. (+info)
Identification of a novel complex between human kallikrein 2 and protease inhibitor-6 in prostate cancer tissue.
(36/1699)
Human kallikrein (hK) 2 is an arginine-selective serine protease expressed predominantly in the prostate that has an 80% sequence identity with prostate-specific antigen. Expression of hK2 is elevated in the tumor epithelium compared to benign prostate tissue. We have purified, sequenced, and identified a novel hK2 complex in prostate tissue consisting of hK2 and a serine protease inhibitor known as protease inhibitor-6 (PI-6). This 64-kDa SDS-PAGE stable complex is elevated in the tumor and is approximately 10% of total hK2. No comparable complex of prostate-specific antigen was detected. PI-6, also known as cytoplasmic antiprotease, has been characterized as an intracellular inhibitor of trypsin and chymotrypsin-like proteases, which has high homology to plasminogen activator inhibitor 1 and 2. The physiological role of PI-6 in the prostate and its relationship to hK2 and prostate cancer are under investigation. (+info)
The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1.
(37/1699)
Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1. (+info)
Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer.
(38/1699)
We have developed sensitive assays for cytokeratin (K) 8, 16, 19, stromelysin 3 (ST3), MUC1 and maspin mRNAs using reverse transcription polymerase chain reaction (RT-PCR) and used these to assess lymph node status in patients undergoing surgery for breast cancer. In addition the RT-PCR assays were tested against lymph nodes from non-cancer patients to determine their specificity. Despite high sensitivity RT-PCR assays for K8, K16, K19, ST3 and maspin were not found to be useful as markers of submicroscopic disease as transcripts of these genes were detected in the great majority of control lymph nodes tested. Expression of MUC1 was also not found to be useful as it was both insensitive and non-specific. The importance of assessing potential markers against an adequately sized control population is demonstrated, as failure to do so can lead to erroneous conclusions. (+info)
Caspase-1 (interleukin-1beta-converting enzyme) is inhibited by the human serpin analogue proteinase inhibitor 9.
(39/1699)
The regulation of caspases, cysteine proteinases that cleave their substrates after aspartic residues, is poorly understood, even though they are involved in tightly regulated cellular processes. The recently discovered serpin analogue proteinase inhibitor 9 (PI9) is unique among human serpin analogues in that it has an acidic residue in the putative specificity-determining position of the reactive-site loop. We measured the ability of PI9 to inhibit the amidolytic activity of several caspases. The hydrolysis of peptide substrates by caspase-1 (interleukin-1beta-converting enzyme), caspase-4 and caspase-8 is inhibited by PI9 in a time-dependent manner. The rate of reaction of caspase-1 with PI9, as well as the rate of substrate hydrolysis of the initial caspase-PI9 complex, shows a hyperbolic dependence on the concentration of PI9, indicative of a two-step kinetic mechanism for inhibition with an apparent second-order rate constant of 7x10(2) M(-1).s(-1). The hydrolysis of a tetrapeptide substrate by caspase-3 is not inhibited by PI9. The complexes of caspase-1 and caspase-4 with PI9 can be immunoprecipitated but no complex with caspase-3 can be detected. No complex can be immunoprecipitated if the active site of the caspase is blocked with a covalent inhibitor. These results show that PI9 is an inhibitor of caspase-1 and to a smaller extent caspase-4 and caspase-8, but not of the more distantly related caspase-3. PI9 is the first example of a human serpin analogue that inhibits members of this class of cysteine proteinases. (+info)
Isolation and characterization of bone morphogenetic protein-binding proteins from the early Xenopus embryo.
(40/1699)
Using a surface plasmon resonance biosensor as a sensitive and specific monitor, we have isolated two distinct bone morphogenetic protein (BMP)-binding proteins, and identified them as lipovitellin 1 and Ep45, respectively. Lipovitellin 1 is an egg yolk protein that is processed from vitellogenin. Both vitellogenin and Ep45 are synthesized under estrogen control in the liver, secreted, and taken up by developing oocytes. In this paper, we have shown that of the TGF-beta family members tested, Ep45 can bind only to BMP-4, whereas lipovitellin 1 can bind to both BMP-4 and activin A. Because of this difference in specificity, we have focused on and further studied Ep45. Kinetic parameters were determined by surface plasmon resonance studies and showed that Ep45 associated rapidly with BMP-4 (k(a) = 1.06 x 10(4) M(-1)s(-1)) and dissociated slowly (k(d) = 1.6 x 10(-4) s(-1)). In Xenopus embryos microinjected with Ep45 mRNA, Ep45 blocked the ability of follistatin to inhibit BMP activity and to induce a secondary body axis in a dose-dependent manner, whereas it had no effect on other BMP antagonists, chordin and noggin. These results support the possibility that Ep45 interacts with BMP to modulate its activities in vivo. (+info)