Volume 384, Issue 2

Signaling by the protease thrombin has started to be appreciated in cell biology, especially since the gene for proteaseactivated receptor-1 (PAR-1) has been cloned. Apart from the central role of thrombin in blood coagulation and wound healing, thrombin also regulates cellular functions in a large variety of cells through PAR-1, PAR-3 and PAR-4. Receptors are activated by a proteolytic cleavage mechanism via G protein-coupled signaling pathways. Accumulating evidence shows that thrombin changes the morphology of neurons and astrocytes, induces glial cell proliferation, and even exerts, depending on the concentration applied, either cytoprotective or cytotoxic effects on neural cells. These effects may be mediated, through either distinct or overlapping signal transduction cascades, by activation of PARs. This review focuses on the underlying signaling events initiated by thrombin in neuronal and glial cells, to summarize our understanding of the intracellular signaling machinery linking thrombin receptors to their potential physiological and pathological functions in the CNS.

Hepatitis B virus (HBV) is a causative agent of chronic and acute hepatitis, and is associated with the development of hepatocellular carcinoma (HCC). We demonstrate here that the Hepatitis B viral core protein (HBc) functions as a repressor on the promoter activity of the human p53 gene. The functional analyses of the promoter of the p53 gene by serial deletion, sitedirected mutagenesis, and the heterologous promoter system revealed that the promoter activity was repressed through the E2F1-binding site (nucleotides -28 to -8) by HBc. An electrophoretic mobility shift assay (EMSA) showed that the HBc reduced the DNA-binding ability of E2F1 to the binding site of the p53 promoter. The interaction of HBc with E2F1 was also observed by glutathione S-transferase (GST) fusion protein binding assay. Furthermore, HBc represses the expression of the p53 gene in the human liver cell line HepG2. Finally, HBc and HBx synergistically repress both the promoter activity and the expression of the p53 gene in HepG2 cells. These results, together with our previous study, strongly suggest that HBc, like HBx, represses the expression of the human p53 tumor suppressor gene.

Cysteinylglycine hydrolysis is a step in the metabolism of glutathione and glutathione S-conjugates. We had previously observed that in rat liver the enzymatic activity is predominantly located in the cytosol. Here we demonstrate that cytosolic leucyl aminopeptidase (EC 3.4.11.1) is the major cysteinylglycine hydrolysing activity in rat liver. Evidence was obtained from the use of peptidase inhibitors and from immunoprecipitation studies using Pansorbin-coupled antibodies raised against hog kidney cytosolic leucyl aminopeptidase. Both isolated cytosolic leucyl aminopeptidase and the cysteinylglycine-hydrolysing activity in rat liver cytosol are bound with equal efficiency to the affinity matrix. We demonstrate that cytosolic leucyl aminopeptidase exhibits leucinamidase and cysteinylglycinase activity. Cysteinylglycine, cystinyl-bis-glycine, S-nitrosocysteinylglycine, and bimane-S-cysteinylglycine are hydrolysed at high rates; low activity is seen with leukotriene D4. Our findings establish a previously unrecognised physiological function of cytosolic leucyl aminopeptidase, participating in glutathione metabolism and in the degradation of glutathione S-conjugates via the mercapturic acid pathway.

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gln-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsintype serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-α and -β. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are instrumental in cellular activities during inflammation, angiogenesis and tumor metastasis. Recent studies suggest that uPA might exert its function on cell proliferation and migration in a uPAR-independent manner or through an adaptor to the uPA-uPAR system. By applying phage display technology, we have identified a putative uPA-binding consensus sequence BXXSSXXB (where B represents a basic amino acid and X represents any amino acid), which has no apparent sequence correlation to uPAR. This uPA-binding motif apparently recognizes the kringle domain of the protease and has an agonistic effect on uPA binding to immobilized uPAR, thereby possibly serving as part of an adaptor component for uPAR signaling. As a result of protein database searches, this motif was found in the extracellular domain of several cell surface proteins, some of which were proposed to be associated with the uPA-uPAR system. Among these, gp130, a common signal transducer for cytokines, was identified as a uPA-binding protein. The specificity of this interaction was demonstrated by inhibition of uPA-binding to immobilized gp130 with soluble gp130. Furthermore, the binding could be partially inhibited by a uPA-binding consensus sequence-containing fusion protein in a dose-dependent manner, with an IC50 of approximately 1 M, indicating that the uPA-binding motif is apparently involved in the uPA-gp130 interaction. The association of gp130 with uPA may link the uPA-uPAR system to various signal transduction pathways.

Peroxynitrite, a potent oxidizing and nitrating species, induces covalent modifications of biomolecules in a number of pathological conditions. In previous studies with S. cerevisiae, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as being especially susceptible to nitration by peroxynitrite. The activity of this enzyme was strongly inhibited by low doses of peroxynitrite in yeast and in cultured rat astrocytes. Here, the sequence of modifications of isolated mammalian GAPDH induced by increasing concentrations of peroxynitrite is demonstrated to be as follows: (i) oxidation, leading to inactivation and to enhanced susceptibility of GAPDH for proteasomal degradation, (ii) oligomer formation, and (iii) nitration. In our study the susceptibility for degradation by isolated 20S proteasome was by far the most sensitive parameter for peroxynitrite-induced damage to GAPDH, implying that this might also occur under pathological conditions where peroxynitrite is generated at low concentrations in vivo.

The prion protein (PrP) plays a central role in prion diseases, and identifying its cellular receptor appears to be of crucial interest. We previously showed in the yeast two-hybrid system that PrP interacts with the 37 kDa precursor (LRP) of the high affinity 67 kDa laminin receptor (LR), which acts as the cellular receptor of PrP in cellular models. However, among the various isoforms of the receptor that have been identified so far, those which are present in the central nervous system and which bind PrP are still unknown. In this study, we have purified mouse brain fractions enriched in the laminin receptor and have performed overlay assays in order to identify those isoforms that interact with the prion protein. We demonstrate (i) the presence, in mouse brain, of several isoforms of the LRP/LR corresponding to different maturation states of the receptor (44, 60, 67 and 220 kDa) and (ii) the binding of all of these isoforms to PrP. Our data strongly support a physiological role of the laminin receptor/ PrP interaction in the brain and highlight its relevance for transmissible spongiform encephalopathies.

The variant VP1-Z of the polyomavirus coat protein VP1 has been recently described as an engineered fusion protein of VP1 and the antibody binding domain protein Z. This construct is able to specifically bind and functionally present antibodies on the surface of viruslike particles of VP1-Z. Here we demonstrate that with the binding of Herceptin, an antibody directed against the receptor tyrosine kinase ErbB2, a cell typespecific targeting was established. ErbB2-positive cell lines were transduced with different plasmids encoding eGFP or β-galactosidase. With both reporter systems functional gene expression in transduced cells could be observed. The transduction was strictly dependent on the use of a ternary complex formed of VLPs of VP1- Z, Herceptin, and the reporter plasmid DNA. The use of single components or ErbB2-negative cell lines did not result in functional gene transfer. The transduction was also completely dependent on the use of chloroquine, a lysosomotropic reagent. This indicates that the complex is internalized by ErbB2-mediated endocytosis.

Membrane type serine protease 1 (MT-SP1) is a representative member of a large family of related enzymes known as type II transmembrane serine proteases or membrane type serine proteases. MTSP1 has been implicated in the selective proteolysis of key extracellular substrates but its physiological role is still not fully understood. MT-SP1 expression at the protein and RNA level has been previously examined by nonquantitative methods such as in situ hybridization, Northern blotting and immunohistochemistry. To establish an introductory understanding of the quantitative mRNA expression of MT-SP1 and to correlate these levels with urokinasetype plasminogen activator receptor (uPAR), a key component of extracellular proteolysis, quantitative RT-PCR was carried out. RNA expression was analyzed in 34 human cancer cell lines, 26 human tissues and 18 primary human breast cancer tissue samples. MT-SP1 mRNA is highly expressed in many breast, ovarian, prostate and colon cancer cell lines and normal human tissues of endodermal origin. At the transcript level, MT-SP1 shows a highly statistically significant correlation (Pearsons product moment correlation r = 0.784, p < 0.001) with uPAR in human breast cancer tissue. The exact role of MT-SP1 in concert with proteins such as uPAR and other members of the plasminogen activator cascade has yet to be ascertained. However, the significant correlation between MT-SP1 and uPAR transcript levels in this initial study suggests further work to establish the role of MT-SP1 as a possible prognostic, diagnostic or therapeutic target for breast cancer.

Full-length Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) both stimulate the cytolytic activity of naive natural killer (NK) cells against Hsp70-positive tumor target cells. Here, we describe the characterization of Hsp70-NK cell interaction with binding studies using the human NK cell line YT. Binding of recombinant Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) to YT cells is demonstrated by immunofluorescence studies. A phenotypic characterization revealed that none of the recently described HSP-receptors (α 2-macroglobulin receptor CD91, Toll-like receptors 2, 4, 9, CD14) are expressed on YT cells. Only the C-type lectin receptor CD94 is commonly expressed by YT cells and Hsp70 reactive NK cells. A correlation of the cell density-dependent, variable CD94 expression and the binding capacity of Hsp70 was detected. Furthermore, Hsp70 binding could be completely abrogated by preincubation of YT cells with a CD94-specific antibody. Competition assays using either unlabeled Hsp70 protein or an unrelated protein (GST) in 20-fold excess and binding studies with escalating doses of Hsp70 protein provide evidence for a specific and concentration-dependent interaction of Hsp70 with YT cells. In addition to Hsp70 and Hsp70C, a 14-mer Hsp70 peptide termed TKD is known to exhibit comparable stimulatory properties on NK cells. Similar to fulllength Hsp70 protein (10 ug/ml-50 ug/ml), a specific binding of this peptide to YT cells was observed at 4C, at equivalent concentrations (2.0 ug/ml-8.0 ug/ml). Following a 30 min incubation period at 37C, membrane-bound Hsp70 protein and Hsp70 peptide TKD were completely taken up into the cytoplasm.

Secretory type 2 cystatins, like cystatins C, E/M and F, are thought to be involved in many pathobiological processes, including vascular amyloidosis, rheumatoid arthritis, Alzheimers disease, osteoporosis, viral and bacterial infections, inflammatory disorders and tumour invasion and metastasis. In order to define the levels of cystatins C, E/M, and F in pleural effusions and to investigate whether these cystatins correlate with diagnostic parameters of pleural and lung diseases, we determined their concentrations in 160 pleural effusions. The median concentration of cystatin C in pleural effusions was 1437 ug/l (95.8 nM), ranging between 18-3967 ug/l. Cystatin C did neither correlate with malignant nor with benign diseases. The concentration of cystatin E/M was significantly higher in effusions of primary pleural tumours (mesotheliomas) compared to secondary pleural tumours and benign diseases. Furthermore, there was a significant correlation between the concentration of cystatin E/M of mesotheliomas and the pleural fluid tumour cell count and of cystatin C. The median values of cystatin F were significantly increased in parapneumonic/ empyema thoracis pleural effusions and tuberculous pleurisy compared to malignant pleural effusions, respectively. The concentration of cystatin F in benign effusions correlated significantly with diagnostic parameters and inflammation (total protein; lactate dehydrogenase; C-reactive protein). Finally, only in the group of parapneumonic/empyema thoracis was there a significant correlation between cystatin F and the neutrophil count. In conclusion, pleural effusions of different origin contain high levels of cystatin C, perhaps constituting the major part of an inhibitor reservoir. The level of cystatin E/M appears to be significantly associated with primary pleural tumours and cystatin F correlates with inflammatory processes of lung disorders.

A homodimeric trypsin inhibitor with a molecular mass of 54 kDa was isolated from the seeds of Clausena lansium (Lour) Skeels with a very simple procedure comprising extraction with an aqueous buffer and ion exchange chromatography on CM-cellulose. It inhibited trypsin with an IC50 of 2.2 nM but was without any inhibitory effect on chymotrypsin and proteinase K. The uptake of MTT by human leukemia HL60 and hepatoma Hep G2 cells was inhibited with an IC50 of 100 uM. Translation in the cellfree rabbit reticulocyte lysate system was inhibited with an IC50 of 3.6 uM. The activity of HIV-1 reverse transcriptase was reduced in the presence of the trypsin inhibitor. The trypsin inhibitor exerted antifungal activity toward Physalospora piricola but not Mycosphaerella arachidicola, Botrytis cinerea, Fusarium oxysporum or Coprinus comatus.

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pI of 5.8. The inhibition constants (K1) for Bacillus licheniformis subtilisin and bovine pancreatic α-chymotrypsin are 3.92×10 -9 M and 7.24×10 -9 M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the CI-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.

In response to insect attack, high levels of proteinase inhibitors are synthesised in potato leaves. This can cause inefficient protein digestion in insects, leading to reduced growth, delayed development and lower fecundity. It has been suggested that Colorado potato beetle overcomes this defence mechanism by inducing the production of a set of cysteine proteases that are resistant to potato proteinase inhibitors. Experiments with gut extracts showed that these proteases have unusual inhibition profiles as they are not inhibited by most of the cystatins but are strongly inhibited by thyropins. In this study we have isolated three cysteine proteases from adapted guts of Colorado potato beetle larvae, named intestains 1, 2 and 3, the first cysteine proteases known to be involved in extracellular protein digestion. The N-terminal sequences suggest their classification into the papain family. Intestains differ in substrate specificities and inhibitory profiles. Their substrate specificities suggest that intestains 1 and 2 are general digestive enzymes, while intestain 3 has a more specific function. The inhibitory profile of intestain 1 is similar to that of proteases of the papain family. However, the K1 values for the interaction of intestain 2 with the same set of inhibitors are several hundred fold higher, which would enable the enzyme to circumvent the potato defence mechanism characterised by high concentrations of protease inhibitors in attacked potato leaves. A further, different strategy of the Colorado potato beetle to avoid potato defence is exhibited by intestain 3, which is able to cleave off the N-terminus of model cystatin and thus inactivate the inhibitor. These results suggest that the Colorado potato beetle combines different strategies to counteract plant defence mechanisms.

Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigusinduced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus.

The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.

A multivariate analysis of the National Cancer Institute gene expression database is reported here. The soft independent modelling of a class analogy approach achieved cell line classification according to histological origin. With the PCA method, based on the expression of 9605 genes and ESTs, classification of colon, leukaemia, renal, melanoma and CNS cells could be performed, but not of lung, breast and ovarian cells. Another multivariate procedure, called partial least squares discriminant analysis (PLS-DA), provides bioinformatic clues for the selection of a limited number of gene transcripts most effective in discriminating different tumoral histotypes. Among them it is possible to identify candidates in the development of new diagnostic tests for cancer detection and unknown genes deserving high priority in further studies. In particular, melan-A, acid phosphatase 5, dopachrome tautomerase, S100-β and acid ceramidase were found to be among the most important genes for melanoma. The potential of the present bioinformatic approach is exemplified by its ability to identify differentiation and diagnostic markers already in use in clinical settings, such as protein S-100, a prognostic parameter in patients with metastatic melanoma and a screening marker for melanoma metastasis.