Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction. (49/8246)

Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.  (+info)

Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter. (50/8246)

The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YlRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YlRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YlRim101p and to override its effects.  (+info)

The catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases. (51/8246)

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.  (+info)

The insect immune protein scolexin is a novel serine proteinase homolog. (52/8246)

Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.  (+info)

Wegener's granulomatosis associated with renal cell carcinoma. (53/8246)

OBJECTIVE: To determine the frequencies and types of malignant neoplasms occurring before or simultaneously with the diagnosis of Wegener's granulomatosis (WG), and to test for the presence of "Wegener's autoantigen," proteinase 3 (PR3), in malignant tissues from WG patients to ascertain whether an association exists between malignancy and WG. METHODS: A retrospective statistical analysis was performed on the medical records of 477 patients with WG as compared with a control group of 479 patients with rheumatoid arthritis (RA). A murine monoclonal antibody was used to test malignant tissues for the presence of PR3. RESULTS: A malignant neoplasm was found in 23 patients in the WG group and in 18 patients in the control group. The odds ratio for malignant neoplasm in the WG group was 1.79 (P = 0.0876, 95% confidence interval [95% CI] 0.92-3.48). Seven patients with renal cell carcinoma were found in the WG group compared with 1 patient in the control group, for an odds ratio of 8.73 (P = 0.0464, 95% CI 1.04-73.69). Simultaneous occurrence of cancer and WG was observed in 14 patients with WG compared with 1 control patient, for an odds ratio of 18.00 (P = 0.0059, 95% CI 230-140.67). Furthermore, the diseases occurred simultaneously in 5 of the 7 patients with both WG and renal cell carcinoma, but not in the single patient in the control group with RA and renal cell carcinoma. PR3 could not be detected in any of the 8 malignant tissue samples (4 renal cell carcinomas) investigated in the patients from the WG group. CONCLUSION: The close temporal association between renal cell carcinoma and WG suggests that malignancy is, in some cases, a trigger for the development of WG. However, since PR3 was not found in malignant tissues from the WG patients, the immunopathologic mechanisms leading to autoimmunity and vasculitis remain unclear.  (+info)

Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis. (54/8246)

A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases.  (+info)

Immunotherapy of human tumors with T-cell-activating bispecific antibodies: stimulation of cytotoxic pathways in vivo. (55/8246)

Bispecific monoclonal antibodies (Bi-mAbs) specific for a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes represent one of the most successful experimental strategies for the immunotherapy of cancer. We report that the in vivo administration of both alpha-CD3/CD30 and alpha-CD28/CD30 Bi-mAbs results in the specific activation of xenotransplanted, resting human T cells infiltrating the CD30-positive Hodgkin's tumor. Bi-mAb treatment resulted in enhanced expression of cytokines such as interleukin 1beta, interleukin 2, tumor necrosis factor type alpha, and activation markers including Ki-67, CD25, and CD45RO in tumor-infiltrating lymphocytes. This antigen-dependent, local T-cell stimulation led to the activation of the cytolytic machinery in T lymphocytes, determined by the up-regulation of mRNA-encoding perforin and the cytotoxic serine-esterases granzymes A and B. The Bi-mAb-induced generation of CTLs depended on the presence of the CD30 antigen and the combined application of both Bi-mAbs. Our findings suggest that the combined application of T-cell-activating Bi-mAbs is able to achieve a tumor site-specific activation of the T-cell cytolytic machinery in vivo. The fact that these cytotoxic cells do not home in tumor-associated antigen-negative tissue and do not enter circulation might explain our previous observations (C. Renner et al., Blood, 87: 2930-2937, 1996) of a high cure rate in preclinical models even at an advanced stage of disease.  (+info)

Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain. (56/8246)

PACE4 is a member of the eukaryotic subtilisin-like endoprotease family. The expression of human PACE4 in RPE.40 cells (furin-null mutants derived from Chinese hamster ovary K1 cells) resulted in the rescue of a number of wild-type characteristics, including sensitivity to Sindbis virus and the ability to process the low-density-lipoprotein receptor-related protein. Expression of PACE4 in these cells failed to restore wild-type sensitivity to Pseudomonas exotoxin A. Co-expression of human PACE4 in these cells with either a secreted form of the human insulin pro-receptor or the precursor form of von Willebrand factor resulted in both proproteins being processed; RPE.40 cells were unable to process either precursor protein in the absence of co-expressed PACE4. Northern analysis demonstrated that untransfected RPE.40 cells express mRNA species for four PACE4 isoforms, suggesting that any endogenous PACE4 proteins produced by these cells are either non-functional or sequestered in a compartment outside of the secretory pathway. In experiments in vitro, PACE4 processed diphtheria toxin and anthrax toxin protective antigen, but not Pseudomonas exotoxin A. The activity of PACE4 in vitro was Ca2+-dependent and, unlike furin, was sensitive to temperature changes between 22 and 37 degrees C. RPE.40 cells stably expressing human PACE4 secreted an endoprotease with the same Ca2+ dependence and temperature sensitivity as that observed in membrane fractions of these cells assayed in vitro. These results, in conjunction with other published work, demonstrate that PACE4 is an endoprotease with more stringent substrate specificity and more limited operating parameters than furin.  (+info)