scholarly journals Endoprotease PACE4 is Ca2+-dependent and temperature-sensitive and can partly rescue the phenotype of a furin-deficient cell strain

1999 ◽  
Vol 339 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Joseph F. SUCIC ◽  
Joan M. MOEHRING ◽  
Noel M. INOCENCIO ◽  
Jason W. LUCHINI ◽  
Thomas J. MOEHRING
Keyword(s):  
Secretory Pathway ◽  
Protective Antigen ◽  
Chinese Hamster ◽  
Northern Analysis ◽  
Temperature Sensitive ◽  
Exotoxin A ◽  
Wild Type ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A

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 °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.

scholarly journals In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells

2017 ◽  
Vol 6 (1) ◽  
pp. 144
Author(s):  
Morteza Karimipoor ◽  
Jahangir Langari ◽  
Majid Golkar ◽  
Hossein Khanahmad ◽  
Sirous Zeinali ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
In Vitro Evaluation ◽  
Pseudomonas Exotoxin A

scholarly journals Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

1989 ◽  
Vol 108 (4) ◽  
pp. 1291-1300 ◽  
Author(s):  
S Schmid ◽  
R Fuchs ◽  
M Kielian ◽  
A Helenius ◽  
I Mellman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Cell Fractionation ◽  
Permissive Temperature ◽  
Wild Type ◽  
Late Endosomes ◽  
Early Endosomes

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.

scholarly journals Pseudomonas Exotoxin A-Mediated Apoptosis Is Bak Dependent and Preceded by the Degradation of Mcl-1

2010 ◽  
Vol 30 (14) ◽  
pp. 3444-3452 ◽  
Author(s):  
Xing Du ◽  
Richard J. Youle ◽  
David J. FitzGerald ◽  
Ira Pastan
Keyword(s):  
Protein Synthesis ◽  
Bacterial Toxin ◽  
Exotoxin A ◽  
Wild Type ◽  
Double Knockout ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A ◽  
Mouse Embryo Fibroblasts ◽  
Induction Of Apoptosis ◽  
Dose Dependent

ABSTRACT Pseudomonas exotoxin A (PE) is a bacterial toxin that arrests protein synthesis and induces apoptosis. Here, we utilized mouse embryo fibroblasts (MEFs) deficient in Bak and Bax to determine the roles of these proteins in cell death induced by PE. PE induced a rapid and dose-dependent induction of apoptosis in wild-type (WT) and Bax knockout (Bax−/−) MEFs but failed in Bak knockout (Bak−/−) and Bax/Bak double-knockout (DKO) MEFs. Also a loss of mitochondrial membrane potential was observed in WT and Bax−/− MEFs, but not in Bak−/− or in DKO MEFs, indicating an effect of PE on mitochondrial permeability. PE-mediated inhibition of protein synthesis was identical in all 4 cell lines, indicating that differences in killing were due to steps after the ADP-ribosylation of EF2. Mcl-1, but not Bcl-xL, was rapidly degraded after PE treatment, consistent with a role for Mcl-1 in the PE death pathway. Bak was associated with Mcl-1 and Bcl-xL in MEFs and uncoupled from suppressed complexes after PE treatment. Overexpression of Mcl-1 and Bcl-xL inhibited PE-induced MEF death. Our data suggest that Bak is the preferential mediator of PE-mediated apoptosis and that the rapid degradation of Mcl-1 unleashes Bak to activate apoptosis.

Proteolysis of Pseudomonas exotoxin A within hepatic endosomes by cathepsins B and D produces fragments displaying in vitro ADP-ribosylating and apoptotic effects

FEBS Journal ◽  
2010 ◽  
Vol 277 (18) ◽  
pp. 3735-3749 ◽  
Author(s):  
Tatiana El Hage ◽  
Séverine Lorin ◽  
Paulette Decottignies ◽  
Mojgan Djavaheri-Mergny ◽  
François Authier
Keyword(s):  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A ◽  
Apoptotic Effects

scholarly journals Furin activates Pseudomonas exotoxin A by specific cleavage in vivo and in vitro.

1994 ◽  
Vol 269 (50) ◽  
pp. 31831-31835
Author(s):  
N M Inocencio ◽  
J M Moehring ◽  
T J Moehring
Keyword(s):  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A

scholarly journals Pseudomonas exotoxin A. Membrane binding, insertion, and traversal.

1986 ◽  
Vol 261 (24) ◽  
pp. 11404-11408 ◽  
Author(s):  
Z T Farahbakhsh ◽  
R L Baldwin ◽  
B J Wisnieski
Keyword(s):  
Membrane Binding ◽  
Exotoxin A ◽  
Pseudomonas Exotoxin ◽  
Pseudomonas Exotoxin A
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