Dissecting the urokinase activation pathway using urokinase-activated anthrax toxin
Shihui Liu1, Thomas H Bugge, Arthur E Frankel
1Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
Methods in Molecular Biology (Clifton, N.J.)
|April 21, 2009
Summary
Anthrax toxin
Area of Science:
- Molecular biology
- Cellular toxicology
- Biochemistry
Background:
- Anthrax toxin, composed of protective antigen (PrAg), edema factor (EF), and lethal factor (LF), intoxicates mammalian cells.
- PrAg binds cells and is activated by furin, forming a heptameric channel for EF and LF translocation into the cytosol.
- The anthrax toxin delivery system's protease specificity can be altered, exemplified by PrAg-U2 activated by urokinase plasminogen activator (uPA).
Purpose of the Study:
- To utilize PrAg-U2 as a molecular reporter to investigate uPAR-mediated pro-uPA activation.
- To determine the essential components for uPAR-dependent cell surface pro-uPA activation.
Main Methods:
- Employing PrAg-U2 as a reporter molecule to assess uPA activation.
- Utilizing western blotting for in vitro analysis of cell lysates.
- Evaluating cell and animal death for in vivo assessments.
Main Results:
- Demonstrated that both uPAR and plasminogen are critical for pro-uPA activation.
- Confirmed the roles of uPAR and plasminogen in both in vitro and in vivo experimental models.
Conclusions:
- uPAR and plasminogen are essential components for uPA activation.
- PrAg-U2 serves as an effective reporter tool for studying protease activation pathways.
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