The structure and function of bacterial protein toxins are analyzed using biochemical, genetic, and cell biology methods. Our previous work showed that the anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of sensitive cells, is cleaved by a cell surface protease, probably furin, and only then captures either of the two other proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and reach an acidic compartment from which LF and EF escape to the cytosol. EF is a calcium- and calmodulin-dependent adenylyl cyclase which causes large and unregulated increases in intracellular cAMP concentrations. Evidence suggests that LF is a metalloprotease, but no substrate has been identified. Through a collaborative study, the structure of PA was solved. The protein contains four functionally distinct domains. The C-terminal region, domain IV, is responsible for binding to the cell-surface receptor. Transport of LF and EF across intracellular membranes appears to require heptamerization of the proteolytically activated 63-kDA fragment of PA. The structure of the heptamer formed by the 63-kDa active fragment was also determined by X-ray diffraction. Interaction of LF and EF with the PA heptamer occurs through the amino-terminal region of LF; 254 amino acids of the 776 total are sufficient. Fusion proteins have been made in which the putative catalytic domain of LF, residues 255-776, were replaced by other polypeptides. These heterologous polypeptides are efficiently translocated to the cytosol of cells when administered with PA. Detailed knowledge of the structure and function of the toxin proteins is being used to design and construct cell-type specific cytotoxic and therapeutic fusion proteins. For example, replacement or modification of PA domain IV with a new ligand is being used to change and restrict the cell types to which the toxin will bind. Creation of fusion proteins containing LF residues 1-254 provides a way to deliver specific polypeptides into cells. As one example, fusion of LF residues 1-254 to gp120, the envelope protein of the HIV-1 virus, has been shown to cause presentation of specific peptide epitopes on the cell surface so as to stimulate cellular immunity. This system may provides a unique and effective way to induce immunity to AIDS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Intramural Research (Z01)
Project #
1Z01DE000677-01
Application #
6161873
Study Section
Special Emphasis Panel (OIIB)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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