Doctor of Philosophy
Berger, Bryan W.
Other advisers/committee members
Lowe-Krentz, Linda J.; Zhang, Xiaohui; Iovine, Kathryn
Fibroblast activation protein (FAP) is a cell-surface serine protease which promotes invasiveness of certain epithelial cancers. In particular, the heightened expression of FAP in tumor cells and the ability of FAP to degrade major components of the extracellular matrix such as type I collagen makes it attractive as a target for anti-cancer therapeutics. FAP is active only when it is in the form of a dimer. Moreover, FAP also can heterodimerize with dipeptidyl peptidase-IV (DPP-IV) to facilitate tumor cells invasion. Despite the importance of FAP to cancer and other disease processes, the mechanism that regulates FAP homo- and heterodimerization, as well as its relation to prolyl peptidase activity is not completely understood. Transmembrane (TM) domain is found to be a key mechanism to regulate homo- and heterodimerization in several transmembrane receptors. Here, I investigate key residues in the FAP TM domain that may be significant for FAP homo- and heterodimerization. Using the TM-CYTO as a mimic for the full-length FAP in the AraTM assay, I found mutations to predicted TM interfacial residues (G10L, S14L, and A18L) in a small-X3-small motif, which has been shown previously to promote TM dimerization, reduced FAP TM-CYTO dimerization relative to wild type, whereas predicted off-interface residues showed no significant change in dimerization from wild type. The results indicate the predicted small-X3-small dimer interface stabilize the FAP TM-CYTO homodimer. Likewise, heterodimerization studies using the DN-AraTM assay between FAP and DPP-IV indicate that heterodimerization occurs; there is no preference in terms of TM-mediated heterodimerization between FAP and DPPIV. In terms of FAP exopeptidase activity when overexpressed in HEK293 cells, the interfacial TM residue G10L significantly decreased FAP exopeptidase activity by more than 25%, and also reduced cell-surface expression relative to other interfacial residues (S14L, A18L). Collectively, my results suggest FAP homodimerization is important for both trafficking and protease activity, and is dependent on a specific, TM small-X3-small interface. In addition to the TM domain, I also investigated the role of hydrophobic residues (W728, F724 and Y239) buried in the homodimer interface of the FAP ectodomain crystal structure in terms of FAP homodimerization and exopeptidase activity. Compared to the off-interface A657V mutant, W728A, F724A and Y239A FAP constructs exhibit decreased exopeptidase activity (74%, 75% and 59% less exopeptidase activity than wild-type, respectively), whereas no decrease in exopeptidase activity occurs for the A657V mutant. Mutants W728A and Y239A also impaired trafficking to the cell surface when overexpressed in HEK293 cells. Collectively, my results indicate that change in dimerization observed for mutants W728A and Y239A affect both enzymatic activity as well as trafficking. In summary, TM interfacial residues (G10L, S14L, and A18L) in a small-X3-small motif and hydrophobic residues (W728, F724 and Y239) buried in the homodimer interface of the FAP ectodomain are important for FAP homodimerization, exopeptidase activity, and localization. My studies provide the insight to understand mechanism of FAP homo- and heterodimerization relating to enzymatic activity and protein localization. They can be a guideline to design an efficient drug delivery system for anti-cancer therapies based on FAP activity. Designed FAP-selective peptides are a next interesting part that I want to use as protease-responsive peptides in delivery systems for targeting cancer cells and accessing the tumor microenvironment (TME).
Wonganu, Benjamaporn, "Understanding the Structural Basis for FAP Homodimerization, Heterodimerization and FAP Substrate Specificity" (2015). Theses and Dissertations. 2880.