Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP)

Abstract

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Prolyl oligopeptidase regulates progesterone secretion via the ERK signaling pathway in murine luteal cells

“…In vitro specificity studies revealed that KYP‐2047 was a potent and selective inhibitor of POP, and currently, it is widely used in in vitro and in vivo studies. (Bainbridge et al, ; Myöhänen, Hannula, et al, ; Svarcbahs, Julku, & Myohanen, ). We found that POP overexpression increased progesterone production in primary luteal cells (Figure d).…”

Altered expression of fibroblast activation protein-α (FAP) in colorectal adenoma-carcinoma sequence and in lymph node and liver metastases

“…In order to determine the levels of soluble FAP, the sFAPDuoSet ELISA kit (R&D Systems, DY3715) was used [ 46 , 47 ]. 100 μl of standards, reagent blank and plasma samples (1/100 dilution) were plated into a 96-well plate and incubated overnight at 4ºC.…”

Pro-tumorigenic roles of fibroblast activation protein in cancer: back to the basics

“…More sophisticated delivery methods designed to increase bioavailability to tumors include loading chemotherapeutics into nanoparticle carriers; in this case FAP can be used to activate the drugs( 130 ) or to help dissemble the carrier to mediate drug release and/or uptake( 131 , 132 ). Similar concepts can be applied to imaging techniques for cancer detection by using FAP-cleavable reporter constructs( 133 , 134 ).…”