Fibroblast activation protein (FAP), also known as seprase, is a cell-surface serine protease that acts on various hormones and extracellular matrix components. FAP is a type II integral serine protease with 97-kDa and is a member of the post-proline dipeptidyl aminopeptidase (DPP) family. Like other DPP enzymes, FAP has postproline exopeptidase activity, but FAP is unique in that it displays additional in vitro endopeptidase, gelatinase, and potentially collagenase activity. Both the postproline dipeptidyl peptidase and the endopeptidase activity of FAP depend on the catalytic triad consisting of serin (S624), aspartate (D702), and histidine (H734) and require homodimerization of the protein. Structurally, FAP consists of 760 amino acids, most of which possess a hydrolytic area exposed laterally of the plasmalemma. ~20 amino acids are anchored in the plasma membrane, and 6 amino acids are located in the cytoplasm. This extracellular domain consists of an eight-bladed β propeller, which acts as a substrate selectivity gate, and an α/β hydrolase domain. FAP monomers are not active but form an active 170 kDa homodimer that contains two N-glycosylated 97 kDa subunits.
Fig.1 Ribbon model of FAP structure. (Fitzgerald, 2020)
FAP’s dual enzymatic activity gives it a range of putative substrates. FAP’s dipeptidyl peptidase activity enables it to cleave neuropeptide Y, peptide YY (PPY), substance P, and brain natriuretic peptide 32 (BNP). However, BNP, substance P, and PYY were far more efficiently cleaved by other proteases in the human plasma, indicating that physiologically, FAP is not likely responsible for the cleavage of these factors. Known substrates of FAP’s endopeptidase activity include denatured collagen type I and III (the components of gelatin), α-2 antiplasmin cleaving enzyme, and recently discovered fibroblast growth factor 21. FAP cleavage of collagen I was shown to enhance macrophage adhesion in vitro.
Multiple environmental and soluble factors have been observed to alter FAP expression, though detailed mechanistic pathways for many of them are unknown. The best characterized is the transforming growth factor β (TGFβ) activation of drosophila mothers against decapentaplegic protein 3 (SMAD3), which binds directly to the FAP promoter. In addition, phosphatase and tensin homolog (PTEN) is a FAP repressor. Downstream signaling targets of FAP include phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT), rat sarcoma/extracellular signal-regulated kinase (RAS/ERK), sonic hedgehog (SHH)/Gli, focal adhesion kinase (FAK), and many others. For example, cells engineered to overexpress FAP have increased proliferation and migration due to activation of the PI3K and the SHH pathways, which are intracellular signaling pathways required for cell cycle and differentiation, respectively. In addition to SHH/Gli pathways’ roles in promoting proliferation, invasion, and migration as previously mentioned, FAP’s effect on epithelial-mesenchymal transition (EMT) may also be due to its activation of the SHH/GLI pathway. FAP might form a complex with the FAK protein and in doing so reduce its phosphorylation, which thus results in the reduction of adhesion and motility ability. FAP is associated with urokinase-type plasminogen activator receptor (uPAR) has been implicated in both the cellular migration and immunosuppression phenotypes associated with FAP.
Fig.2 Potential signaling pathways affected by FAP. (Fitzgerald, 2020)
FAP is expressed during development, but only rarely in healthy adult tissues. However, it is highly upregulated especially on fibroblasts at sites of active tissue remodeling, including wound healing, fibrosis, and cancer. FAP can directly enhance proliferation, migration, and invasion of cells by which it is expressed. FAP expression also displays enrichment of gene pathways for immune regulation, angiogenesis, and cell differentiation and growth. Therefore, FAP has been linked to multiple human pathologies including fibrosis, arthritis, atherosclerosis, autoimmune diseases, metabolic diseases, and cancer. FAP is overexpressed in many cancers, including 90% of carcinomas, such as breast, colorectal, pancreatic, lung, bladder, ovarian, and other cancers. In these cancers, FAP is usually heavily expressed in the stroma and has thus become a universal marker of cancer-associated fibroblasts (CAFs). In most instances, FAP is associated with progression and heightened severity of the disease.
The physiological role of FAP has expanded from simple collagen degradation to functions including activation of tumorigenic signaling cascades, angiogenesis, EMT, and even immunosuppression. Creative Biolabs provides a full set of FAP assay portfolio services, such as cell viability assays, cell apoptotic assay, EMT assay (cell proliferation assay, cell migration assay, cell invasion assay, EMT marker assay, etc.), ELISA, colony formation assay, Masson Trichrome staining, gelatin zymogram, collagen zymogram, hydroxyproline assay, immunoblotting, immunoprecipitation, immunofluorescence and immunohistochemistry, and so on.
Creative Biolabs provides a powerful and excellent tool to aid in custom tumor marker assay services. Our professional and experienced scientists are pleased to assist you in your project. If you don’t find profiling that suits your needs, don’t hesitate to contact us and let us know what you are looking for. We’ll discuss details with you to ensure our assay meets your unique needs.
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