RESUMEN
Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.
Asunto(s)
Fibroblastos/citología , Gelatinasas/genética , Gelatinasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Adipoquinas/sangre , Adipoquinas/química , Aminoácido Oxidorreductasas/sangre , Aminoácido Oxidorreductasas/química , Animales , Técnicas de Cultivo de Célula , Línea Celular , Quimiocina CXCL5/sangre , Quimiocina CXCL5/química , Endopeptidasas , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Factor Estimulante de Colonias de Macrófagos/sangre , Factor Estimulante de Colonias de Macrófagos/química , Ratones , Mapas de Interacción de Proteínas , Proteolisis , Especificidad por SustratoRESUMEN
Fibroblast activation protein (FAP) is best known for its heightened expression in tumour stroma. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. FAP expression is difficult to detect in non-diseased adult organs, but is greatly upregulated in sites of tissue remodelling, which include liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This review highlights this imbalance, emphasising the need to better define the substrate repertoire and expression patterns of FAP to elucidate its role in biological and pathological processes.