Proteomic analysis of the fibroblast activation protein alpha (FAPα) interactome in activated human fibroblasts: identification of potential partners, substrates, and cooperation with integrin signaling

Background. Fibroblast activation protein alpha (FAPα) is a transmembrane serine protease whose expression is markedly upregulated in stromal cells during fibrosis and tumor development. Although FAPα is known to participate in extracellular matrix remodeling, the full repertoire of its potential protein partners and enzymatic substrates in activated fibroblasts remains incompletely characterized. This study aimed to identify proteins that form complexes with FAPα in activated human fibroblasts, to evaluate their substrate status, and to confirm cooperation with integrin signaling.
Materials and methods. Primary human dermal fibroblasts were activated by treatment with TGF-β1 (10 ng/mL, 24 h). Immunoprecipitation of FAPα was performed using two independent antibodies (Cell Signaling and R&D Systems), and specificity was confirmed by Western blotting. Proteins eluted from immunoprecipitates were precipitated with trichloroacetic acid, subjected to trypsin digestion, and analyzed by mass spectrometry on a Q Exactive Plus instrument. Data were processed using the MaxQuant and Perseus software suites. Functional annotation of identified proteins was carried out using GO Enrichment Analysis. The presence of putative FAPα cleavage sites was assessed bioinformatically. Interactions with integrins β1, αV, and TGFβRII were verified by Western blotting.
Results. Immunoprecipitation specificity was confirmed for both antibody types. In two independent replicates, a total of 47 proteins were identified, of which 16 specifically co-precipitated with FAPα. Functional annotation revealed significant enrichment among these proteins for peptidases (chymase, carboxypeptidase D), protease inhibitors (annexin A2), and cell adhesion molecules (tenascin). Cleavage site analysis demonstrated that only ZC3H4 simultaneously contains GP, PPGP, and GPA motifs, whereas carboxypeptidase D contains only the GPA motif. Western blotting confirmed the formation of complexes between FAPα and integrins β1 and αV, as well as reduced co-precipitation of TGFβRII following cell activation.
Conclusions. In this study, we identified 16 potential protein partners of FAPα in activated fibroblasts, among which ZC3H4 emerges as the most likely direct substrate. Cooperation between FAPα and integrins β1 and αV was experimentally confirmed. Collectively, these findings expand our understanding of the functional role of FAPα and provide a foundation for further investigation into its contribution to the pathogenesis of fibrosis and tumor progression.

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