Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity.
Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.
Department of Molecular Oncology, Genentech, Inc., 1 DNA Way - MS42, South San Francisco, CA 94080, USA.
Conrad Yap
CY
Edosada
Clifford
C
Quan
Thuy
T
Tran
Victoria
V
Pham
Christian
C
Wiesmann
Wayne
W
Fairbrother
Beni B
BB
Wolf
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000737:chemistry*; D014408:Biomarkers, Tumor / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000187:drug effects; D015195:Drug Design; D010450:Endopeptidases; D018093:Gelatinases; D006801:Humans; D008565:Membrane Proteins; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000187:drug effects*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D013379:Substrate Specificity; D000979:alpha-2-Antiplasmin / Q000737:chemistry; D000099167:Fibroblast Activation Protein Alpha
D000590:Amino Acid Chloromethyl Ketones; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D018819:Dipeptidyl Peptidase 4; D010450:Endopeptidases; D018093:Gelatinases; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; D000979:alpha-2-Antiplasmin; D000099167:Fibroblast Activation Protein Alpha; C061903:glycyl-glycyl-proline