title: Reverse biochemistry: use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue.
abstract: Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a "fold-specific" inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription-PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA.
authors.forename: T
authors.initials: T
authors.lastname: Takeuchi
authors.forename: M A
authors.initials: MA
authors.lastname: Shuman
authors.forename: C S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000201:enzymology*; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000209:etiology; D012333:RNA, Messenger / Q000032:analysis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D012333:RNA, Messenger; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases