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 chymotrypsinfold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membranetype 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.

Coordinate expression and functional profiling identify an extracellular proteolytic signaling pathway

“…Targeted overexpression of MT-SP1 in squamous epithelia in mice results in skin-limited nodules of squamous cell carcinoma that become metastatic in the presence of the chemical carcinogen DMBA (10). Small molecule and macromolecular inhibitors of MT-SP1 have been developed and applied in a mouse model of cancer, resulting in growth suppression of androgen-independent prostate cancer xenografts (2,11). Taken together, these findings suggest a role for MT-SP1 in cancer.…”

TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial–mesenchymal transition

“…Matriptase/MT-SP1, a TTSP with gelatinase activity, is highly expressed in breast, prostate, ovarian and colorectal cancers (Lin et al, 1999;Benaud et al, 2002;NetzelArnett et al, 2003;List et al, 2006). Matriptase/MT-SP1 is also implicated in tumor growth and metastasis in a mouse model of prostate cancer (Takeuchi et al, 1999;Galkin et al, 2004) and induces skin tumorigenesis in a transgenic mouse model (List et al, 2005). Another TTSP, TMPRSS1/hepsin, is highly expressed in hepatomas and ovarian and prostate cancers .…”

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

“…2a) (Zhang et al 1998;Kim et al 1999;Takeuchi et al 1999). This protease is co-expressed with HAI-1 in a variety of epithelial cells (Oberst et al 2003a;Szabo et al 2008).…”

“…The activation of this protease (i.e., conversion to disulfide-linked two-chain form via cleavage after Arg614, Fig. 2a) is known to occur via a mechanism requiring its catalytic triad (Takeuchi et al 1999;Oberst et al 2003b;Désilets et al 2008;Miyake et al 2009). The activated two-chain protease cleaves to activate a number of substrates, including pro-HGF, possibly at the cell surface (Lee et al 2000;Satomi et al 2001;Yamasaki et al 2003;Kojima et al 2009a).…”