David Deperthes

The humanKLK14gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members.KLK14, like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated thatKLK14gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin α-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities towa...

Although the cellular steps required for metastasis are similar for all cancer cells, proteases involved in this process and their expression levels vary greatly between different cancer types. Thus, the identification of these proteolytic activities represents a crucial issue in the understanding of cancer development. Until now, phage display substrate technology has been successfully employed for the characterization of purified proteases but was never used with a mix of proteases. In the present work, we report an easy protocol to identify multiple proteolytic activities secreted by cancer cells. We selected substrates from a phage display library of high diversity using secreted media of three established prostate cancer cell lines (DU-145, LNCaP and PC-3) with variable degrees of invasive capability. Some of these selected peptide substrates were hydrolyzed by the secreted proteins of all three prostatic cancer cell lines, demonstrating similarities in their proteolytic activities. On the other hand, a few substrates were cancer cell specific, indicating differences in the phenotypes of protease expression in prostate cancer. This work reports for the first time the selection of substrates from a mix of proteases using phage display technology and opens a new avenue for the direct identification of proteolytic activities for tumor extracts.