The human KLK14 gene 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 that KLK14 gene 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 alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P¢1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.Keywords: cyan fluorescent protein; human kallikrein; phage display; prostate cancer; substrate.The human prostatic kallikreins hK3, or prostate specific antigen (PSA), is considered the gold standard for prostate cancer diagnosis and screening; however, hK2, the second prostatic kallikrein to be discovered [1], has recently emerged as a complementary marker for its positive correlation with prostate cancer grade and progression. PSA is more highly expressed in benign hyperplasia (BHP) than in cancer thus hK2 is helpful to further distinguish malignant from benign disease [2][3][4]. The recent discovery of 12 new members of the kallikrein family [5][6][7] could provide additional prostate cancer markers.In the seminal plasma, hK2 is mostly recovered complexed with protein C inhibitor [1]. Because hK2 cleaves, with trypsin-like specificity, certain components of the semen coagulum (fibronectin and semenogelins), it is possible that it has a role in the early stages of semen liquefaction, a biological process which immediately follows ejaculation [8]. In addition, in vitro studies have shown that hK2 can activate urokinase-type plasminogen activator [9] and inactivate plasminogen activator inhibitor-1 [10] leading to the activation of urokinase system. Moreover, hK2 degrades insulin-like growth factor binding proteins (IGF-BP) to release IGF, a putative local mitogenic signal for prostate cancer cells [11].Despi...

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of α1‐antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607–613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display‐selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgoño CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291–298], we studied the importance of the scaffold, serpins α1‐antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACTE8, ACTG9, AATE8 and AATG9 showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8‐based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin‐like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.

Our data illustrate that telomerase activity may be detected in voided urine or washing after prostatic massage in patients with prostate cancer. Sensitivity was higher for poorly differentiated tumors. This approach is not currently available for detecting prostate cancer in clinical practice. However, these results are promising and further studies are ongoing.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747Biochem. 269, -2754. Selected substrates were then transplanted into the reactive site loop of a1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific a1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.Keywords: phage-display; protease; human kallikrein; inhibitor; a1-antichymotrypsin.Prostate cancer is currently the most commonly diagnosed cancer in American men. This pathology is the second leading cause of cancer death after lung cancer and the majority of the patients with locally advanced prostate cancer have an increased risk for disease progression. In this progression, proteases are believed to play a pivotal role in the malignant behaviour of cancer cells, including rapid tumor growth, invasion and metastasis. Human glandular kallikrein (hK2) protein is a trypsin-like serine protease expressed predominantly in the prostate epithelium. First isolated from human seminal plasma [1], hK2 has emerged recently as a diagnostic marker for prostate cancer. When tested in combination with assays for various forms of prostate specific antigen (PSA), hK2 seemed to be better suited to distinguish malignant from benign prostate disease than the well established marker PSA (prostate specific antigen or hK3) [2][3][4]. In addition to its role as a marker, the proteolytic activities suggest that hK2 could contribute to cancer progression. Several potential functions for this enzyme have been proposed, including the activation of urokinase-type plasminogen activator Taking into account its prostate tissue-specific expression and the involvement of all its potential substrates in cancer development, hK2 can be considered as a potential therapeutic target.The serpins (serine protease inhibitors) are a large family of proteins implicated in the regulation of complex physiological processes. These proteins of about 45 kDa can be subdivided into two groups, one being inhibitory and the other noninhibitory. Serpins contain an exposed flexible reactive-site loop (RSL), which is implicated in the interaction with the putative target protease. Following the binding to the enzyme and cleavage of the P1-P'1 scissile bond of the RSL, a covalent com...