Proteolytic cascades of human tissue kallikreins

4368 Proteases are a family of enzymes responsible for hydrolysis of peptide bonds and protein disassembly. Understanding impaired activation pathways of proteases, particularly through proteolytic cascades, has been the center of extensive cancer research in the past few years. It is now evident that perturbed protease activity directly contributes to tumorogenesis and metastasis via aberrant extracellular matrix (ECM) remodeling, as well as, targeting non-ECM proteins of tumor microenvironment. The human tissue kallikrein (hK) family is a subgroup of secreted serine proteases, comprised of fifteen members. There is accumulating evidence that expression and proteolytic activity of various members of the hK family are dysregulated in a wide range of tumors, including ovarian, breast, and prostate cancers. Recent findings suggest that, analogous to other proteases, hKs exert their physiologic and pathologic functions through highly regulated proteolytic cascades. For instance, hK5 was shown to be autoactivated and sequentially activating hK7 in stratum corneum (SC) layer of skin. Similarly, hK2 was reported to activate hK3 in seminal plasma. Moreover, various hKs are known to be co-expressed and are coordinately up- or down- regulated in various cancer cell lines and/or tissues. Furthermore, inactive pro-hKs are activated by cleavage at their trypsin-like cleavage motifs, suggesting activation of chymotrypsin-like by trypsin-like hKs. To elucidate proteolytic cascades involved in activation of hKs, synthetic heptapeptides representing the activation sites of the fifteen hKs were designed. The ability of the active recombinant hKs to cleave the heptapeptides was examined by reversed phase HPLC. Cleavage efficiency was quantified as a time-dependent percent reduction of the peak representing uncleaved heptapeptide. Synthetic peptides representing the two cleaved fragments of each heptapeptide were used as controls to eliminate peak reduction due to non-specific degradation. We confirmed previously reported hK3 and hK7 cleavage by hK2 and hK5, respectively. In addition, we demonstrated possible activation of hKs 1-3, 5,7,8,9, and 11 by both hK5 and hK2. hK5 cleaves additional heptapeptides representing hKs 8 and 15. Activation of pro-hK3 was further confirmed using the hK3-specific flourogenic substrate, AAPF-AMC. Interestingly, we observed that excess pro- hK2 and hK3 are internally cleaved and deactivated in a dose-dependent manner by active hK5. Analogous to many other proteases, hK5 seems to be bidirectionally involved in the regulation of certain hK proteolytic cascades. experiments to elucidate the dynamic process of hK proteolytic cascades and their complex tumor-associated dysregulation are ongoing. Therapeutic manipulation of hK regulatory pathways can supplement the current clinical treatments of certain carcinomas.

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