Regulatory mechanisms of KLK proteases from prostate

Kallikrein-related peptidases (KLKs) form a family of (chymo)trypsin-like serine proteases that are produced in an enzymatically inactive zymogen form. Four KLKs are predominantly expressed in the prostate: KLK2, 3, 4, and 11 are secreted into the prostate fluid, where the prostatic KLKs are converted in a cascade-like activation web from the zymogen state to proteolytically activated KLKs. In addition to its physiological role in degrading gel forming proteins in seminal fluid, KLK3 (aka PSA) has become a widely used clinical marker for diagnosis of prostate cancer. Moreover, KLK2 and KLK4 are promising prognostic markers for prostate cancer. Both the activation and the regulation of proteolytic activity in the prostatic KLK2, 3, 4 & 11 are in the focus of our study. Along this line, we will measure enzyme kinetic parameters depending on physiological regulators, such as Zn2+ or Ca2+-ions, glycosylation, and synthetic and natural inhibitors. We will further substantiate these investigations by their structural determination with X-ray crystallography. We will validate mechanistic details of zymogen activation, enzymatic activity, and its regulation by mutational analysis, flanked with biophysical studies. Previously, we observed tetramer and octamer formation for KLKs 3 and 4, which appears to reduce the protease activity dramatically and may, therefore, constitute an essential regulation principle within prostatic KLKs. Consequently, we propose to elucidate the structural and mechanistic basis of this oligomerization process. Moreover, the available prostate KLK variants and complexes should allow for developing antibodies that recognize imbalanced KLK levels occurring during prostate cancer and may eventually allow for improved etiopathological prognosis. Concomitantly, we will extend the understanding of KLK activity regulation by functional studies in human prostate cancer cell lines, such as DU-145. These cells will be transfected with wild-type or mutant KLK expression plasmids. The effects of individual and combined KLK protease activity for the proliferative, adhesive, migratory, and invasive potential of prostate cancer cells will be elucidated. Finally, overexpression and/or modulation of the KLK activity in animal models can delineate the prostate cancer activome in vivo and help to identify new targets for therapy concepts of prostate cancer. KLK crystal structures should aid the rational design of inhibitory compounds that may eventually result in future pharmaceutical drugs. In summary, our project exploits the synergistic effects of interdisciplinary research spanning KLK activity regulation in living organisms to the atomic resolution of our target prostatic proteases.

Kallikrein-related peptidases (KLKs) are a family of serine proteases that are active outside of cells. Four members, KLK2, 3, 4 & 11 are mainly produced by the prostate and ensure the motility of sperms by degrading gel-forming proteins. The best known example is KLK3 (PSA), which is used as a clinical marker for prostate cancer. Using X-ray crystallography, the structure of the KLK2 was determined and its function in activation, specific substrate binding, enzymatic activity was elucidated. In particular, the so-called 99-loop, a flexible stretch near the active site was found to be an important regulatory element, which, e.g., binds the inhibitory, physiologically important Zn2+ ions. A KLK2 variant from eukaryotic Leishmania cell cultures was obtained, in which the sugar molecules were attached to the 99-loop. Such modifications (glycosylation) occur naturally, but cannot be generated by E. coli bacteria cultures. A comparison of the glycosylated with the sugar-free KLK2 explained a regulatory function of the sugar for the protease activity and its protective effect against self degradation of the protease. All four KLKs were purified as inactive proforms from bacterial cultures, which were then refolded, before they were activated by the enzyme enterokinase with propeptide cleavage at the N-terminus. In addition, a simple and efficient method for the purification of recombinant enterokinase was established. Accordingly, KLKs 3, 4 and 11 were activated, while a KLK2 variant was also capable of self-activation. Similarly, purification of the four KLK proforms was successful, but it turned out that generation of the correct N-terminal amino acid residues was difficult with a variety of activating proteases. As for KLK2 the purification of glycosylated variants of KLK3, 4 and 11 was successful. Mass spectrometry confirmed the attachment of the sugars even at specific sites, which will be important for further biochemical analyses. In a cooperation with the Clinical Research Unit of the University of Munich (V. Magdolen) the regulation of KLK activity was investigated in human prostate cancer cells, for example with PC-3. The stable transfection with KLK expression plasmids had a significant effect on proliferation, adhesion, migration and invasion of prostate cancer cells. In addition, very specific synthetic KLK4 inhibitors could be found in cooperation with the University of Antwerp (K. Augustyns, J. Joossens) that might serve in the future to prevent the formation of metastases in prostate cancer. Also, the role of the sugar molecules of prostatic KLKs becomes more comprehensible and important, especially since they exhibit distinct changes in cancer, and, thus, may be employed similar to the KLK inhibitors in medical application for the diagnosis and treatment of prostate cancer.