Viktor Magdolen

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences.

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.

CELLULAR GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE D REGULATES UROKINASE RECEPTOR SHEDDING AND CELL SURFACE EXPRESSION

The glycosylphosphatidylinositol (GPI) ‐ anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane‐anchored and soluble isoforms (s‐uPAR) in vitro and in vivo. Recent evidence indicates that s‐uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s‐uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI‐specific phospholipase D (GPI‐PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI‐PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI‐PLD activity by GPI‐PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI‐PLD also regulated uPAR expression, possibly by releasing a GPI‐anchored growth factor. Our data suggest that cellular GPI‐PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI‐anchored proteins by generating functionally distinct, soluble counterparts. J. Cell. Physiol. 180:225–235, 1999.

Natural and synthetic inhibitors of kallikrein-related peptidases (KLKs)

Including the true tissue kallikrein KLK1, kallikrein-related peptidases (KLKs) represent a family of fifteen mammalian serine proteases. While the physiological roles of several KLKs have been at least partially elucidated, their activation and regulation remain largely unclear. This obscurity may be related to the fact that a given KLK fulfills many different tasks in diverse fetal and adult tissues, and consequently, the timescale of some of their physiological actions varies significantly. To date, a variety of endogenous inhibitors that target distinct KLKs have been identified. Among them are the attenuating Zn2+ ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α2-macroglobulin. Failure of these inhibitory systems can lead to certain pathophysiological conditions. One of the most prominent examples is the Netherton syndrome, which is caused by dysfunctional domains of the Kazal-type inhibitor LEKTI-1 which fail to appropriately regulate KLKs in the skin. Small synthetic inhibitory compounds and natural polypeptidic exogenous inhibitors have been widely employed to characterize the activity and substrate specificity of KLKs and to further investigate their structures and biophysical properties. Overall, this knowledge leads not only to a better understanding of the physiological tasks of KLKs, but is also a strong fundament for the synthesis of small compound drugs and engineered biomolecules for pharmaceutical approaches. In several types of cancer, KLKs have been found to be overexpressed, which makes them clinically relevant biomarkers for prognosis and monitoring. Thus, down regulation of excessive KLK activity in cancer and in skin diseases by small inhibitor compounds may represent attractive therapeutical approaches.

High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells.

Overproduction of actin is lethal to yeast cells. In contrast, overexpression of the profilin gene, PFYI, encoding an actin‐binding protein, leads to no very obvious phenotype. Interestingly, profilin over‐production can compensate for the deleterious effects of too much actin in a profilin concentration‐dependent manner. Our results, thus, document that actin and profilin interact in vivo. Immunofluorescence studies suggest that suppression works by reducing actin assembly. We observed, however, that even massive overproduction of profilin fails to fully restore the wild‐type phenotype (e.g. the wild‐type appearance of the actin microfilament system). This may indicate that actin monomer sequestration is not the only mechanism by which the balance of actin polymerization is controlled.

Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by Neutrophil Elastase and Cathepsin G

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)—the constitutive α-secretase—is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin−/− mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Structures and specificity of the human kallikrein-related peptidases KLK 4, 5, 6, and 7

Abstract Human kallikrein-related peptidases (KLKs) are (chymo)-trypsin-like serine proteinases that are expressed in a variety of tissues such as prostate, ovary, breast, testis, brain, and skin. Although their physiological functions have been only partly elucidated, many of the KLKs appear to be useful prognostic cancer markers, showing distinct correlations between their expression levels and different stages of cancer. Recent advances in the purification of ‘new type’ recombinant KLKs allowed solution of the crystal structures of KLK4, KLK5, KLK6, and KLK7. Along with these data, enzyme kinetic studies and extended substrate specificity profiling have led to an understanding of the non-prime-side substrate preferences of KLK4, 5, 6, and 7. The shape and polarity of the specificity pockets S1–S4 explain well their substrate preferences. KLK4, 5, and 6 exhibit trypsin-like specificity, with a strong preference for Arg at the P1 position of substrates. In contrast, KLK7 displays a unique chymotrypsin-like specificity for Tyr, which is also preferred at P2. All four KLKs show little specificity for P3 residues and have a tendency to accept hydrophobic residues at P4. Interestingly, for KLK4, 5, and 7 extended charged surface regions were observed that most likely serve as exosites for physiological substrates.

Urokinase-type Plasminogen Activator (uPA) and its Receptor (uPAR): Development of Antagonists of uPA / uPAR Interaction and their Effects In Vitro and In Vivo

In cancer, increased levels of the tumor-associated serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are linked to tumor progression, metastasis, and shortened survival in patients afflicted with this disease. Strong clinical and experimental evidence has accumulated that the cell surface interaction of uPA with uPAR facilitates extravasation and intravasation of cancer cells by regulating local proteolysis and attachment of the cells to components of the extracellular matrix. Moreover, the uPA/uPAR system is also implicated in proliferation of some tumor cells and migration of tumor and endothelial cells. Thus, metastasis formation is facilitated via tumor cell spread through the blood circulation system and neovascularization at the metastatic site. This multifunctional potential has rendered the uPA/uPAR system an attractive novel target for anti-metastatic therapy. Consequently, inhibitors of the uPA/uPAR interaction have been and are currently developed for suppression of tumor growth and angiogenesis. In addition to antibodies and recombinant uPA- or uPAR-derived proteins, various linear and cyclic peptides as well as small molecules have been designed and synthesized which potently interfere with the uPA/uPAR interaction, leading to reduced tumor progression in experimental animals. Such compounds affecting the uPA/uPAR system represent novel tumor biology-based therapeutic agents, thereby opening new ways for patient optimized and individualized cancer therapy.

Suppression of rat breast cancer metastasis and reduction of primary tumour growth by the small synthetic urokinase inhibitor WX-UK1.

The serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are often elevated in malignant tumours, hence, inhibition of this tumour-associated plasminogen activation system provides an attractive target for therapeutic strategies. WX-UK1, a derivative of 3-aminophenylalanine in the L-conformation with inhibitory antiproteolytic properties, was tested for its specificity spectrum using specific chromogenic paranitroanilide peptide substrates. The corresponding D-enantiomer of WX-UK1 was used as a control. The anti-tumour and anti-metastatic (number of lung foci and weight of the axillary lymph nodes) properties were studied by subcutaneous administration of WX-UK1 to Brown Norwegian (BN) rats carrying orthotopically transplanted BN472 rat breast tumours. WX-UK1 selectively inhibited tumour-related proteases from rats and humans such as uPA, plasmin, or thrombin in the sub or low micromolar range. The activity was stereoselective as the D-enantiomer of WX-UK1 inhibited uPA and plasmin at approximately 70-fold higher Ki values than the active L-form. Chronical administration of the L-enantiomer of WXUK1 impaired primary tumour growth and metastasis of BN472 rat breast cancer in a dose-dependent manner. The minimum inhibitory dosage with maximal effect was between 0.15 and 0.3 mg/kg/day. The inactive D-enatiomer of WX-UK1 was not active in this respect. Daily treatment with WX-UK1 for up to 35 days was well tolerated as judged by the unchanged body and organ weight development. In conclusion, our results provide evidence that WX-UK1 as a single agent inhibits breast tumour growth and metastasis in vivo, and thus is a promising candidate drug to treat human cancer.

Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function

Ovarian cancer metastasis is associated with an increase in the urokinase‐type plasminogen activator (uPA) and its receptor uPAR. We present evidence that binding of uPA to uPAR provokes a mitogenic response in the human ovarian cancer cell line OV‐MZ‐6 in which endogenous uPA production had been significantly reduced by stable uPA ‘antisense’ transfection. High molecular weight (HMW) uPA, independent of its enzymatic activity, produced an up to 95% increase in cell number concomitant with 2‐fold elevated [3H]thymidine incorporation as did the catalytically inactive but uPAR binding amino‐terminal fragment of uPA, ATF. uPA‐induced cell proliferation was significantly decreased by blocking uPA/uPAR interaction by the monoclonal antibody IIIF10 and by soluble uPAR. The efficiency of the uPAR binding synthetic peptide cyclo19,31uPA19–31 to enhance OV‐MZ‐6 cell growth proved this molecular domain to be the minimal structural determinant for uPA mitogenic activity. Dependence of uPA‐provoked cell proliferation on uPAR was further demonstrated in Raji cells which do not express uPAR and were thus not induced by uPA. However, upon transfection with full‐length uPAR, Raji cells acquired a significant growth response to HMW uPA and ATF.