Viktor Magdolen - Academia.edu (original) (raw)

Papers by Viktor Magdolen

Biochemical and biophysical research communications, Jan 30, 2016

Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved ... more Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.

Journal of Biological Chemistry, 2014

Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation. Res... more Background: Serine proteases KLK2 and KLK3 clear the way for spermatozoa before impregnation. Results: Enzymatic assays and structures of KLK2 elucidate its catalytic action, especially when compared with conformations of similar proteases. Conclusion: Flexible loops around the active site of serine proteases open concertedly upon substrate binding. Significance: This mechanistic model will stimulate the design of pharmaceutical inhibitors.

Proceedings of the National Academy of Sciences, 2007

hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) ser... more hK7 or human stratum corneum chymotryptic enzyme belongs to the human tissue kallikrein (hKs) serine proteinase family and is strongly expressed in the upper layers of the epidermis. It participates in skin desquamation but is also implicated in diverse skin diseases and is a potential biomarker of ovarian cancer. We have solved x-ray structures of recombinant active hK7 at medium and atomic resolution in the presence of the inhibitors succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone and Ala-Ala-Phe-chloromethyl ketone. The most distinguishing features of hK7 are the short 70 -80 loop and the unique S1 pocket, which prefers P1 Tyr residues, as shown by kinetic data. Similar to several other kallikreins, the enzyme activity is inhibited by Zn 2؉ and Cu 2؉ at low micromolar concentrations. Biochemical analyses of the mutants H99A and H41F confirm that only the metal-binding site at His 99 close to the catalytic triad accounts for the noncompetitive Zn 2؉ inhibition type. Additionally, hK7 exhibits large positively charged surface patches, representing putative exosites for prime side substrate recognition. 99 loop ͉ desquamation ͉ metal-binding proteinase ͉ Netherlon syndrome

Journal of Molecular Biology, 2007

Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has... more Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 Å resolution were obtained with and without Zn 2+ . While the overall structure and the specificity of S1 pocket for basic sidechains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn 2+ . Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn 2+ . Instead, Zn 2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn 2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metalbinding rat trypsin, rotate out of its triad position to provide the third coordination site of the bound Zn 2+ , rendering Zn 2+ -bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn 2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn 2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn 2+ . The His96-99-57 triad is thus suggested to be responsible for the Zn 2+mediated inhibition of hK5 catalysis.

Journal of Molecular Biology, 2006

Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinas... more Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition.

Journal of Biological Chemistry, 2006

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine pro... more 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. The abbreviations used are: PSA, prostate-specific antigen; hK, human tissue kallikrein; EK, enterokinase; AMC, 7-amino-4-methylcoumarine; uPA, urokinase-type plasminogen activator; PS-SCL, positional scanning with a synthetic combinatorial peptide library; BPTI, bovine pancreatic trypsin inhibitor.

FEBS Letters, 1993

Electrospray mass s~trometry of the purified isoforms of the hypusine-containing protein of ~ucc~... more Electrospray mass s~trometry of the purified isoforms of the hypusine-containing protein of ~ucc~ro~yces cerevisiae HypZp suggested a phospho~lation of the acidic isoform, which was confirmed by phosphatase treatment. The phospho~lation site was mapped to the N-acetylated se&e residue in position no. 1 by mass spectrometric analysis of enzymatic fragments. Mutation of this serine residue gives rise to only the basic isoform, confirming our protein chemical data. As this mutation has no effect on cell viability or growth rate. the unphosphorylated isoform is sufficient to exert the essential in vivo function of Hyp2p.

Biological Chemistry, 2000

Human kallikrein-related peptidases (KLKs) are (chymo)trypsin-like serine proteinases that are ex... more 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.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1993

We have purified bleomycin hydrolase from yeast (molecular mass 55000 Da). Using protein sequence... more We have purified bleomycin hydrolase from yeast (molecular mass 55000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blhl mutant cells.

The American Journal of Pathology, 2010

Certain serine proteases are considered to be signaling molecules that act through protease-activ... more Certain serine proteases are considered to be signaling molecules that act through protease-activated receptors (PARs). Our recent studies have implicated PAR1 and PAR4 (thrombin receptors) and PAR2 (trypsin receptor) in human colon cancer growth. Here we analyzed the expression of KLK4 , a member of the kallikrein-related peptidase (KLK) family of serine proteases and explored whether this member can activate PAR1 and PAR2 in human colon cancer cells. Immunohistochemistry showed KLK4 expression in human colon adenocarcinomas and its absence in normal epithelia. KLK4 (1 mol/L) initiated loss of PAR1 and PAR2 from the HT29 cell surface as well as increased intracellular calcium transients in HT29 cells. This KLK4-induced Ca 2؉ flux was abrogated after an initial challenge of the cells with TRAP (SFLLR-NH 2 ; 100 mol/L), which is known to desensitize PAR1 and PAR2. Interestingly , PAR1 blocking antibody , which inhibits cleavage and activation by thrombin , dramatically reduced KLK4-induced Ca 2؉ influx , but blocking cleavage of PAR2 failed to attenuate the KLK4-induced Ca 2؉ flux. Consistently, desensitization with AP1 (TFFLR-NH 2 ) , targeting PAR1, attenuated most of the Ca 2؉ flux induced by KLK4. KLK4 also induced a rapid and significant ERK1/2 phosphorylation in HT29 cells. Our results demon-strate, for the first time, that KLK4 is aberrantly expressed in colon cancer and capable of inducing PAR1 signaling in cancer cells. These data suggest that KLK4 signaling via PAR1 may represent a novel pathway in colon tumorigenesis.

International Journal of Molecular Medicine, 2008

Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type... more Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) play a key role in tumor-associated processes such as the degradation of extracellular matrix proteins, tissue remodeling, cell adhesion, migration, and invasion. High antigen levels of uPA and PAI-1 in tumor tissue of various solid malignant tumors, including breast cancer, are associated with poor patient prognosis. In

The human tissue kallikrein gene family (KLK1 to KLK15) encodes a group of 15 serine proteases (h... more The human tissue kallikrein gene family (KLK1 to KLK15) encodes a group of 15 serine proteases (hK1 to hK15), several of which have been implicated in cancer-related processes. Methods: We established a specific quantitative reverse transcription-PCR assay for full-length KLK7 mRNA that excluded amplification of the exon 2 deletion splice variant (the latter does not encode a functional protease), and evaluated full-length KLK7 mRNA expression [normalized to human glucose-6-phosphate dehydrogenase (h-G6PDH)] in tumor tissue specimens from 155 breast cancer patients. Results: High KLK7 mRNA expression (continuous) was significantly associated with a better patient outcome according to both univariate (P ‫؍‬ 0.005) and multivariate (P ‫؍‬ 0.046) Cox survival analysis. Separation of patients by optimized dichotomization revealed a significantly better prognosis patients with high KLK7 mRNA status (n ‫؍‬ 89) compared with patients with low KLK7 mRNA status [n ‫؍‬ 66; univariate hazard ratio (HR) ‫؍‬ 0.45 (P ‫؍‬ 0.001); multivariate HR ‫؍‬ 0.50 (P ‫؍‬ 0.005)]. In the subgroup of patients not receiving adjuvant treatment (n ‫؍‬ 69), KLK7 mRNA status was a significant prognosticator [univariate HR ‫؍‬ 0.29 (P ‫؍‬ 0.002); multivariate HR ‫؍‬ 0.40 (P ‫؍‬ 0.034)]. This subgroup was least influenced by postoperative treatment and thus best showed the impact of KLK7 expression on the natural course of breast cancer disease. Conclusion: Expression of full-length KLK7 mRNA may represent a new prognostic marker in breast cancer disease.

Cancer research, Jan 15, 2001

Overexpression of matrix metalloproteinases (MMPs) facilitates tumor cell invasion. Synthetic MMP... more Overexpression of matrix metalloproteinases (MMPs) facilitates tumor cell invasion. Synthetic MMP inhibitors such as batimastat have been designed to treat cancer. We report that because of batimastat treatment, human breast carcinoma cells metastasized to the liver in nude mice and that an increase of liver metastases of murine T-cell lymphoma cells was observed in syngeneic mice. Batimastat treatment also caused liver-specific overexpression of MMPs-2, -9, and mRNA up-regulation of angiogenesis factors and caspase-1, even in tumor-free animals. Induction of organ-specific side effects need to be taken into account regarding further development and clinical use of synthetic MMP inhibitors.

Advances in experimental medicine and biology, 2000

In the present overview, the multifunctional capacity of the uPA-system in tumor biological proce... more In the present overview, the multifunctional capacity of the uPA-system in tumor biological processes was emphasized. In addition to its central role in pericellular proteolysis, uPA/uPAR-mediated activities contribute to many different processes like cell proliferation, adhesion, migration, and angiogenesis. Whereas extracellular PAI-2 solely acts as an inhibitor of uPA (and tPA), PAI-1 clearly exerts additional functions, e. g. involvement in

Thrombosis and haemostasis, 2003

The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in bot... more The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-spec...

Recent results in cancer research. Fortschritte der Krebsforschung. Progrès dans les recherches sur le cancer, 2003

Tumor cell invasion and metastasis depend on the coordinated and temporal expression of proteolyt... more Tumor cell invasion and metastasis depend on the coordinated and temporal expression of proteolytic enzymes to degrade the surrounding extracellular matrix and of adhesion molecules to remodel cell-cell and/or cell-matrix attachments. The tumor cell-associated urokinase-type plasminogen activator system, consisting of the serine protease uPA, its substrate plasminogen, its membrane-bound receptor uPAR, as well as its inhibitors PAI-1 and PAI-2, plays an important role in these pericellular processes. Especially, association of the proteolytic activity of uPA with the cell surface via interaction with uPAR significantly increases the invasive capacity of tumor cells. Consequently, various approaches have been pursued to interfere with the expression or activity of uPA and/or uPAR, including antisense strategies and the development of active-site inhibitors of uPA or inhibitors of uPA/uPAR interaction. In this review, we focus on the results obtained in vitro and in vivo with tumor ce...

Thrombosis and haemostasis, 2004

Urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-I, play a key role in tumor inv... more Urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-I, play a key role in tumor invasion and metastasis. They were the first novel tumor biological factors to be validated at the highest level of evidence (LOE I) regarding their clinical utility in breast cancer. Their antigen levels are determined in tumor tissue extracts by standardized, quality-assured immunometric assays (ELISA). Since the late 1980s, numerous independent studies have demonstrated that patients with low levels of uPA and PAI-I in their primary tumor tissue have a significantly better survival than patients with high levels of either factor. These prognostic data have recently been validated by an EORTC (European Organization for Research and Treatment of Cancer) pooled analysis comprising more than 8,000 breast cancer patients. In addition, results from a multicenter prospective randomized therapy trial in node-negative breast cancer ("Chemo N(0)") showed that node-negative breast cancer ...

Radiology and oncology, 2013

Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers ... more Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer-directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only. Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic dru...

Oncotarget, Jan 10, 2015

miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associa... more miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221-/-222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3' UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results d...

The serine protease urokinase-type plasminogen activator (uPA) mediates cancer invasion and metas... more The serine protease urokinase-type plasminogen activator (uPA) mediates cancer invasion and metastasis by binding to a cell surface receptor (uPA-R, CD87) on both tumor and stromal cells. In the present study we assessed uPA-R distribution in formalin-fixed, paraffin-embedded breast cancer specimens (n=50) and benign lesions (n=10) by immunohistochemistry employing a newly developed polyclonal chicken antibody to uPA-R (pAb HU277) in parallel with established monoclonal antibody (mAb) 3936 to uPA-R. In addition, uPA-R mRNA synthesis was investigated by in situ hybridization. In all of the sections analyzed, macrophage-like cells reacted with either antibody type. In 22 of the 50 cancer specimens, tumor cells reacted with pAb HU277 in contrast to mAb 3936 which only stained 9 of the 22 positive cases. Nevertheless, in 49 of the 50 cases, uPA-R mRNA was detected in cancer and in stromal cells by in situ hybridization suggesting posttranscriptional regulation of uPA-R expression in breast cancer cells. In 18 of 50 cases, uPA-R mRNA was also visualized in blood vessel lining endothelial cells by in situ hybridization and applying pAb HU277 in 14 of these 18 cases by immunohistochemistry. mAb 3936 did not stain any endothelial cells. pAb HU277 reacted with the breast gland epithelial cells of benign lesions as well, in contrast to mAb 3936 which did not. As for the cancer tissue, in benign lesions, endothelial cells were sporadically stained by pAb HU277. This antibody, but not mAb 3936, also stained myoepithelial cells in intraductal areas of invasive breast carcinoma. The results presented demonstrate the usefulness of pAb HU277 in locating uPA-R in tumor and normal cells with high sensitivity in formalin-fixed, paraffin-embedded breast tissue.