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Eleftherios P Diamandis - One of the best experts on this subject based on the ideXlab platform.
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kallikrein related peptidases protein expression in lymphoid tissues suggests potential implications in immune response
Clinical Biochemistry, 2020Co-Authors: Antoninus Soosaipillai, Eleftherios P Diamandis, Panagiota S Filippou, Annie H Ren, Roaa Safar, Ioannis Prassas, James ConnerAbstract:Abstract Objectives Kallikrein-related peptidases (KLKs) are a subgroup of 15 secreted chymotrypsin- and trypsin-like serine proteases that have been reported to possess novel functions in innate immunity and inflammation. Since the potential role of KLKs in immunity has not been studied in detail at the protein level, we examined the expression pattern of 12 members of the KLK family in immune-related tissues. Design & Methods Protein expression in tissue extracts was evaluated using immunoassays (ELISA). Immunohistochemistry (IHC) was performed on representative sections of tonsil and lymph nodes to determine the cellular localization of the KLK family members. Results ELISA profiling of KLK3-KLK15 (except KLK12) revealed higher protein levels in the tonsil, compared to the lymph nodes and spleen. Relatively high protein levels in the tonsil were observed for KLK7, KLK9, KLK10 and KLK13. Expression of these KLKs was significantly lower in lymph nodes and spleen. IHC analysis in tonsil unveiled that KLK9 and KLK10 were differentially expressed in lymphoid cells. KLK9 was strongly expressed in the germinal center of lymphoid follicles where activated B-cells reside, whereas KLK10 was expressed in the follicular dendritic cells (FDCs) that are vital for maintaining the cycle of B cell maturation. Conclusion Overall, our study revealed the possible implications of KLK expression and regulation in the immune cells of lymphoid tissues.
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Kallikrein-related peptidases 6 and 10 are elevated in cerebrospinal fluid of patients with Alzheimer’s disease and associated with CSF-TAU and FDG-PET
Translational Neurodegeneration, 2019Co-Authors: Oliver Goldhardt, Viktor Magdolen, Antoninus Soosaipillai, Eleftherios P Diamandis, Inanna Warnhoff, Igor Yakushev, Ilijana Begcevic, Hans Forstl, Panagiotis Alexopoulos, Timo GrimmerAbstract:Background Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer’s disease (AD). We elucidated the suitability of KLK6, KLK8 and KLK10 to distinguish AD from NC and explored associations with established AD biomarkers. Methods KLK levels in cerebrospinal fluid (CSF), as determined by ELISA, were compared between 32 AD patients stratified to A/T/(N) system with evidence for amyloid pathology and of 23 normal controls with normal AD biomarkers. Associations between KLK levels and clinical severity, CSF and positron emission tomography (PET) based AD biomarkers were tested for. Results Levels of KLK6 and KLK10 were significantly increased in AD. KLK6 differed significantly between AD A+/T+/N+ and AD A+/T−/N+ or NC with an AUC of 0.922. CSF pTau and tTau levels were significantly associated with KLK6 in AD. Conclusions KLK6 deserves further investigations as a potential biomarker of Tau pathology in AD.
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kallikrein related peptidases 6 and 10 are elevated in cerebrospinal fluid of patients with alzheimer s disease and associated with csf tau and fdg pet
Translational neurodegeneration, 2019Co-Authors: Oliver Goldhardt, Viktor Magdolen, Antoninus Soosaipillai, Eleftherios P Diamandis, Inanna Warnhoff, Igor Yakushev, Ilijana Begcevic, Hans Forstl, Panagiotis AlexopoulosAbstract:Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer’s disease (AD). We elucidated the suitability of KLK6, KLK8 and KLK10 to distinguish AD from NC and explored associations with established AD biomarkers. KLK levels in cerebrospinal fluid (CSF), as determined by ELISA, were compared between 32 AD patients stratified to A/T/(N) system with evidence for amyloid pathology and of 23 normal controls with normal AD biomarkers. Associations between KLK levels and clinical severity, CSF and positron emission tomography (PET) based AD biomarkers were tested for. Levels of KLK6 and KLK10 were significantly increased in AD. KLK6 differed significantly between AD A+/T+/N+ and AD A+/T−/N+ or NC with an AUC of 0.922. CSF pTau and tTau levels were significantly associated with KLK6 in AD. KLK6 deserves further investigations as a potential biomarker of Tau pathology in AD.
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quantification of human kallikrein related peptidases in biological fluids by multiplatform targeted mass spectrometry assays
Molecular & Cellular Proteomics, 2016Co-Authors: Antoninus Soosaipillai, Eleftherios P Diamandis, Theano D Karakosta, Ihor Batruch, Andrei P DrabovichAbstract:Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.
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novel biological substrates of human kallikrein 7 identified through degradomics
Journal of Biological Chemistry, 2015Co-Authors: Eleftherios P Diamandis, Ioannis Prassas, Yijing Yu, Apostolos DimitromanolakisAbstract:Abstract Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.
Andreas Scorilas - One of the best experts on this subject based on the ideXlab platform.
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discovery of novel transcripts of the human tissue kallikrein klk1 and kallikrein related peptidase 2 klk2 in human cancer cells exploiting next generation sequencing technology
Genomics, 2019Co-Authors: Panagiotis G Adamopoulos, Christos K. Kontos, Andreas ScorilasAbstract:Abstract Tissue kallikrein, kallikrein-related peptidases (KLKs), and plasma kallikrein form the largest group of serine proteases in the human genome, sharing many structural and functional properties. Several KLK transcripts have been found aberrantly expressed in numerous human malignancies, confirming their prognostic or/and diagnostic values. However, the process of alternative splicing can now be studied in-depth due to the development of Next-Generation Sequencing (NGS). In the present study, we used NGS to discover novel transcripts of the KLK1 and KLK2 genes, after nested touchdown PCR. Bioinformatics analysis and PCR experiments revealed a total of eleven novel KLK transcripts (two KLK1 and nine KLK2 transcripts). In addition, the expression profiles of each novel transcript were investigated with nested PCR experiments using variant-specific primers. Since KLKs are implicated in human malignancies, qualifying as potential biomarkers, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.
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Molecular cloning of novel transcripts of human kallikrein-related peptidases 5, 6, 7, 8 and 9 (KLK5 - KLK9), using Next-generation sequencing.
Scientific reports, 2017Co-Authors: Panagiotis G Adamopoulos, Christos K. Kontos, Andreas ScorilasAbstract:Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity and affecting cell cycle control, proliferation, apoptosis, angiogenesis, invasion, and metastasis. In this study, we describe the discovery and molecular cloning of thirty novel transcripts of the human KLK5, KLK6, KLK7, KLK8 and KLK9 genes, using 3′ rapid amplification of cDNA ends (3′ RACE) and NGS technology, as well as their expression analysis in many established cell lines, originating from several distinct cancerous and normal tissues. Extensive bioinformatic analysis revealed novel splice variants of these five members of the KLK family, comprising entirely new exons, previously unknown boundaries of the already annotated exons (extensions and truncations) as well as alternative splicing events between these exons. Nested RT-PCR in a panel of human cell lines originating from seventeen cancerous and two normal tissues with the use of variant-specific pairs of primers was carried out for expression analysis of these novel splice variants, and Sanger sequencing of the respective amplicons confirmed our NGS results. Given that some splice variants of KLK family members possess clinical value, novel alternatively spliced transcripts appear as new candidate biomarkers for diagnostic and/or prognostic purposes and as targets for therapeutic strategies.
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emerging clinical importance of the cancer biomarkers kallikrein related peptidases klk in female and male reproductive organ malignancies
Radiology and Oncology, 2013Co-Authors: Manfred Schmitt, Viktor Magdolen, George M Yousef, Andreas Scorilas, Eleftherios P Diamandis, Feng Yang, M Kiechle, Jane Bayani, Julia DornAbstract: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 drugs to interfere with tumor progression and metastasis.
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kallikrein related peptidase 4 KLK4 mrna predicts short term relapse in colorectal adenocarcinoma patients
Cancer Letters, 2013Co-Authors: Christos K. Kontos, Dimitrios Chantzis, Iordanis N Papadopoulos, Andreas ScorilasAbstract:The members of the kallikrein-related peptidase (KLK) family are aberrantly expressed in cancer, including colorectal adenocarcinoma. KLK4 is an endogenous activator of protease-activated receptor 1 (PAR1) in HT-29 colorectal adenocarcinoma cells, inducing PAR1 signaling and subsequent ERK1/2 activation. The aim of this study was to analyze KLK4 mRNA expression in colorectal adenocarcinoma and to examine its prognostic value as a novel molecular tissue biomarker in this malignancy. Therefore, total RNA was isolated from primary tumors of 81 colorectal adenocarcinoma patients, cDNA was prepared, and KLK4 mRNA expression analysis was performed using quantitative real-time PCR. KLK4 mRNA was significantly associated with the Dukes stage, tumor invasion, size, and histological grade. Survival analysis demonstrated that KLK4 mRNA expression constitutes an unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free survival (DFS), independently of the nodal status and tumor size. Furthermore, KLK4 mRNA predicts short-term relapse of lymph node-negative patients or those with tumors of early Dukes stage. In conclusion, KLK4 mRNA expression can be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma.
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Significant alterations in the expression pattern of kallikrein-related peptidase genes KLK4, KLK5 and KLK14 after treatment of breast cancer cells with the chemotherapeutic agents epirubicin, docetaxel and methotrexate
Tumor Biology, 2013Co-Authors: Georgia Papachristopoulou, M Talieri, Andreas ScorilasAbstract:Given that 1.3 million new cases of breast cancer are universally registered among women and approximately 36 % of the patients die annually, the revelation of new predictive markers for treatment efficiency is of vital importance. Recently, our group has depicted that KLK4 , KLK5 , and KLK14 are differentially expressed in breast carcinoma. The objective of this study was to determine and investigate the expression pattern of the KLK4 , KLK5 , and KLK14 genes in breast cancer cells after treatment with established chemotherapeutic agents. We evaluated these genes’ expression after treatment of the BT-20 cells with epirubicin, docetaxel and methotrexate, determining their cytotoxic effect by MTT and trypan blue assays. The relative quantification of genes’ mRNA levels was performed by using the SYBR Green® chemistry, and the HPRT1 served as an endogenous control gene. The drugs triggered apoptosis in treated cells and induced deregulations in the expression of the above KLKs . The most significant alterations were a 12-fold and tenfold increase of KLK5 in docetaxel and methotrexate-treated cells, respectively, while the KLK4 levels decreased by ten-fold in epirubicin, five-fold in docetaxel and twenty-fold in methotrexate treated-cells, compared to the untreated ones. In the case of KLK14 levels, a twofold increase in epirubicin and threefold decrease in methotrexate-treated cells were observed. Present significant alterations in the expression pattern of KLK4 , KLK5 , and KLK14 could comprise an initial stage for predicting chemotherapy response in breast cancer and should be further investigated as predictive markers in the future.
Judith A. Clements - One of the best experts on this subject based on the ideXlab platform.
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Pericellular regulation of prostate cancer expressed kallikrein-related peptidases and matrix metalloproteinases by cell surface serine proteases.
American journal of cancer research, 2017Co-Authors: Janet C. Reid, Viktor Magdolen, Judith A. Clements, Admire Matsika, Claire M. Davies, Amy Broomfield, Nigel C. Bennett, Bhuvana Srinivasan, John D HooperAbstract:We provide evidence of a pericellular network of proteases that are elevated and co-expressed in prostate cancer. The network involves the membrane bound serine proteases hepsin and TMPRSS2, the secreted kallikrein-related peptidases KLK4 and KLK14, and the secreted matrix metalloproteinases MMP-3 and MMP-9. Western blot analysis of cell lysates, conditioned cell culture media, immunoprecipitates and cell surface proteins, demonstrates a network of interactions centred largely at the plasma membrane, with the Arg/Lys specific proteases hepsin and TMPRSS2 key regulators of the network. Our data demonstrate that like TMPRSS2, hepsin is able to autoactivate. Active hepsin degrades KLK4, generating a cell associated degradation product with corresponding reduction in levels of cell-free KLK4. In contrast hepsin activates KLK14. TMPRSS2 appears to cleave amino terminal to the KLK4 activation site such that it is available for further processing to generate the active KLK4 protease. In contrast with hepsin, TMPRSS2 degrades KLK14. In addition to these direct mechanisms of regulation, hepsin and TMPRSS2 indirectly modulate KLK4 activity by cleaving the KLK4-activating protease MMP-3. Hepsin and TMPRSS2 also activate MMP-9, which similar to MMP-3, associates with the cell surface. Interestingly our data also show that proteolysis occurs between the membrane spanning and catalytic domains of hepsin and TMPRSS2. Hepsin cleavage occurs via an autoproteolytic mechanism, whereas TMPRSS2 cleavage is mediated by KLK14. Hepsin and TMPRSS2 are not shed from the cell surface but proteolysis likely disrupts domains that regulate the proteolytic activity of these proteases. Immunocytochemical analyses demonstrate that hepsin and TMPRSS2 colocalize on the cell surface with the secreted serine proteases KLK4 and KLK14, only in membrane protrusions, suggesting that reciprocal proteolytic interactions occur in defined cellular structures that are important during cancer dissemination for cell migration, invasion and survival. Also of note, immunohistochemical analysis of serial sections of prostate tumor demonstrated significant overlapping expression of the six proteases in vivo. Collectively these data suggest the possibility that the novel proteolytic network identified by us, will be most important during active dissemination of prostate cancers, and that its disruption could inhibit metastasis.
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Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum.
The Journal of investigative dermatology, 2016Co-Authors: Simon J. De Veer, Maria Brattsand, Judith A. Clements, Alain Hovnanian, Laetitia Furio, Joakim E. Swedberg, Christopher A. Munro, Jonathan M. HarrisAbstract:Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (Ki = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis.
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Combined expression of KLK4, KLK5, KLK6, and KLK7 by ovarian cancer cells leads to decreased adhesion and paclitaxel-induced chemoresistance.
2012Co-Authors: Daniela Loessner, Viktor Magdolen, Verena M.c. Quent, Julia Kraemer, Eva C. Weber, Dietmar W. Hutmacher, Judith A. ClementsAbstract:OBJECTIVE: Chemoresistance is a critical feature of advanced ovarian cancer with only 30% of patients surviving longer than 5 years. We have previously shown that four kallikrein-related (KLK) peptidases, KLK4, KLK5, KLK6 and KLK7 (KLK4-7), are implicated in peritoneal invasion and tumour growth, but underlying mechanisms were not identified. We also reported that KLK7 overexpression confers chemoresistance to paclitaxel, and cell survival via integrins. In this study, we further explored the functional consequenses of overexpression of all four KLKs (KLK4-7) simultaneously in the ovarian cancer cell line, OV-MZ-6, and its impact on integrin expression and signalling, cell adhesion and survival as contributors to chemoresistance and metastatic progression. METHODS: Quantitative gene and protein expression analyses, confocal microscopy, cell adhesion and chemosensitivity assays were performed. RESULTS: Expression of α5β1/αvβ3 integrins was downregulated upon combined stable KLK4-7 overexpression in OV-MZ-6 cells. Accordingly, the adhesion of these cells to vitronectin and fibronectin, the extracellular matrix binding proteins of α5β1/αvβ3 integrins and two predominant proteins of the peritoneal matrix, was decreased. KLK4-7-transfected cells were more resistant to paclitaxel (10-100 nmol/L: 38-54%), but not to carboplatin, which was associated with decreased apoptotic stimuli. However, the KLK4-7-induced paclitaxel resistance was not blocked by the MEK1/2 inhibitor, U0126. CONCLUSIONS: This study demonstrates that combined KLK4-7 expression by ovarian cancer cells promotes reduced integrin expression with consequently less cell-matrix attachment, and insensitivity to paclitaxel mediated by complex integrin and MAPK independent interactions, indicative of a malignant phenotype and disease progression suggesting a role for these KLKs in this process.
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Kallikreins on Steroids: Structure, Function, and Hormonal Regulation of Prostate-Specific Antigen and the Extended Kallikrein Locus
Endocrine Reviews, 2010Co-Authors: Mitchell G. Lawrence, Judith A. ClementsAbstract:The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and putative proteolytic functions. The kallikrein family is also emerging as a rich source of disease biomarkers with KLK3, commonly known as prostate-specific antigen, being the current serum biomarker for prostate cancer. The kallikrein locus is also notable because it is extraordinarily responsive to steroids and other hormones. Indeed, at least 14 functional hormone response elements have been identified in the kallikrein locus. A more comprehensive understanding of the transcriptional regulation of kallikreins may help the field make more informed hypotheses about the physiological functions of kallikreins and their effectiveness as biomarkers. In this review, we describe the organization of the kallikrein locus and the structure of kallikrein genes and proteins. We also focus on the transcriptional regulation of kallikreins by androgens, progestins, glucocorticoids, mineralocorticoids, estrogens, and other hormones in animal models and human prostate, breast, and reproductive tract tissues. The interaction of the androgen receptor with androgen response elements in the promoter and enhancer of KLK2 and KLK3 is also summarized in detail. There is evidence that all kallikreins are regulated by multiple nuclear receptors. Yet, apart from KLK2 and KLK3, it is not clear whether all kallikreins are direct transcriptional targets. Therefore, we argue that gaining more detailed information about the mechanisms that regulate kallikrein expression should be a priority of future studies and that the kallikreinlocuswillcontinuetobeanimportantmodelintheeraofgenome-wideanalyses.(EndocrineReviews 31: 407–446, 2010)
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Tissue-specific promoter utilisation of the kallikrein-related peptidase genes, KLK5 and KLK7, and cellular localisation of the encoded proteins suggest roles in exocrine pancreatic function
2008Co-Authors: Ying Dong, Tracey J. Harvey, John D Hooper, Nicholas Matigian, Hemamali Samaratunga, Judith A. ClementsAbstract:Tissue kallikrein (kallikrein 1) was first identified in pancreas and is the namesake of the kallikrein-related peptidase (KLK) family. KLK1 and the other 14 members of the human KLK family are encoded by 15 serine protease genes clustered at chromosome 19q13.4. Our Northern blot analysis of 19 normal human tissues for expression of KLK4 to KLK15 identified pancreas as a common expression site for the gene cluster spanning KLK5 to KLK13, as well as for KLK15 which is located adjacent to KLK1. Consistent with previous reports detailing the ability of KLK genes to generate organ- and disease-specific transcripts, detailed molecular and in silico analyses indicated that KLK5 and KLK7 generate transcripts in pancreas variant from those in skin or ovary. Consistently, we identified in the promoters of these KLK genes motifs which conform with consensus binding sites for transcription factors conferring pancreatic expression. In addition, immunohistochemical analysis revealed predominant localisation of KLK5 and KLK7 in acinar cells of the exocrine pancreas, suggesting roles for these enzymes in digestion. Our data also support expression patterns derived from gene duplication events in the human KLK cluster. These findings suggest that, in addition to KLK1, other related KLK enzymes will function in the exocrine pancreas.
Viktor Magdolen - One of the best experts on this subject based on the ideXlab platform.
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kallikrein related peptidases 6 and 10 are elevated in cerebrospinal fluid of patients with alzheimer s disease and associated with csf tau and fdg pet
Translational neurodegeneration, 2019Co-Authors: Oliver Goldhardt, Viktor Magdolen, Antoninus Soosaipillai, Eleftherios P Diamandis, Inanna Warnhoff, Igor Yakushev, Ilijana Begcevic, Hans Forstl, Panagiotis AlexopoulosAbstract:Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer’s disease (AD). We elucidated the suitability of KLK6, KLK8 and KLK10 to distinguish AD from NC and explored associations with established AD biomarkers. KLK levels in cerebrospinal fluid (CSF), as determined by ELISA, were compared between 32 AD patients stratified to A/T/(N) system with evidence for amyloid pathology and of 23 normal controls with normal AD biomarkers. Associations between KLK levels and clinical severity, CSF and positron emission tomography (PET) based AD biomarkers were tested for. Levels of KLK6 and KLK10 were significantly increased in AD. KLK6 differed significantly between AD A+/T+/N+ and AD A+/T−/N+ or NC with an AUC of 0.922. CSF pTau and tTau levels were significantly associated with KLK6 in AD. KLK6 deserves further investigations as a potential biomarker of Tau pathology in AD.
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Kallikrein-related peptidases 6 and 10 are elevated in cerebrospinal fluid of patients with Alzheimer’s disease and associated with CSF-TAU and FDG-PET
Translational Neurodegeneration, 2019Co-Authors: Oliver Goldhardt, Viktor Magdolen, Antoninus Soosaipillai, Eleftherios P Diamandis, Inanna Warnhoff, Igor Yakushev, Ilijana Begcevic, Hans Forstl, Panagiotis Alexopoulos, Timo GrimmerAbstract:Background Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer’s disease (AD). We elucidated the suitability of KLK6, KLK8 and KLK10 to distinguish AD from NC and explored associations with established AD biomarkers. Methods KLK levels in cerebrospinal fluid (CSF), as determined by ELISA, were compared between 32 AD patients stratified to A/T/(N) system with evidence for amyloid pathology and of 23 normal controls with normal AD biomarkers. Associations between KLK levels and clinical severity, CSF and positron emission tomography (PET) based AD biomarkers were tested for. Results Levels of KLK6 and KLK10 were significantly increased in AD. KLK6 differed significantly between AD A+/T+/N+ and AD A+/T−/N+ or NC with an AUC of 0.922. CSF pTau and tTau levels were significantly associated with KLK6 in AD. Conclusions KLK6 deserves further investigations as a potential biomarker of Tau pathology in AD.
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characterization of kallikrein related peptidase 4 KLK4 mrna expression in tumor tissue of advanced high grade serous ovarian cancer patients
PLOS ONE, 2019Co-Authors: Weiwei Gong, Julia Dorn, Yueyang Liu, Christof Seidl, Tobias Dreyer, Enken Drecoll, Matthias Kotzsch, Holger Bronger, Viktor MagdolenAbstract:Overexpression of several members of the kallikrein-related peptidase (KLK) family, including KLK4, has been reported in ovarian cancer tissue, consistent with the fact that elevated levels of KLK protein are often also found in serum and in effusion fluids of ovarian cancer patients. In the present study, we quantitatively analyzed KLK4 tumor tissue mRNA expression levels in a homogeneous cohort including 138 patients of advanced high-grade serous ovarian cancer (FIGO stage III/IV). Age as well as ascites fluid volume were found to be significantly associated with KLK4 mRNA expression levels. In univariate Cox regression analysis, the clinical factors residual tumor mass and ascites fluid volume represented univariate predictors for both overall survival (OS) and progression-free survival (PFS). Furthermore, elevated KLK4 mRNA expression levels were significantly linked with reduced OS (p = 0.001), but not with PFS. The results concerning the association of KLK4 mRNA expression with OS were validated in a publicly available Affymetrix-based mRNA data set from The Cancer Genome Atlas (n = 252) applying the Kaplan-Meier Plotter tool (p = 0.047). In multivariable analyses, elevated KLK4 mRNA values turned out as an additional, independent predictive marker for shortened OS (p = 0.006), whereas residual tumor mass, but not ascites fluid volume, remained an independent indicator for both OS and PFS (p < 0.001 and p = 0.002, respectively). The results of the present study, obtained in a well-defined, homogenous cohort of patients afflicted with advanced high-grade serous ovarian cancer, are in line with previous reports describing high KLK4 levels as an unfavorable marker in ovarian cancer patients.
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Activation and activity of glycosylated KLKs 3, 4 and 11
Biological Chemistry, 2018Co-Authors: Peter Briza, Hans Brandstetter, Viktor Magdolen, Peter GoettigAbstract:Human kallikrein-related peptidases 3, 4, 11, and KLK2, the activator of KLK3/PSA, belong to the prostatic group of the KLKs, whose major physiological function is semen liquefaction during the fertilization process. Notably, these KLKs are upregulated in prostate cancer and are used as clinical biomarkers or have been proposed as therapeutic targets. However, this potential awaits a detailed characterization of these proteases. In order to study glycosylated prostatic KLKs resembling the natural proteases, we used Leishmania (LEXSY) and HEK293 cells for secretory expression. Both systems allowed the subsequent purification of soluble pro-KLK zymogens with correct propeptides and of the mature forms. Periodic acid-Schiff reaction, enzymatic deglycosylation assays, and mass spectrometry confirmed the glycosylation of these KLKs. Activation of glycosylated pro-KLKs 4 and 11 turned out to be most efficient by glycosylated KLK2 and KLK4, respectively. By comparing the glycosylated prostatic KLKs with their non-glycosylated counterparts from Escherichia coli, it was observed that the N-glycans stabilize the KLK proteases and change their activation profiles and their enzymatic activity to some extent. The functional role of glycosylation in prostate-specific KLKs could pave the way to a deeper understanding of their biology and to medical applications.
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Pericellular regulation of prostate cancer expressed kallikrein-related peptidases and matrix metalloproteinases by cell surface serine proteases.
American journal of cancer research, 2017Co-Authors: Janet C. Reid, Viktor Magdolen, Judith A. Clements, Admire Matsika, Claire M. Davies, Amy Broomfield, Nigel C. Bennett, Bhuvana Srinivasan, John D HooperAbstract:We provide evidence of a pericellular network of proteases that are elevated and co-expressed in prostate cancer. The network involves the membrane bound serine proteases hepsin and TMPRSS2, the secreted kallikrein-related peptidases KLK4 and KLK14, and the secreted matrix metalloproteinases MMP-3 and MMP-9. Western blot analysis of cell lysates, conditioned cell culture media, immunoprecipitates and cell surface proteins, demonstrates a network of interactions centred largely at the plasma membrane, with the Arg/Lys specific proteases hepsin and TMPRSS2 key regulators of the network. Our data demonstrate that like TMPRSS2, hepsin is able to autoactivate. Active hepsin degrades KLK4, generating a cell associated degradation product with corresponding reduction in levels of cell-free KLK4. In contrast hepsin activates KLK14. TMPRSS2 appears to cleave amino terminal to the KLK4 activation site such that it is available for further processing to generate the active KLK4 protease. In contrast with hepsin, TMPRSS2 degrades KLK14. In addition to these direct mechanisms of regulation, hepsin and TMPRSS2 indirectly modulate KLK4 activity by cleaving the KLK4-activating protease MMP-3. Hepsin and TMPRSS2 also activate MMP-9, which similar to MMP-3, associates with the cell surface. Interestingly our data also show that proteolysis occurs between the membrane spanning and catalytic domains of hepsin and TMPRSS2. Hepsin cleavage occurs via an autoproteolytic mechanism, whereas TMPRSS2 cleavage is mediated by KLK14. Hepsin and TMPRSS2 are not shed from the cell surface but proteolysis likely disrupts domains that regulate the proteolytic activity of these proteases. Immunocytochemical analyses demonstrate that hepsin and TMPRSS2 colocalize on the cell surface with the secreted serine proteases KLK4 and KLK14, only in membrane protrusions, suggesting that reciprocal proteolytic interactions occur in defined cellular structures that are important during cancer dissemination for cell migration, invasion and survival. Also of note, immunohistochemical analysis of serial sections of prostate tumor demonstrated significant overlapping expression of the six proteases in vivo. Collectively these data suggest the possibility that the novel proteolytic network identified by us, will be most important during active dissemination of prostate cancers, and that its disruption could inhibit metastasis.
Iacovos P Michael - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome
2016Co-Authors: Laetitia Furio, Iacovos P Michael, Georgios Pampalakis, Andras NagyAbstract:Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desqua-mation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in SPINK5 encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aber-rantly increased proteolysis in the epidermis. Spink5-/- mice recapitulate the NS pheno-type, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which Klk5 and Spink5 were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in Spink5-/-mice. By repeated intercrossing between Klk5-/- mice with Spink5-/-mice, we generated Spink5-/-Klk5-/- animals. We showed that Klk5 knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epiderma
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klk5 inactivation reverses cutaneous hallmarks of netherton syndrome
PLOS Genetics, 2015Co-Authors: Laetitia Furio, Iacovos P Michael, Georgia Sotiropoulou, Georgios Pampalakis, Andras Nagy, Alain HovnanianAbstract:Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in SPINK5 encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. Spink5⁻/⁻ mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which Klk5 and Spink5 were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in Spink5⁻/⁻ mice. By repeated intercrossing between Klk5⁻/⁻ mice with Spink5⁻/⁻ mice, we generated Spink5⁻/⁻Klk5⁻/⁻ animals. We showed that Klk5 knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using in situ zymography and specific protease substrates, we showed that Klk5 knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in Spink5⁻/⁻Klk5⁻/⁻. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in Spink5⁻/⁻Klk5⁻/⁻ skin. Notably, Il-1β, Il17A and Tslp levels were normalized. Our results provide in vivo evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in Spink5⁻/⁻ skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.
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a potential role for multiple tissue kallikrein serine proteases in epidermal desquamation
Journal of Biological Chemistry, 2006Co-Authors: Carla Borgoňo, Nahoko Komatsu, Ravi M Kapadia, Gary L Clayman, Iacovos P Michael, Georgia Sotiropoulou, Arumugam Jayakumar, Eleftherios P DiamandisAbstract:Abstract Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1–6 (rLEKTI(1–6)), domains 6–8 and partial domain 9 (rLEKTI(6–9′)), domains 9–12 (rLEKTI(9–12)), and domains 12–15 (rLEKTI(12–15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1–6), rLEKTI(6–9′), and rLEKTI(9–12) with Ki values in the range of 2.3–28.4 nm, 6.1–221 nm, and 2.7–416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12–15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.
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intron retention a common splicing event within the human kallikrein gene family
Clinical Chemistry, 2005Co-Authors: Iacovos P Michael, George M Yousef, Lisa Kurlender, Nader Memari, Linda Grass, Carsten Stephan, Klaus JungAbstract:Background: All human kallikrein ( KLK ) genes have at least one splice variant, some of which possess clinical utility in cancer diagnostics/prognostics. Given that introns <100 bp in length are retained in 95% of human genes and that splice variants of KLK3 and KLK4 retain intron III, we hypothesized that other proteins in this family, with a small intron III, may also retain it. Methods: Variant-specific reverse transcription-PCRs (RT-PCRs) for KLK1 , KLK2 , KLK5 , and KLK15 were used to identify and clone the full coding sequence of intron III-containing splice variants. In addition, variant-specific RT-PCRs for the cloned KLK3 and KLK4 variants as well as for the “classical” forms of the six genes were used to determine their expression profiles in healthy tissues, their regulation by steroids, and their differential expression in prostate cancer. Results: KLK1 , KLK2 , KLK3 , KLK4 , KLK5 , and KLK15 showed a common type of splice variant in which intron III is retained. Expression profiling of these splice variants revealed expression profiles similar to those of the classical mRNA forms, although the pattern of hormonal regulation was different. The KLK15 splice variant was up-regulated in 8 of 12 cancerous prostate tissues. All encoded variant proteins were predicted to be truncated and catalytically inactive because of a lack of the serine residue of the catalytic triad. Conclusions: The first six centromeric members of the KLK gene family have splice variants that retain intron III. Some variants show tissue-specific expression. The KLK15 splice variant appears to be a candidate biomarker for prostate cancer.