The solution structure of the kallikrein-related peptidases inhibitor SPINK6.
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ABSTRACT:
SUBMITTER: Jung S
PROVIDER: S-EPMC5553623 | biostudies-literature | 2016 Feb
REPOSITORIES: biostudies-literature
Publications
The solution structure of the kallikrein-related peptidases inhibitor SPINK6.
Jung Sascha S Fischer Jan J Spudy Björn B Kerkow Tim T Sönnichsen Frank D FD Xue Li L Bonvin Alexandre M J J AM Goettig Peter P Magdolen Viktor V Meyer-Hoffert Ulf U Grötzinger Joachim J
Biochemical and biophysical research communications 20160130 1
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 under ...[more]
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Project description:Kallikrein-related peptidases (KLKs) play a central role in skin desquamation. They are tightly controlled by specific inhibitors, including the lymphoepithelial Kazal-type inhibitor (LEKTI) encoded by SPINK5 and LEKTI-2 encoded by SPINK9. Herein, we identify SPINK6 as a selective inhibitor of KLKs in the skin. Unlike LEKTI but similar to LEKTI-2, SPINK6 possesses only one typical Kazal domain. Its mRNA was detected to be expressed at low levels in several tissues and was induced during keratinocyte differentiation. Natural SPINK6 was purified from human plantar stratum corneum extracts. Immunohistochemical analyses revealed SPINK6 expression in the stratum granulosum of human skin at various anatomical localizations and in the skin appendages, including sebaceous glands and sweat glands. SPINK6 expression was decreased in lesions of atopic dermatitis. Using KLK5, KLK7, KLK8, KLK14, thrombin, trypsin, plasmin, matriptase, prostasin, mast cell chymase, cathepsin G, neutrophil elastase, and chymotrypsin, inhibition with recombinant SPINK6 was detected only for KLK5, KLK7, and KLK14, with apparent K(i) values of 1.33, 1070, and 0.5 nm, respectively. SPINK6 inhibited desquamation of human plantar callus in an ex vivo model. Our findings suggest that SPINK6 plays a role in modulating the activity of KLKs in human skin. A selective inhibition of KLKs by SPINK6 might have therapeutic potential when KLK activity is elevated.
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Project description:Kallikrein-related peptidases constitute a single family of 15 (chymo)trypsin-like proteases (KLK1-15) with pleiotropic physiological roles. Aberrant regulation of KLKs has been associated with diverse diseases such as hypertension, renal dysfunction, skin disorders, inflammation, neurodegeneration, and cancer. Recent studies suggested that coordinated activation and regulation of KLK activity are achieved via a complex network of interactions referred to as the "KLK activome." However, it remains to be validated whether these hypothetical KLK activation cascade pathways are operative in vivo. In addition, KLKs have emerged as versatile signaling molecules. In summary, KLKs represent attractive biomarkers for clinical applications and potential therapeutic targets for common human pathologies.
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Project description: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 Zn(2+) ions, active site-directed proteinaceous inhibitors, such as serpins and the Kazal-type inhibitors, or the huge, unspecific compartment forming α(2)-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.
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Project description:The 15 human kallikrein-related peptidases (KLKs) are clinically important biomarkers and therapeutic targets of interest in inflammation, cancer, and neurodegenerative disease. KLKs are secreted as inactive pro-forms (pro-KLKs) that are activated extracellularly by specific proteolytic release of their amino-terminal pro-peptide, and this is a key step in their functional regulation. Physiologically relevant KLK regulatory cascades of activation have been described in skin desquamation and semen liquefaction, and work by a large number of investigators has elucidated pairwise and autolytic activation relationships among the KLKs with the potential for more extensive activation cascades. More recent work has asked whether functional intersection of KLKs with other types of regulatory proteases exists. Such studies show a capacity for members of the thrombostasis axis to act as broad activators of pro-KLKs. In the present report, we ask whether such functional intersection is possible between the KLKs and the members of the matrix metalloproteinase (MMP) family by evaluating the ability of the MMPs to activate pro-KLKs. The results identify MMP-20 as a broad activator of pro-KLKs, suggesting the potential for intersection of the KLK and MMP axes under pathological dysregulation of MMP-20 expression.
| S-EPMC3709557 | biostudies-literature
Project description:Human kallikrein-related peptidases (KLKs) are a subgroup of serine proteases that participate in proteolytic pathways and control protein levels in normal physiology as well as in several pathological conditions. Their complex network of stimulatory and inhibitory interactions may induce inflammatory and immune responses and contribute to the neoplastic phenotype through the regulation of several cellular processes, such as proliferation, survival, migration, and invasion. This family of proteases, which includes one of the most useful cancer biomarkers, kallikrein-related peptidase 3 or PSA, also has a protective effect against cancer promoting apoptosis or counteracting angiogenesis and cell proliferation. Therefore, they represent attractive therapeutic targets and may have important applications in clinical oncology. Despite being intensively studied, many gaps in our knowledge on several molecular aspects of KLK functions still exist. This review aims to summarize recent data on their involvement in different processes related to health and disease, in particular those directly or indirectly linked to the neoplastic process.
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Project description:Several members of the human tissue kallikrein-related peptidase (KLK) family are emerging cancer biomarkers. The aim of this study was to analyse the expression of a panel of KLKs in colorectal cancer and to find out if the multiparametric combination of them can increase the accuracy of prediction of patients survival beyond the traditional clinical information. Nine KLKs (KLK5-8, KLK10, KLK11, KLK13-15) were measured using ELISA assays in cytosolic extracts of 122 colon cancer tissues and their nearby normal mucosa, obtained during surgery. The mean levels of almost all KLKs in tumour tissues were significantly different from their counterparts of normal tissue (P<0.0001). KLK 5, 6, 7, 13, 14 were significantly associated with overall survival in univariate analysis, but after adjusting for age, TNM and differentiation stage, only KLK5 (HR: 1.24 (95% CI: 1.05-1.47)), KLK7 (HR: 1.57 (95% CI: 1.04-2.37)) and KLK14 (HR: 1.43 (95% CI: 1.05-1.94)) remained significant. Addition of a panel of selected KLK markers to clinical parameters gave an increment in AUC of 0.86 beyond the clinical factors at year 1, showing that it can increase the accuracy of prediction of overall survival beyond the traditional clinical information, particularly the short-term (1 year) survival after surgery.
| S-EPMC2696752 | biostudies-literature
Project description:Background: Tissue kallikrein-related peptidases 4, 5, 6 and 7 (KLK4-7) strongly increase the malignancy of ovarian cancer cells. By further deciphering the downstream effectors of KLK4-7, we aimed at finding new potential prognostic biomarkers and treatment targets for ovarian cancer patients. KLK4-7-transfected and vector-control OVMZ-6 (OV-KLK4-7 and OV-VC) ovarian cancer cells were established to select differentially regulated factors. Methods: Three independent approaches, PCR arrays, genome-wide microarray and proteome analyses, were pursued leading to the identification of ten candidates (MSN, KRT19, COL5A2, COL1A2, BMP5, F10, KRT7, JUNB, BMP4, MMP1). Differential gene expression in OV-KLK4-7 versus OV-VC cells was validated by qPCR. To determine differential protein expression, Western blot analyses, immunofluorescence and immunohistochemistry were applied for four candidates (MSN, KRT19, KRT7, JUNB) in both cell lines and tumor xenografts. Results: These experiments demonstrated that KLK4-7 distinctly regulate expression of MSN, KRT19, KRT7 and JUNB on the mRNA and protein levels in ovarian cancer cells and tissues. Protein expression of the top upregulated effectors, MSN and KRT19, was investigated by immunohistochemistry in a cohort of patients (n=66) afflicted with high-grade serous ovarian cancer and related to the immuno-expression levels of KLK4-7. Significant positive associations were found for KRT19/KLK4 (P=0.010), KRT19/KLK5 (P=0.030) and MSN/KLK7 (P=0.001); KRT19 and MSN immuno-expression showed a trend towards significance with KLK6. Conclusions: These findings imply that KLK4-7 exert key modulatory effects on other cancer-related genes and proteins in ovarian cancer. The key downstream effectors of KLK4-7, MSN and KRT19, may represent important therapeutic targets associated with serous ovarian cancer.
2018-12-26 | GSE109261 | GEO
Project description: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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Project description:Background:Recent studies have demonstrated that the kallikrein and kallikrein-related peptidases (KLKs) exhibit aberrant expression in patients with colorectal cancer (CRC) and might be considered as potential prognostic biomarkers of CRC. However, inconsistent findings have been reported, which promote us to summarize the global prognostic roles of KLKs for survival in CRC patients. Methods:Eligible published studies were identified by searching electronic databases with several search strategies. The patients' baseline characteristics and survival results were extracted from enrolled studies and pooled as combined hazard ratio (HR) with 95% confidence interval (95% CI) to estimate the effect size. Results:A total of 25 and 22 eligible studies were included in the meta-analysis to evaluate the prognostic roles of KLKs on overall survival (OS) and disease-free survival (DFS), respectively. KLKs overexpression was significantly associated with worse OS (pooled HR?=?1.43, 95% CI 1.27-1.60, P?<?0.001) and short DFS (pooled HR?=?1.35, 95% CI 1.21-1.51, P?<?0.001). Importantly, subgroup and meta-regression analyses revealed the survival differences among different races and detection methods of KLKs. Furthermore, several specific members of KLKs were identified to be more significantly related to worse OS and DFS compared with other members. Conclusion:The present study demonstrated that KLKs may have the potential to serve as promising biomarkers to monitor CRC prognosis and progression. The promising results concerning the utility of KLKs in clinical practice encourage the further investigation of their clinical utility applicability as tumor markers of CRC.
| S-EPMC7310231 | biostudies-literature
Project description:BackgroundTissue kallikrein-related peptidases 4, 5, 6 and 7 (KLK4-7) strongly increase the malignancy of ovarian cancer cells. Deciphering their downstream effectors, we aimed at finding new potential prognostic biomarkers and treatment targets for ovarian cancer patients. KLK4-7-transfected (OV-KLK4-7) and vector-control OV-MZ-6 (OV-VC) ovarian cancer cells were established to select differentially regulated factors.MethodsWith three independent approaches, PCR arrays, genome-wide microarray and proteome analyses, we identified 10 candidates (MSN, KRT19, COL5A2, COL1A2, BMP5, F10, KRT7, JUNB, BMP4, MMP1). To determine differential protein expression, we performed western blot analyses, immunofluorescence and immunohistochemistry for four candidates (MSN, KRT19, KRT7, JUNB) in cells, tumour xenograft and patient-derived tissues.ResultsWe demonstrated that KLK4-7 clearly regulates expression of MSN, KRT19, KRT7 and JUNB at the mRNA and protein levels in ovarian cancer cells and tissues. Protein expression of the top-upregulated effectors, MSN and KRT19, was investigated by immunohistochemistry in patients afflicted with serous ovarian cancer and related to KLK4-7 immunoexpression. Significant positive associations were found for KRT19/KLK4, KRT19/KLK5 and MSN/KLK7.ConclusionThese findings imply that KLK4-7 exert key modulatory effects on other cancer-related genes and proteins in ovarian cancer. These downstream effectors of KLK4-7, MSN and KRT19 may represent important therapeutic targets in serous ovarian cancer.
| S-EPMC6189062 | biostudies-literature