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Publications (10 of 37) Show all publications
Chen, X., Riley, B. T., de Veer, S. J., Hoke, D. E., Van Haeften, J., Leahy, D., . . . Harris, J. M. (2019). Potent, multi-target serine protease inhibition achieved by a simplified beta-sheet motif. PLoS ONE, 14(1), Article ID e0210842.
Open this publication in new window or tab >>Potent, multi-target serine protease inhibition achieved by a simplified beta-sheet motif
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2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 1, article id e0210842Article in journal (Refereed) Published
Abstract [en]

Engagement of an extended beta-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like beta-sheet to enzyme inhibition. Here we report the crystal structure of an simplified beta-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by beta-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design.

Place, publisher, year, edition, pages
Public Library Science, 2019
National Category
Medicinal Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-156314 (URN)10.1371/journal.pone.0210842 (DOI)000456306400019 ()30668585 (PubMedID)
Available from: 2019-02-20 Created: 2019-02-20 Last updated: 2019-02-20Bibliographically approved
Haddada, M., Draoui, H., Deschamps, L., Walker, F., Delaunay, T., Brattsand, M., . . . Darmoul, D. (2018). Kallikrein-related peptidase 7 overexpression in melanoma cells modulates cell adhesion leading to a malignant phenotype. Biological chemistry (Print), 399(9), 1099-1105
Open this publication in new window or tab >>Kallikrein-related peptidase 7 overexpression in melanoma cells modulates cell adhesion leading to a malignant phenotype
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2018 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 399, no 9, p. 1099-1105Article in journal (Refereed) Published
Abstract [en]

We recently reported that human melanoma cells, but not benign melanocytes, aberrantly express kallikrein-related peptidase 7 (KLK7). Here, we show a KLK7 overexpression-mediated decrease of cell adhesion to extracellular matrix binding proteins, associated with downregulation of alpha 5/beta 1/alpha v/beta 3 integrin expression. We also report an up-regulation of MCAM/CD146 and an increase in spheroid formation of these cells. Our results demonstrate that aberrant KLK7 expression leads to a switch to a more malignant phenotype suggesting a potential role of KLK7 in melanoma invasion. Thus, KLK7 may represent a biomarker for melanoma progression and may be a potential therapeutic target for melanoma.

Place, publisher, year, edition, pages
Walter de Gruyter, 2018
Keywords
E-cadherin, integrins, kallikrein-related peptidase 7, MCAM/CD146, melanoma, spheroid
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-151387 (URN)10.1515/hsz-2017-0339 (DOI)000441526200018 ()29498930 (PubMedID)
Available from: 2018-09-06 Created: 2018-09-06 Last updated: 2018-09-06Bibliographically approved
Brattsand, M. (2018). Kan KLK7 vara en användbar biomarkör för progression av melanom?. BestPractice Dermatologi, 9(25)
Open this publication in new window or tab >>Kan KLK7 vara en användbar biomarkör för progression av melanom?
2018 (Swedish)In: BestPractice Dermatologi, Vol. 9, no 25Article, review/survey (Refereed) Published
Place, publisher, year, edition, pages
Ballerup, Danmark: BestPractice ApS, 2018
National Category
Dermatology and Venereal Diseases
Research subject
Dermatology and Venerology
Identifiers
urn:nbn:se:umu:diva-154115 (URN)
Available from: 2018-12-12 Created: 2018-12-12 Last updated: 2019-01-16Bibliographically approved
Delaunay, T., Deschamps, L., Haddada, M., Walker, F., Soosaipillai, A., Soualmia, F., . . . Darmoul, D. (2017). Aberrant expression of kallikrein-related peptidase 7 is correlated with human melanoma aggressiveness by stimulating cell migration and invasion. Molecular Oncology, 11(10), 1330-1347
Open this publication in new window or tab >>Aberrant expression of kallikrein-related peptidase 7 is correlated with human melanoma aggressiveness by stimulating cell migration and invasion
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2017 (English)In: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 11, no 10, p. 1330-1347Article in journal (Refereed) Published
Abstract [en]

Members of the tissue kallikrein-related peptidase (KLK) family not only regulate several important physiological functions, but aberrant expression has also been associated with various malignancies. Clinically, KLKs have been suggested as promising biomarkers for diagnosis and prognosis in many types of cancer. As of yet, expression of KLKs and their role in skin cancers are, however, poorly addressed. Malignant melanoma is an aggressive disease associated with poor prognosis. Hence, diagnostic biomarkers to monitor melanoma progression are needed. Herein, we demonstrate that although mRNA of several KLKs are aberrantly expressed in melanoma cell lines, only the KLK7 protein is highly secreted in vitro. In line with these findings, ectopic expression of KLK7 in human melanomas and its absence in benign nevi were demonstrated by immunohistochemistry in vivo. Interestingly, overexpression of KLK7 induced a significant reduction in melanoma cell proliferation and colony formation. Moreover, KLK7 overexpression triggered an increase in cell motility and invasion associated with decreased expression of E-cadherin and an upregulation of MCAM/CD146. Our results demonstrate, for the first time, that aberrant KLK7 expression leads to a switch from proliferative to invasive phenotype, suggesting a potential role of KLK7 in melanoma progression. Thus, we hypothesize that KLK7 may represent a potential biomarker for melanoma progression.

Keywords
E-cadherin, invasion, kallikrein-related peptidase 7, melanoma, migration, proliferation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-142381 (URN)10.1002/1878-0261.12103 (DOI)000417440300002 ()28636767 (PubMedID)
Available from: 2017-11-29 Created: 2017-11-29 Last updated: 2018-06-09Bibliographically approved
Djusberg, E., Jernberg, E., Thysell, E., Golovleva, I., Lundberg, P., Crnalic, S., . . . Wikström, P. (2017). High Levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases. The Prostate, 77(6), 625-638
Open this publication in new window or tab >>High Levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases
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2017 (English)In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 77, no 6, p. 625-638Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. METHODS: Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. RESULTS: AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. CONCLUSIONS: AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 2017
Keywords
androgen receptor variant 7, AKR1C3, extracellular vesicles, exosomes, blood coagulation
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-131817 (URN)10.1002/pros.23307 (DOI)000397496300007 ()28144969 (PubMedID)
Available from: 2017-02-22 Created: 2017-02-22 Last updated: 2018-06-09Bibliographically approved
Kasparek, P., Ileninova, Z., Zbodakova, O., Kanchev, I., Benada, O., Chalupsky, K., . . . Sedlacek, R. (2017). KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype. PLoS Genetics, 13(1), Article ID e1006566.
Open this publication in new window or tab >>KLK5 and KLK7 Ablation Fully Rescues Lethality of Netherton Syndrome-Like Phenotype
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2017 (English)In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 13, no 1, article id e1006566Article in journal (Refereed) Published
Abstract [en]

Netherton syndrome (NS) is a severe skin disease caused by the loss of protease inhibitor LEKTI, which leads to the dysregulation of epidermal proteases and severe skin-barrier defects. KLK5 was proposed as a major protease in NS pathology, however its inactivation is not sufficient to rescue the lethal phenotype of LEKTI-deficient mice. In this study, we further elucidated the in vivo roles of the epidermal proteases in NS using a set of mouse models individually or simultaneously deficient for KLK5 and KLK7 on the genetic background of a novel NS-mouse model. We show that although the ablation of KLK5 or KLK7 is not sufficient to rescue the lethal effect of LEKTI-deficiency simultaneous deficiency of both KLKs completely rescues the epidermal barrier and the postnatal lethality allowing mice to reach adulthood with fully functional skin and normal hair growth. We report that not only KLK5 but also KLK7 plays an important role in the inflammation and defective differentiation in NS and KLK7 activity is not solely dependent on activation by KLK5. Altogether, these findings show that unregulated activities of KLK5 and KLK7 are responsible for NS development and both proteases should become targets for NS therapy.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-132846 (URN)10.1371/journal.pgen.1006566 (DOI)000394147700035 ()
Available from: 2017-04-04 Created: 2017-04-04 Last updated: 2018-06-09Bibliographically approved
Brattsand, M. (2017). Missing factors in human skin equivalent models?. British Journal of Dermatology, 176(1), 11-12
Open this publication in new window or tab >>Missing factors in human skin equivalent models?
2017 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 176, no 1, p. 11-12Article in journal, Editorial material (Refereed) Published
National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:umu:diva-131646 (URN)10.1111/bjd.15108 (DOI)000392474100021 ()28098372 (PubMedID)
Available from: 2017-02-27 Created: 2017-02-27 Last updated: 2018-06-09Bibliographically approved
de Veer, S. J., Furio, L., Swedberg, J. E., Munro, C. A., Brattsand, M., Clements, J. A., . . . Harris, J. M. (2017). Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum. Journal of Investigative Dermatology, 137(2), 430-439
Open this publication in new window or tab >>Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum
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2017 (English)In: Journal of Investigative Dermatology, ISSN 0022-202X, E-ISSN 1523-1747, Vol. 137, no 2, p. 430-439Article in journal (Refereed) Published
Abstract [en]

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 (K-i = 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.

National Category
Dermatology and Venereal Diseases
Identifiers
urn:nbn:se:umu:diva-131631 (URN)10.1016/j.jid.2016.09.017 (DOI)000392469700024 ()27697464 (PubMedID)
Available from: 2017-03-03 Created: 2017-03-03 Last updated: 2018-06-09Bibliographically approved
de Veer, S. J., Swedberg, J. E., Brattsand, M., Clements, J. A. & Harris, J. M. (2016). Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors. Paper presented at 6th International Symposium on Kallikreins and Kallikrein-Related Peptidases (ISK), SEP 28-OCT 01, 2015, Brisbane, AUSTRALIA. Biological chemistry (Print), 397(12), 1237-1249
Open this publication in new window or tab >>Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors
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2016 (English)In: Biological chemistry (Print), ISSN 1431-6730, E-ISSN 1437-4315, Vol. 397, no 12, p. 1237-1249Article in journal (Refereed) Published
Abstract [en]

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (k(cat) and K-M) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (K-i = 4.2 +/- 0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

Keywords
inhibitor engineering, peptide, serine protease, sparse matrix library, sunflower trypsin inhibitor-1
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-129684 (URN)10.1515/hsz-2016-0112 (DOI)000387888700005 ()26894578 (PubMedID)
Conference
6th International Symposium on Kallikreins and Kallikrein-Related Peptidases (ISK), SEP 28-OCT 01, 2015, Brisbane, AUSTRALIA
Available from: 2017-01-13 Created: 2017-01-09 Last updated: 2018-06-09Bibliographically approved
de Veer, S. J., Swedberg, J. E., Akcan, M., Rosengren, K. J., Brattsand, M., Craik, D. J. & Harris, J. M. (2015). Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition. Biochemical Journal, 469(2), 243-253
Open this publication in new window or tab >>Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition
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2015 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 469, no 2, p. 243-253Article in journal (Refereed) Published
Abstract [en]

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.

Keywords
cyclic peptides, drug design, kallikrein-related peptidase, Laskowski mechanism, protease inhibitors
National Category
Medical Bioscience
Identifiers
urn:nbn:se:umu:diva-109817 (URN)10.1042/BJ20150412 (DOI)000361051800009 ()25981970 (PubMedID)
Available from: 2015-10-07 Created: 2015-10-06 Last updated: 2018-06-07Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0001-7175-1336

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