ORCID Profile
0000-0002-5535-5760
Current Organisation
Jagiellonian University
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Publisher: Walter de Gruyter GmbH
Date: 2010
DOI: 10.1515/BC.2010.009
Abstract: Proteases of Tannerella forsythia , a pathogen associated with periodontal disease, are implicated as virulence factors. Here, we characterized a matrix metalloprotease (MMP)-like enzyme of T. forsythia referred to as karilysin. Full-length (without a signal peptide) recombinant karilysin (49.9 kDa) processed itself into the mature 18-kDa enzyme through sequential autoproteolytic cleavage at both N- and C-terminal profragments. The first cleavage at the Asn14-Tyr15 peptide bond generated the fully active enzyme (47.9 kDa) and subsequent truncations at the C-terminus did not affect proteolytic activity. Mutation of Tyr15 to Ala generated a prokarilysin variant that processed itself into the final 18-kDa form with greatly reduced kinetics. Inactive prokarilysin with the mutated catalytic Glu residue (E136A) was processed by active karilysin at the same sites as the active enzymes. Karilysin proteolytic activity and autoprocessing were inhibited by 1,10-phenanthroline and EDTA. Calcium ions were found to be important for both the activity and thermal stability of karilysin. Using CLiPS technology, the specificity of karilysin was found to be similar to that of MMPs with preference for Leu/Tyr/Met at P1′ and Pro/Ala at P3. This specificity and the ability to degrade elastin, fibrinogen and fibronectin may contribute to the pathogenicity of periodontitis.
Publisher: Wiley
Date: 10-10-2017
DOI: 10.1002/LARY.26949
Abstract: Staphylococcus aureus (S. aureus) infection is known to contribute to the severity and recalcitrance of chronic rhinosinusitis (CRS), and its secreted products have been shown to alter the airway barrier. Extracellular proteases secreted by S. aureus are thought to be important in epithelial infection and immune evasion however, their effect on airway mucosal barrier function is not known. To investigate the impact of extracellular proteases on airway epithelial integrity, the purified S. aureus proteases V8 protease, Staphopain A, Staphopain B, Exfoliative toxin A, and serine protease-like A-F were applied to human nasal epithelial cell air-liquid interface (HNEC-ALI) cultures. Transepithelial electrical resistance (TEER), permeability (Papp) measurements, and immuno-localization of the tight junction proteins claudin-1 and ZO-1 were used to assess barrier integrity. Effects of the proteases on inflammation and cell viability were measured using interleukin-6 (IL-6) ELISA and a lactate dehydrogenase assay. Application of V8 protease to HNEC-ALI cultures caused a significant concentration and time-dependent decrease in TEER (22.67%, P < 0.0001), a reciprocal Papp increase (20.14-fold, P < 0.05), and a discontinuous ZO-1 immuno-localization compared to control. IL-6 production was significantly reduced in V8 protease-treated cells (153.5 pg/mL, P = 0.0069) compared to control (548.3 pg/mL), whereas no difference in cell viability was observed. S. aureus V8 protease causes dysfunction of mucosal barrier structure and function indicative of a leaky barrier. A reduction in IL-6 levels suggests that the mucosal immunity is impaired by this protease and thus has the potential to contribute to CRS recalcitrance. NA. Laryngoscope, 128:E8-E15, 2018.
Publisher: Springer Science and Business Media LLC
Date: 23-03-2016
DOI: 10.1038/SREP23123
Abstract: In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis , the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis , alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.
Publisher: MDPI AG
Date: 19-06-2020
DOI: 10.3390/IJMS21124383
Abstract: Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.
No related grants have been discovered for Grzegorz Dubin.