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1.
Int J Mol Sci ; 21(12)2020 Jun 19.
Article in English | MEDLINE | ID: mdl-32575583

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.


Subject(s)
Enzyme Precursors/metabolism , Kallikreins/genetics , Kallikreins/metabolism , Matrix Metalloproteinase 14/metabolism , Animals , Cell Line , Cell Membrane/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Hydrolysis , Kallikreins/chemistry , Matrix Metalloproteinase 14/genetics , Mice , Porphyromonas gingivalis , Protein Engineering , Recombinant Proteins/metabolism
2.
Int J Mol Sci ; 20(7)2019 Mar 28.
Article in English | MEDLINE | ID: mdl-30925705

ABSTRACT

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.


Subject(s)
Enzyme Assays/methods , Kallikreins/metabolism , Peptides/metabolism , Amino Acid Sequence , Cell Line , Humans , Kinetics , Neoplasms/enzymology , Peptide Library , Peptides/chemistry , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Substrate Specificity
3.
J Biol Chem ; 291(36): 18753-64, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27354280

ABSTRACT

Periodontitis, a chronic inflammation driven by dysbiotic subgingival bacterial flora, is linked on clinical levels to the development of a number of systemic diseases and to the development of oral and gastric tract tumors. A key pathogen, Porphyromonas gingivalis, secretes gingipains, cysteine proteases implicated as the main factors in the development of periodontitis. Here we hypothesize that gingipains may be linked to systemic pathologies through the deregulation of kallikrein-like proteinase (KLK) family members. KLKs are implicated in cancer development and are clinically utilized as tumor progression markers. In tissues, KLK activity is strictly controlled by a limited number of tissue-specific inhibitors, including SPINK6, an inhibitor of these proteases in skin and oral epithelium. Here we identify gingipains as the only P. gingivalis proteases responsible for SPINK6 degradation. We further show that gingipains, even at low nanomolar concentrations, cleaved SPINK6 in concentration- and time-dependent manner. The proteolysis was accompanied by loss of inhibition against KLK13. We also mapped the cleavage by Arg-specific gingipains to the reactive site loop of the SPINK6 inhibitor. Moreover, we identified a significant fraction of SPINK6-sensitive proteases in healthy saliva and confirmed the ability of gingipains to inactivate SPINK6 under ex vivo conditions. Finally, we demonstrate the double-edge action of gingipains, which, in addition, can activate KLKs because of gingipain K-mediated proteolytic processing of the zymogenic proform of KLK13. Altogether, the results indicate the potential of P. gingivalis to disrupt the control system of KLKs, providing a possible mechanistic link between periodontal disease and tumor development.


Subject(s)
Adhesins, Bacterial/metabolism , Cysteine Endopeptidases/metabolism , Porphyromonas gingivalis/enzymology , Proteinase Inhibitory Proteins, Secretory/metabolism , Saliva/metabolism , Salivary Proteins and Peptides/metabolism , Adhesins, Bacterial/chemistry , Cysteine Endopeptidases/chemistry , Gingipain Cysteine Endopeptidases , Humans , Kallikreins/antagonists & inhibitors , Kallikreins/chemistry , Kallikreins/metabolism , Protein Stability , Proteinase Inhibitory Proteins, Secretory/chemistry , Saliva/chemistry , Salivary Proteins and Peptides/antagonists & inhibitors , Salivary Proteins and Peptides/chemistry , Serine Peptidase Inhibitors, Kazal Type
4.
J Innate Immun ; 8(2): 185-98, 2016.
Article in English | MEDLINE | ID: mdl-26613585

ABSTRACT

Aspiration pneumonia is a life-threatening infectious disease often caused by oral anaerobic and periodontal pathogens such as Porphyromonas gingivalis. This organism produces proteolytic enzymes, known as gingipains, which manipulate innate immune responses and promote chronic inflammation. Here, we challenged mice with P. gingivalis W83 and examined the role of gingipains in bronchopneumonia, lung abscess formation, and inflammatory responses. Although gingipains were not required for P. gingivalis colonization and survival in the lungs, they were essential for manifestation of clinical symptoms and infection-related mortality. Pathologies caused by wild-type (WT) P. gingivalis W83, including hemorrhage, necrosis, and neutrophil infiltration, were absent from lungs infected with gingipain-null isogenic strains or WT bacteria preincubated with gingipain-specific inhibitors. Damage to lung tissue correlated with systemic inflammatory responses, as manifested by elevated levels of TNF, IL-6, IL-17, and C-reactive protein. These effects were unequivocally dependent on gingipain activity. Gingipain activity was also implicated in the observed increase in IL-17 in lung tissues. Furthermore, gingipains increased platelet counts in the blood and activated platelets in the lungs. Arginine-specific gingipains made a greater contribution to P. gingivalis-related morbidity and mortality than lysine-specific gingipains. Thus, inhibition of gingipain may be a useful adjunct treatment for P. gingivalis-mediated aspiration pneumonia.


Subject(s)
Adhesins, Bacterial/immunology , Bacteroidaceae Infections/immunology , Cysteine Endopeptidases/immunology , Pneumonia, Aspiration/immunology , Porphyromonas gingivalis/immunology , Adhesins, Bacterial/genetics , Animals , Bacteroidaceae Infections/genetics , Bacteroidaceae Infections/microbiology , Bacteroidaceae Infections/pathology , Blood Platelets/immunology , Cysteine Endopeptidases/genetics , Cytokines/immunology , Female , Gingipain Cysteine Endopeptidases , Hemorrhage/genetics , Hemorrhage/immunology , Hemorrhage/microbiology , Hemorrhage/pathology , Mice , Mice, Inbred BALB C , Necrosis , Neutrophil Infiltration/immunology , Neutrophils/immunology , Neutrophils/pathology , Platelet Activation/immunology , Platelet Count , Pneumonia, Aspiration/genetics , Pneumonia, Aspiration/pathology , Porphyromonas gingivalis/genetics
5.
J Innate Immun ; 5(3): 251-60, 2013.
Article in English | MEDLINE | ID: mdl-23235402

ABSTRACT

The pulmonary surfactant is a complex mixture of lipids and proteins that is important for respiratory lung functions, which also provides the first line of innate immune defense. Pulmonary surfactant protein-A (SP-A) is a major surfactant component with immune functions with importance during Staphylococcus aureus infections that has been demonstrated in numerous studies. The current study showed that S. aureus can efficiently cleave the SP-A protein using its arsenal of proteolytic enzymes. This degradation appears to be mediated by cysteine proteases, in particular staphopain A (ScpA). The staphopain-mediated proteolysis of SP-A resulted in a decrease or complete abolishment of SP-A biological activity, including the promotion of S. aureus phagocytosis by neutrophils, aggregation of Gram-negative bacteria and bacterial cell adherence to epithelium. Significantly, ScpA has also efficiently degraded SP-A in complete bronchi-alveolar lavage fluid from human lungs. This indicates that staphopain activity in the lungs is resistant to protease inhibitors, thus suggesting that SP-A can be cleaved in vivo. Collectively, this study showed that the S. aureus protease ScpA is an important virulence factor that may impair innate immunity of the lungs.


Subject(s)
Cysteine Endopeptidases/immunology , Immunity, Innate , Lung/immunology , Neutrophils/immunology , Pneumonia, Staphylococcal/immunology , Pulmonary Surfactant-Associated Protein A/immunology , Staphylococcus aureus/immunology , Cysteine Endopeptidases/metabolism , Humans , Lung/metabolism , Lung/microbiology , Neutrophils/metabolism , Phagocytosis/immunology , Pneumonia, Staphylococcal/enzymology , Pulmonary Surfactant-Associated Protein A/metabolism , Staphylococcus aureus/enzymology , Staphylococcus aureus/pathogenicity
6.
Biol Chem ; 392(5): 483-9, 2011 May.
Article in English | MEDLINE | ID: mdl-21476872

ABSTRACT

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.


Subject(s)
Antigens, Neoplasm/pharmacology , Cysteine Endopeptidases/metabolism , Serpins/pharmacology , Antigens, Neoplasm/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/drug effects , Humans , Protein Binding , Serpins/metabolism
7.
Biol Chem ; 391(5): 581-7, 2010 May.
Article in English | MEDLINE | ID: mdl-20302517

ABSTRACT

Human kallikrein-related peptidases (KLKs) are 15 homologous serine proteases involved in several (patho)physiological processes, including cancer. Secreted as precursors, they are activated upon proteolytic release of a short pro-peptide. We searched for interconnection of KLKs within extracellular proteolytic networks leading to activation of protease zymogens and found that (i) pro-KLK activation by other KLKs is scarce, with the exception of pro-KLK11, which is efficiently activated by KLK4 and 5; (ii) pro-KLK4 is activated by matrix metalloproteinase 3; and (iii) trypsin-like KLKs efficiently activate the serine protease urokinase. Our observations provide new insights into the regulation of these important tumor-associated proteases.


Subject(s)
Enzyme Precursors/metabolism , Kallikreins/metabolism , Enzyme Activation , Gene Expression Regulation, Enzymologic , Humans , Kallikreins/genetics , Matrix Metalloproteinase 3/metabolism , Neoplasms/enzymology , Serine Endopeptidases/metabolism , Urokinase-Type Plasminogen Activator
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