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Laboratory of Proteolysis and Post-translational Modification of Proteins

Group leader
Tomasz Kantyka, PhD
email: tomasz.kantyka@uj.edu.pl

Research
The scientific interests in our research group are focused on posttranslational modifications (PTMs) of proteins and peptides, which coincide with infection, inflammation and development of civilization-related diseases. The underlying hypothesis is that PTMs impact the structure and activity of proteins and bioactive peptides. Our aim is to correlate PTMs with observed activity and structural changes in the pathophysiological context in the effort to delineate mechanisms of diseases development and to identify novel potential therapeutic targets. Although our focus is on basic research, we always keep our sight on the pathophysiological importance of our findings and their applicability for translational developments. 

Our efforts are divided among three closely cooperating, interwoven groups dedicating their research to:
 
Human proteolytic enzymes in cell function regulation
This study is concentrated on the analysis of human proteases – MMPs and kallikreins involvement in cell and tissue function, development and progress of inflammation and their role in bacterial infections. We are focused on the epithelial tissue, which forms the first and arguably the most important barrier of defense against potentially deleterious external factors. Our research includes involvement of proteases in posttranslational processing of proteins during infection, wound healing, chronic inflammation and obesity. Tomasz Kantyka, PhD, the Malopolska Centre of Biotechnology UJ full employee, is the group leader. 
 
Microbial proteases, their inhibitors and bacterial virulence factors
The main aim of this group is the identification and analysis of bacterial virulence factors which play a role in the development of the gum diseases. This includes the analysis of a novel protein secretion system in bacteria, investigation of bacterial enzymes, their inhibitors and newly identified virulence factors in the context of their involvement in the initiation and progress of the systemic diseases, such as rheumatoid arthritis, COPD, atherosclerosis, aspiration pneumonia or Sjogren’s syndrome. The research is broad and involves genetic analysis, activity of the isolated virulence factors and animal model evaluations. The group leader is Jan Potempa, PhD, who is involved in the parallel research at Faculty of Biochemistry, Biophysics and Biotechnology UJ, Malopolska Centre of Biotechnology UJ and University of Louisville, Ky, USA.
 
Posttranslational modifications in the inflammatory diseases
This team’s interests are focused on the analysis of the biological function of peptides and proteins modified as a result of the inflammatory reaction associated with RA, periodontitis, aspiration pneumonia or Sjogren’s syndrome. Research aims include analysis on how citrullination and carbamylation impact the immune system components, including antibacterial peptides, proteolytic cascades, antibodies and ECM proteins, in context of the pathological conditions. In addition to the basic research, the group is interested in identification and characterization of novel biomarkers associated with early subclinical stages of diseases development. The group is led by Piotr Mydel, PhD, who supervises the investigations at MCB UJ and University of Bergen in Norway.

Research techniques and equipment

As a whole, the research group has a broad experience in expression and purification of recombinant and native human and bacterial proteins, their biochemical characterization, PTM analysis, including effects on biological functions of peptides and proteins. The core of the laboratory machine park encompasses several FPLC and HPLC chromatography systems, Seismos NT.X Instrument from NanoTemper Technologies, which offers the Surface Acoustic Wave (SAW) method to measure binding kinetics and conformational changes, and microscale thermophoresis (MST) analyzers, including Monolith NT.115 and Monolith NT.FreeLabel (both from NanoTemper Technologies). Our research embraces many collaborators, local (WBBiB UJ, Collegium Medicum UJ, Institute of Pharmacology, PAN), national (Wroclaw Polytechnic, Gdansk University) and international (Bremen, Barcelona, Aarhus, Brno and many others), both academic and translational (Cortexyme, Inc., USA and Fraunhofer Institute, Germany) creating a truly interdisciplinary and international group very well linked, with high potentials to carry out ambitious research projects.

Research grants: 
PhD Jan Potempa 
SYMFONIA 1: " Extracellular proteolytic activity of human epithelium – role in the modulation of signal transduction. "
OPUS 11 : " Structure and function characterization of newly discovered unique inhibitors of proteases " 

PhD Tomasz Kantyka 
SONATA BIS: " Processing and posttranslational modifications of ghrelin - an appetite modulating peptide as the mechanistic link between prolonged inflammation, chronic bacterial infection and obesity development" 

PhD Piotr Mydel
SONATA BIS: " Carbamylation as a Modulator of Immune Response"
OPUS 12: " Serum PAD-activity: Risk Factor in Development and Novel Biomarker in Rheumatoid Arthritis "

PhD Mirosław Książek 
OPUS 9 : " Insight into biochemistry, structure, evolution and biology of bacterial serpins on example of miropin from human pathogen Tannerella forsythia.”

MSc Mariusz Madej
PRELUDIUM 10: „The role of PorX and PorY proteins in regulating the expression of proteins responsible for secreting major virulence factors of Porphyromonas gingivalis”

MSc Katherine Falkowski
PRELUDIUM 12: „Investigating the role of human tissue kallikreins in regulating matrix metalloproteinase activity.”

PhD Dominika Staniec
PRELUDIUM 13: „The structural and functional characterization of Tpr, calpain-like peptidase and important virulence factor of Porphyromonas gingivalis”

Total number of publications from 2013-2018: 424

Publications from 2014-2018

1.        Bochtler, M. et al. The Bacteroidetes Q-Rule: Pyroglutamate in Signal Peptidase I Substrates. Front. Microbiol. 9, 230 (2018).

2.        Chaiyarit, P. et al. Proteolytic effects of gingipains on trefoil factor family peptides. Clin. Oral Investig. 22, 1009–1018 (2018).

3.        Wong, A. et al. A Novel Biological Role for Peptidyl-Arginine Deiminases: Citrullination of Cathelicidin LL-37 Controls the Immunostimulatory Potential of Cell-Free DNA. J. Immunol. 200, 2327–2340 (2018).

4.        Stach, N. et al. Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureus. Structure 26, 572–579.e4 (2018).

5.        Karkowska-Kuleta, J. et al. The activity of bacterial peptidylarginine deiminase is important during formation of dual-species biofilm by periodontal pathogen Porphyromonas gingivalis and opportunistic fungus Candida albicans. Pathog. Dis. (2018). doi:10.1093/femspd/fty033

6.        Grabiec, A. M. et al. Epigenetic regulation in bacterial infections: targeting histone deacetylases. Crit. Rev. Microbiol. 44, 336–350 (2018).

7.        Eckert, M. et al. In vivo expression of proteases and protease inhibitor, a serpin, by periodontal pathogens at teeth and implants. Mol. Oral Microbiol. 33, 240–248 (2018).

8.        Binder, V. et al. Impact of fibrinogen carbamylation on fibrin clot formation and stability. Thromb. Haemost. 117, 899–910 (2017).

9.        Jentsch, H. F. R. et al. Salivary, gingival crevicular fluid and serum levels of ghrelin and chemerin in patients with periodontitis and overweight. J. Periodontal Res. 52, 1050–1057 (2017).

10.      Koneru, L. et al. Mirolysin, a LysargiNase from Tannerella forsythia, proteolytically inactivates the human cathelicidin, LL-37. Biol. Chem. 398, (2017).

11.      Naylor, K. L. et al. Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells. Microbiologyopen 6, e00401 (2017).

12.      Kariu, T. et al. Inhibition of gingipains and Porphyromonas gingivalis growth and biofilm formation by prenyl flavonoids. J. Periodontal Res. 52, 89–96 (2017).

13.      Godlewska, U. et al. Antimicrobial and Attractant Roles for Chemerin in the Oral Cavity during Inflammatory Gum Disease. Front. Immunol. 8, 353 (2017).

14.      Wilensky, A. et al. Vaccination with recombinant RgpA peptide protects against Porphyromonas gingivalis -induced bone loss. J. Periodontal Res. 52, 285–291 (2017).

15.      Pomowski, A. et al. Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis , a causative agent of gum disease from the human oral microbiome. J. Biol. Chem. 292, 5724–5735 (2017).

16.      Glowczyk, I. et al. Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner. Front. Cell. Infect. Microbiol. 7, 140 (2017).

17.      Sochalska, M. et al. Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis. Front. Cell. Infect. Microbiol. 7, 197 (2017).

18.      Lasica, A. M. et al. The Type IX Secretion System (T9SS): Highlights and Recent Insights into Its Structure and Function. Front. Cell. Infect. Microbiol. 7, 215 (2017).

19.      Goulas, T. et al. A structure-derived snap-trap mechanism of a multispecific serpin from the dysbiotic human oral microbiome. J. Biol. Chem. 292, 10883–10898 (2017).

20.      Steiger, S. et al. Immunomodulatory Molecule IRAK-M Balances Macrophage Polarization and Determines Macrophage Responses during Renal Fibrosis. J. Immunol. 199, 1440–1452 (2017).

21.      Sonesson, A. et al. Identification of bacterial biofilm and the Staphylococcus aureus derived protease, staphopain, on the skin surface of patients with atopic dermatitis. Sci. Rep. 7, 8689 (2017).

22.      Miller, D. P. et al. Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments. Front. Cell. Infect. Microbiol. 7, 378 (2017).

23.      Gawron, K. et al. Analysis of mutations in the SOS-1 gene in two Polish families with hereditary gingival fibromatosis. Oral Dis. 23, 983–989 (2017).

24.      Potempa, J. et al. The case for periodontitis in the pathogenesis of rheumatoid arthritis. Nat. Rev. Rheumatol. 13, 606–620 (2017).

25.      Wichert, R. et al. Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB. Cell Rep. 21, 2090–2103 (2017).

26.      Schwenzer, A. et al. Association of Distinct Fine Specificities of Anti−Citrullinated Peptide Antibodies With Elevated Immune Responses to Prevotella intermedia in a Subgroup of Patients With Rheumatoid Arthritis and Periodontitis. Arthritis Rheumatol. 69, 2303–2313 (2017).

27.      Eriksson, K. et al. Effects by periodontitis on pristane-induced arthritis in rats. J. Transl. Med. 14, 311 (2016).

28.      Stanford, S. M. et al. Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice. Arthritis Rheumatol. 68, 359–369 (2016).

29.      Nepal, C. et al. Transcriptional, post-transcriptional and chromatin-associated regulation of pri-miRNAs, pre-miRNAs and moRNAs. Nucleic Acids Res. 44, 3070–3081 (2016).

30.      Delaleu, N. et al. Sjögren’s syndrome patients with ectopic germinal centers present with a distinct salivary proteome. Rheumatology 55, 1127–1137 (2016).

31.      Bergum, B. et al. Antibodies against carbamylated proteins are present in primary Sjögren’s syndrome and are associated with disease severity. Ann. Rheum. Dis. 75, 1494–1500 (2016).

32.      Hellvard, A. et al. Inhibition of CDK9 as a therapeutic strategy for inflammatory arthritis. Sci. Rep. 6, 31441 (2016).

33.      Siddiqui, H. et al. Microbiological and bioinformatics analysis of primary Sjogren’s syndrome patients with normal salivation. J. Oral Microbiol. 8, 31119 (2016).

34.      Sandal, I. et al. Bone loss and aggravated autoimmune arthritis in HLA-DRβ1-bearing humanized mice following oral challenge with Porphyromonas gingivalis. Arthritis Res. Ther. 18, 249 (2016).

35.      Benedyk, M. et al. Gingipains: Critical Factors in the Development of Aspiration Pneumonia Caused by Porphyromonas gingivalis. J. Innate Immun. 8, 185–198 (2016).

36.      Dobosz, E. et al. MCPIP-1, Alias Regnase-1, Controls Epithelial Inflammation by Posttranscriptional Regulation of IL-8 Production. J. Innate Immun. 8, 564–578 (2016).

37.      Gao, S. et al. Presence of Porphyromonas gingivalis in esophagus and its association with the clinicopathological characteristics and survival in patients with esophageal cancer. Infect. Agent. Cancer 11, 3 (2016).

38.      Gawron, K. et al. Gingival fibromatosis: clinical, molecular and therapeutic issues. Orphanet J. Rare Dis. 11, 9 (2016).

39.      Kalinska, M. et al. Kallikreins – The melting pot of activity and function. Biochimie 122, 270–282 (2016).

40.      Kharlamova, N. et al. Antibodies to Porphyromonas gingivalis Indicate Interaction Between Oral Infection, Smoking, and Risk Genes in Rheumatoid Arthritis Etiology. Arthritis Rheumatol. 68, 604–613 (2016).

41.      de Diego, I. et al. The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain. Sci. Rep. 6, 23123 (2016).

42.      Koro, C. et al. Carbamylated LL-37 as a modulator of the immune response. Innate Immun. 22, 218–229 (2016).

43.      Laugisch, O. et al. Citrullination in the periodontium—a possible link between periodontitis and rheumatoid arthritis. Clin. Oral Investig. 20, 675–683 (2016).

44.      Plaza, K. et al. Gingipains of Porphyromonas gingivalis Affect the Stability and Function of Serine Protease Inhibitor of Kazal-type 6 (SPINK6), a Tissue Inhibitor of Human Kallikreins. J. Biol. Chem. 291, 18753–18764 (2016).

45.      Johansson, L. et al. Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis. Arthritis Res. Ther. 18, 201 (2016).

46.      Gawron, K. et al. Gingival fibromatosis with significant de novo formation of fibrotic tissue and a high rate of recurrence. Am. J. Case Rep. 17, 671–675 (2016).

47.      Jusko, M. et al. FACIN, a Double-Edged Sword of the Emerging Periodontal Pathogen Filifactor alocis : A Metabolic Enzyme Moonlighting as a Complement Inhibitor. J. Immunol. 197, 3245–3259 (2016).

48.      Widziolek, M. et al. Zebrafish as a new model to study effects of periodontal pathogens on cardiovascular diseases. Sci. Rep. 6, 36023 (2016).

49.      Lasica, A. M. et al. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis. Sci. Rep. 6, 37708 (2016).

50.      Goulas, T. et al. Structure of RagB, a major immunodominant outer-membrane surface receptor antigen of Porphyromonas gingivalis. Mol. Oral Microbiol. 31, 472–485 (2016).

51.      Surmiak, E. et al. A Unique Mdm2-Binding Mode of the 3-Pyrrolin-2-one- and 2-Furanone-Based Antagonists of the p53-Mdm2 Interaction. ACS Chem. Biol. 11, 3310–3318 (2016).

52.      Balarini, G. M. et al. Serum calprotectin is a biomarker of carotid atherosclerosis in patients with primary Sjögren’s syndrome. Clin. Exp. Rheumatol. 34, 1006–1012 (2016).

53.      Valim, V. et al. Atherosclerosis in Sjögren’s syndrome: evidence, possible mechanisms and knowledge gaps. Clin. Exp. Rheumatol. 34, 133–42 (2016).

54.      Delaleu, N. et al. High Fidelity Between Saliva Proteomics and the Biologic State of Salivary Glands Defines Biomarker Signatures for Primary Sjögren’s Syndrome. Arthritis Rheumatol. 67, 1084–1095 (2015).

55.      Koziel, J. et al. The Janus Face of a-Toxin: A Potent Mediator of Cytoprotection in Staphylococci-Infected Macrophages. J. Innate Immun. 7, 187–198 (2015).

56.      Ksiazek, M. et al. Miropin, a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia , Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop. J. Biol. Chem. 290, 658–670 (2015).

57.      Tancharoen, S. et al. Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients. PLoS One 10, e0117775 (2015).

58.      Zuwała, K. et al. The Nucleocapsid Protein of Human Coronavirus NL63. PLoS One 10, e0117833 (2015).

59.      López-Pelegrín, M. et al. A Novel Mechanism of Latency in Matrix Metalloproteinases. J. Biol. Chem. 290, 4728–4740 (2015).

60.      Benedyk, M. et al. Pyocycanin, a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung. PLoS One 10, e0118319 (2015).

61.      Ksiazek, M. et al. Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia. Biol. Chem. 396, 261–75 (2015).

62.      Veillard, F. et al. Purification and characterisation of recombinant His-tagged RgpB gingipain from Porphymonas gingivalis. Biol. Chem. 396, 377–84 (2015).

63.      Ksiazek, M. et al. KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure. Front. Microbiol. 6, 312 (2015).

64.      Goulas, T. et al. Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase. Sci. Rep. 5, 11969 (2015).

65.      Burmistrz, M. et al. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems. J. Bacteriol. 197, 2631–2641 (2015).

66.      Zhou, Y. et al. Noncanonical Activation of β-Catenin by Porphyromonas gingivalis. Infect. Immun. 83, 3195–3203 (2015).

67.      Jusko, M. et al. A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System. J. Immunol. 195, 2231–2240 (2015).

68.      Ramos-Junior, E. S. et al. A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection. J. Dent. Res. 94, 1233–1242 (2015).

69.      Byrne, D. P. et al. Breakdown of albumin and haemalbumin by the cysteine protease interpain A, an albuminase of Prevotella intermedia. BMC Microbiol. 15, 185 (2015).

70.      Fisher, B. A. et al. Smoking, Porphyromonas gingivalis and the immune response to citrullinated autoantigens before the clinical onset of rheumatoid arthritis in a Southern European nested case–control study. BMC Musculoskelet. Disord. 16, 331 (2015).

71.      Staniec, D. et al. Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase. J. Biol. Chem. 290, 27248–27260 (2015).

72.      Fischer, J. et al. Characterization of Spink6 in Mouse Skin: The Conserved Inhibitor of Kallikrein-Related Peptidases Is Reduced by Barrier Injury. J. Invest. Dermatol. 134, 1305–1312 (2014).

73.      Jusko, M. et al. Staphylococcal Proteases Aid in Evasion of the Human Complement System. J. Innate Immun. 6, 31–46 (2014).

74.      Morandini, A. C. et al. Porphyromonas gingivalis Fimbriae Dampen P2X7-Dependent Interleukin-1β Secretion. J. Innate Immun. 6, 831–845 (2014).

75.      Quirke, A.-M. et al. Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis. Ann. Rheum. Dis. 73, 263–269 (2014).

76.      Burchacka, E. et al. Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus. Protein Sci. 23, 179–189 (2014).

77.      Wang, Q. et al. Filifactor alocis Infection and Inflammatory Responses in the Mouse Subcutaneous Chamber Model. Infect. Immun. 82, 1205–1212 (2014).

78.      Pustelny, K. et al. Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation. J. Biol. Chem. 289, 15544–15553 (2014).

79.      Akiyama, T. et al. Porphyromonas gingivalis -derived Lysine Gingipain Enhances Osteoclast Differentiation Induced by Tumor Necrosis Factor-α and Interleukin-1β but Suppresses That by Interleukin-17A. J. Biol. Chem. 289, 15621–15630 (2014).

80.      Koziel, J. et al. Citrullination Alters Immunomodulatory Function of LL-37 Essential for Prevention of Endotoxin-Induced Sepsis. J. Immunol. 192, 5363–5372 (2014).

81.      Moelants, E. A. V. et al. Citrullination and Proteolytic Processing of Chemokines by Porphyromonas gingivalis. Infect. Immun. 82, 2511–2519 (2014).

82.      Gawron, K. et al. In vitro testing the potential of a novel chimeric IgG variant for inhibiting collagen fibrils formation in recurrent hereditary gingival fibromatosis: chimeric antibody in a gingival model. J. Physiol. Pharmacol. 65, 585–91 (2014).

83.      Olsen, I. et al. Strategies for the inhibition of gingipains for the potential treatment of periodontitis and associated systemic diseases. J. Oral Microbiol. 6, 24800 (2014).

84.      Florek, D. et al. Stability of infectious human coronavirus NL63. J. Virol. Methods 205, 87–90 (2014).

85.      Siddiqui, H. et al. Genome Sequence of Porphyromonas gingivalis Strain HG66 (DSM 28984). Genome Announc. 2, e00947-14-e00947-14 (2014).

86.      Eick, S. et al. Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis. Orphanet J. Rare Dis. 9, 148 (2014).

87.      Milewska, A. et al. Human Coronavirus NL63 Utilizes Heparan Sulfate Proteoglycans for Attachment to Target Cells. J. Virol. 88, 13221–13230 (2014).

88.      Koro, C. et al. Carbamylation of immunoglobulin abrogates activation of the classical complement pathway. Eur. J. Immunol. 44, 3403–3412 (2014).

89.      de Diego, I. et al. Structure and Mechanism of Cysteine Peptidase Gingipain K (Kgp), a Major Virulence Factor of Porphyromonas gingivalis in Periodontitis. J. Biol. Chem. 289, 32291–32302 (2014).

90.      Bielecka, E. et al. Peptidyl Arginine Deiminase from Porphyromonas gingivalis Abolishes Anaphylatoxin C5a Activity. J. Biol. Chem. 289, 32481–32487 (2014).

91.      Tomek, M. B. et al. The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O -glycosylation. Mol. Oral Microbiol. 29, 307–320 (2014).

92.      Bryzek, D. et al. A pathogenic trace of Tannerella forsythia - shedding of soluble fully active tumor necrosis factor α from the macrophage surface by karilysin. Mol. Oral Microbiol. 29, 294–306 (2014).

93.      Gawron, K. et al. Peptidylarginine deiminase from Porphyromonas gingivalis contributes to infection of gingival fibroblasts and induction of prostaglandin E 2 -signaling pathway. Mol. Oral Microbiol. 29, 321–332 (2014).

94.      Wilensky, A. et al. Porphyromonas gingivalis Gingipains Selectively Reduce CD14 Expression, Leading to Macrophage Hyporesponsiveness to Bacterial Infection. J. Innate Immun. 7, 127–135 (2014).

95.       Łysek, R. P. et al. Relationship between past myocardial infarction, periodontal disease and Porphyromonas gingivalis serum antibodies: A case-control study. Cardiol. J. (2013). doi:10.5603/CJ.a2017.0015