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Protein Crystallography Research Group

Group Leader

 

 

Grzegorz Dubin, PhD

email: grzegorz.dubin@uj.edu.pl

http://www.crystallab.pl

 

 

 

 

 

Project Leaders within Protein Crystallography Research Group

dr Anna Czarna

Polish Returns” Project financed by NAWA

http://www.crystallab.pl/nawa.html

 

 

 

 

 

dr Przemysław Grudnik

Project „SONATA 10” from NCN

Deputy Project Manager - "Structural Biology Core Facility" from FNP

http://structuralbiology.pl/

 

Team members

Post-doc:

  • Przemysław Grudnik
    Email: przemyslaw.grudnik@uj.edu.pl
  • Stanisław Malicki
    Email: stanislaw.malicki@uj.edu.pl
  • Krzysztof Rembacz
    Email: krzysztof.rembacz@uj.edu.pl
  • Małgorzata Romanowska
    Email: malgorzata.1.romanowska@uj.edu.pl

 

PhD students:

  • Bozena Boczkus
  • Józefina Bogusz
  • Aleksandra Pęcak
  • Natalia Stach
  • Karol Źrubek

and masters and bachelor students.

Research themes

We are primarily interesting study in interactions of proteins and proteins and low molecular weight substances within the subject of carcinogenesis and bacterial pathogenesis. Currently were realize our own projects involving structural characterization of kinases of essential role cancer, p53 pathway, bacterial proteases of unknown catalytic mechanism and staphylococcal proteases. In parallel to the crystallographic studies we also develop aptamer selection techniques (especially single-stranded DNA) against targets of importance in cancer. In addition to our own research, we are also involved in broad scientific collaboration and cooperation with the industry on other subjects whenever problem scan be solved by X-ray crystallography of proteins. We invite anyone interested in collaboration.

Research methodology and specialized equipment

The X-ray Crystallography Laboratory of MCB has extensive experience in solving protein structures and the structures of complexes. The primary laboratory equipment consists of an X-ray diffractometer (RigakuMicroMax-007 HF) with CCD and IP detectors. Crystallization screening is carried out using an automatic pipetting station (Phoenix RE). Laboratory is also exceptionally well equipped for expression, purification and analysis of proteins.

Research grants

We are primarily interesting study in interactions of proteins and proteins and low molecular weight substances within the subject of carcinogenesis and bacterial pathogenesis. Currently were realize our own projects involving structural characterization of kinases of essential role cancer, p53 pathway, bacterial proteases of unknown catalytic mechanism and staphylococcal proteases. In parallel to the crystallographic studies we also develop aptamer selection techniques (especially single-stranded DNA) against targets of importance in cancer. In addition to our own research, we are also involved in broad scientific collaboration and cooperation with the industry on other subjects whenever problem scan be solved by X-ray crystallography of proteins. We invite anyone interested in collaboration. Research methodology and specialized equipment The X-ray Crystallography Laboratory of MCB has extensive experience in solving protein structures and the structures of complexes. The primary laboratory equipment consists of an X-ray diffractometer (RigakuMicroMax-007 HF) with CCD and IP detectors. Crystallization screening is carried out using an automatic pipetting station (Phoenix RE). Laboratory is also exceptionally well equipped for expression, purification and analysis of proteins.

Research papers

1. Szelazek B, Kabala W, Kus K, Zdzalik M, Twarda-Clapa A, Golik P, Burmistrz M, Florek D, Wladyka B, Pyrc K, Dubin G. (2017). Structural Characterization of Human Coronavirus NL63 N Protein. J. Virol. (in press)

2. Surmiak E, Neochoritis CG, Musielak B, Twarda-Clapa A, Kurpiewska K, Dubin G, Camacho C, Holak TA, Domling A. (2017). Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction. Eur. J. Med. Chem 126, 384-407.

3. Magiera K, Tomala M, Kubica K, De Cesare V, Trost M, Zieba BJ, Kachamakova-Trojanowska N, Les M, Dubin G, Holak TA, Skalniak L. (2017). Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a. Cell Chem. Biol. 24, 1–13.

4. Surmiak E, Twarda-Clapa A, Zak KM, Musielak B, Tomala MD, Kubica K, Grudnik P, Madej M, Jablonski M, Potempa J, Kalinowska-Tluscik J, Domling A, Dubin G, Holak TA. (2016). A Unique Mdm2-Binding Mode of the 3‑Pyrrolin-2-one- and 2‑Furanone-Based Antagonists of the p53-Mdm2 Interaction. Chem. Biol.

5. Gieldon A, Zurawa-Janicka D, Jarzab M, Wenta T, Golik P, Dubin G, Lipinska B, Ciarkowski J. (2016). Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants. PLoS One 11(8):e0161526

6. Zarganes-Tzitzikas T, Konstantinidou M, Gao Y, Krzemien D, Zak K, Dubin G, Holak TA, Dömling A. (2016) Inhibitors of programmed cell death 1 (PD-1): a patent review (2010-2015). Expert Opin Ther Pat. 26(9):973-7

7. Bonar E, Wojcik I, Jankowska U, Kedracka-Krok S, Bukowski M, Polakowska K, Lis MW, Kosecka-Strojek M, Sabat AJ, Dubin G, Friedrich AW, Miedzobrodzki J, Dubin A, Wladyka B. (2016) Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains. Front Cell Infect Microbiol. 6:51

8. Zak KM, Grudnik P, Guzik K, Zieba BJ, Musielak B, Dömling A, Dubin G, Holak TA. (2016) Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1). Oncotarget 7(21):30323-35

9. de Diego I, Ksiazek M, Mizgalska D, Koneru L, Golik P, Szmigielski B, Nowak M, Nowakowska Z, Potempa B, Houston JA, Enghild JJ, Thøgersen IB, Gao J, Kwan AH, Trewhella J, Dubin G, Gomis-Rüth FX, Nguyen KA, Potempa J. (2016) 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.

10. Zak KM, Kitel R, Przetocka S, Golik P, Guzik K, Musielak B, Dömling A, Dubin G, Holak TA. (2015) Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1. Structure 23(12):2341-8

11. Karna N, Łęgowska A, Malicki S, Dębowski D, Golik P, Gitlin A, Grudnik P, Wladyka B, Brzozowski K, Dubin G, Rolka K. (2015). Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI-1). Chembiochem. 16(14):2036-45

12. Horwacik, I., Golik, P., Grudnik, P., Kolinski, M., Zdzalik, M., Rokita, H., Dubin, G. (2015). Structural Basis of GD2 Ganglioside and Mimetic Peptide Recognition by 14G2a Antibody. Molecular & Cellular Proteomics, 14: 2577-2590.

13. Grudnik, P., Debowski, D., Legowska, A., Malicki, S., Golik, P., Karna, N., Rolka, K., Dubin, G. (2015). Atomic resolution crystal structure of HV-BBI protease inhibitor from amphibian skin in complex with bovine trypsin. Proteins – structure, function and bioinformatics, 83:582-589.

14. Janczak, M., Bukowski, M., Gorecki, A., Dubin, G., Dubin, A., Wladyka, B. (2015). A systematic investigation of the stability of green fluorescent protein fusion proteins. Acta Biochimica Polonica, 62:407-411.

15. Zuwala, K., Golda, A., Kabala, W., Burmistrz, M., Zdzalik, M., Nowak, P., Kedracka-Krok, S., Zarebski, M., Dobrucki, J., Florek, D., Zeglen, S., Wojarski, J., Potempa, J., Dubin, G., Pyrc, K., (2015). The Nucleocapsid Protein of Human Coronavirus NL63. Plos ONE, 10:e0117833.

16. Jusko, M., Potempa, J., Kantyka, T., Bielecka, E., Miller, HK., Kalinska, M., Dubin, G., Garred, P., Shaw, LN., Blom, AM. (2014). Staphylococcal proteases aid in evasion of the human complement system. Journal of Innate Immunity, 6:31-46.

17. Pustelny, K., Stach, N., Wladyka, B., Dubin, A., Dubin, G. (2014). Evaluation of P1' substrate specificity of staphylococcal SplB protease. Acta Biochim. Pol. 61:149-52.

18. Pustelny, K., Zdzalik, M., Stach, N., Stec-Niemczyk, J., Cichon, P., Czarna, A., Popowicz, G., Mak, P., Drag, M., Salvesen, G.S., Wladyka, B., Potempa, J., Dubin, A., Dubin, G. (2014). Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation. J. Biol. Chem. 289:15544-15553.

19. Burchacka, E., Zdzalik, M., Stec-Niemczyk, J., Pustelny, K., Popowicz, G., Wladyka, B., Dubin, A., Potempa, J., Sienczyk, M., Dubin, G., Oleksyszyn, J. (2014). Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus. Prot. Sci. 23:179-189.

20. Wladyka, B., Wielebska, K., Wloka, M., Bochenska, O., Dubin, G., Dubin, A., Mak, P. (2012). Isolation, biochemical characterization, and cloning of a bacteriocin from the poultry-associated Staphylococcus aureus strain CH-91. (2013). Appl. Microbiol. Biotechnol. 97: 7229-7239.

21. Bukowski, M., Lyzen, R., Helbin, WM., Bonar, E., Szalewska-Palasz, A., Wegrzyn, G., Dubin, G., Dubin, A., Wladyka. B. (2013). A regulatory role for Staphylococcus aureus toxin-antitoxin system pemIKSa. Nature Communications, 4:2012. doi:10.1038/ncomms3012

22. Zubko, M., Kusz, J., Prodan, A., Sturm, S., van Midden, HJP., Bennett, JC., Dubin, G., Zupanic, E., Bohm, H. (2013). Structural phase transition and related electronic properties in quasi one-dimensional (NbSe4)10/3I. Acta Crystallographica Section B. 69:229-237

23. Kolesinski, P., Golik, P., Grudnik, P., Piechota, J., Markiewicz, M., Tarnawski, M., Dubin, G., Szczepaniak, A. (2013). Insights into eukaryotic Rubisco assembly - crystal structures of RbcX chaperones from Arabidopsis thaliana. Biochim. Biophys. Acta. 1830:2899-2906.

24. Zak, K., Pecak, A., Rys, B., Wladyka, B., Domling, A., Weber, L., Holak, TA., Dubin, G. (2013). MDM2 and MdmX inhibitors for the treatment of cancer: a patent review (2011-present). Expert Opinion on Therapeutic Patents 23:425-448.

25. Dubin, G., Koziel, J., Pyrc, K., Wladyka, B., Potempa. J. (2013). Bacterial proteases in disease - role in intracellular survival, evasion of coagulation/fibrinolysis innate defenses, toxicoses and viral infections. Curr. Pharm. Des. 19:1090-113.

26. Zdzalik, M., Kalinska, M.,Wysocka, M.,Stec-Niemczyk, J., Cichon, P., Stach, N.,Gruba, N., Stennicke, H.R., Jabaiah, A.,Markiewicz, M., Kedracka-Krok, S., Wladyka, B., Daugherty, P.S., Lesner, A., Rolka, K., Dubin, A.,Potempa, J., Dubin, G. (2013). Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus. PLoS One, 8: e76812

27. Polakowska, K., Lis, M., Helbin, W.M., Dubin, G., Dubin, A., Niedziolka, J., Miedzobrodzki, J., Wladyka, B. (2012). The virulence of Staphylococcus aureus correlates with strain genotype in a chicken embryo model but not a nematode model. Microbes Infect. 14: 1352-1362.

28. Burchacka, E. Walczak, M., Sienczyk, M., Dubin, G., Zdzalik, M., Potempa, J., Oleksyszyn, J. (2012). The development of first Staphylococcus aureus SplB protease inhibitors: phosphonic analogues of glutamine. Bioorg. Med. Chem. Lett. 22: 5574-5578.

29. Bista, M., Smithson, D., Pecak, A., Salinas, G., Pustelny, K., Min, J., Pirog, A., Finch, K., Zdzalik, M., Waddell, B., Wladyka, B., Kedracka-Krok, S., Dyer, M.A., Dubin, G., Guy, R.K. (2012). On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53. PLoS One, 7:e37518.

30. Zdzalik, M., Karim, A.Y., Wolski, K., Buda, P., Wojcik, K., Brueggemann, S., Wojciechowski, P., Eick, S., Calander, A.M., Jonsson, I.M., Kubica, M., Polakowska, K., Miedzobrodzki, J., Wladyka, B., Potempa, J., Dubin, G. (2012). Prevalence of genes encoding extracellular proteases in Staphylococcus aureus – important targets triggering immune response in vivo. FEMS Immunol. Med. Microbiol. 66:220-229

31. Kalinska, M., Kantyka, T., Greenbaum, D.C., Larsen, K.S., Wladyka, B., Jabaiah, A., Bogyo, M., Daugherty, P.S., Wysocka, M., Jaros, M., Lesner, A., Rolka, K., Schaschke, N., Stennicke, H., Dubin, A., Potempa, J., Dubin, G. (2012). Substrate specificity of Staphylococcus aureus cysteine proteases – Staphopains A, B and C. Biochimie, 94:318-327

32. Wladyka, B., Kozik, A.J., Bukowski, M., Rojowska, A., Kantyka, T., Dubin, G., Dubin, A. (2011) Alpha-1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition. Biochimie, 93: 948-953.

33. Wladyka, B., Dubin, G., Dubin, A. (2011). Activation mechanism of thiol protease precursor from broiler chicken specific Staphylococcus aureus strain CH-91. Vet. Microbiol. 147:195-199.

34. Zdzalik, M., Pustelny, K., Kedracka-Krok, S., Huben, K., Pecak, A., Wladyka, B., Jankowski, S., Dubin, A., Potempa, J., Dubin, G. (2010). Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73. Cell Cycle 9:4584-4591.

35. Bukowski, M., Wladyka, B., Dubin, G. (2010). Exfoliative toxins of Staphylococcus aureus. Toxins 2: 1148-1165.

36. Karim, AY., Kulczycka, M., Kantyka, T., Dubin, G., Jabaiah, A., Daugherty, PS., Thogersen, IB, Enghild, JJ., Nguyen, KA., Potempa, J. (2010). A Novel Matrix Metalloprotease-like Enzyme (Karilysin) of the Periodontal Pathogen Tannerella forsythia ATCC 43037. Biol. Chem. 391: 105-117.

37. Kantyka, T., Latendorf, T., Wiedow, O., Bartels J., Glaser, R., Dubin, G., Schroder, J-M., Potempa, J., Meyer-Hoffert, U. (2009). Elafin is specifically inactivated by RgpB from Porphyromonas gingivalis by distinct proteolytic cleavage. Biol. Chem. 390: 1313-1320.

38. Czarna, A., Popowicz, GM., Pecak, A., Wolf, S., Dubin, G., Holak, TA. (2009). High affinity interaction of the p53 peptide-analogue with human Mdm2 and Mdmx. Cell Cycle. 8: 1176-1184.

39. Stec-Niemczyk, J., Pustelny, K., Kisielewska, M., Bista, M., Boulware, K.T., Stennicke, H.R., Thogersen, I.B., Daugherty, P.S., Enghild, J.J., Baczynski, K., Popowicz, G.M., Dubin, A., Potempa, J., Dubin, G. (2009). Structural and functional characterization of SplA, an exclusively specific protease of Staphylococcus aureus. Biochem. J. 419: 555-564.

40. Calander, AM., Dubin, G., Potempa, J., Tarkowski, A. (2008). Staphylococcus aureus infection triggers production of neutralizing, V8 protease-specific antibodies. FEMS Immunol. Med. Mic. 52: 267-272

41. Dubin, G. , Stec-Niemczyk, J., Kisielewska, M., Pustelny, K., Popowicz, G., Bista, M., Kantyka, K., Boulware, K.T., Stennicke, H.R., Czarna, A., Phopaisarn, M., Daugherty, P.S., Thøgersen, I.B., Enghild, J.J, Thornberry, N., Dubin, A., Potempa, J. (2008). Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln. J. Mol. Biol. 379: 343-356

42. Beaufort, N., Wojciechowski, P., Sommerhoff, CP., Szmyd, G., Dubin, G., Eick, S., Kellermann, J., Schmitt, M., Potempa, J., Magdolen, V. (2008). The human fibrinolytic system is a target for the staphylococcal metalloprotease aureolysin. Biochem. J. 410: 157-165.

43. Kulig, P., Zabel, BA., Dubin, G., Allen, SJ., Ohyama, T., Potempa, J., Handel, TM., Butcher, EC., Cichy, J. (2007). Staphylococcus aureus-derived staphopain B, a potent cysteine protease activator of plasma chemerin. J. Immunol. 178: 3713-3720.

44. Dubin, G., Wladyka, B., Stec-Niemczyk, J., Chmiel, D., Zdzalik, M., Dubin, A., Potempa J. (2007) The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity. Biol. Chem. 388: 227-235.

45. Hirsch, T., von Peter, S., Dubin, G., Mittler, D., Jacobsen, F., Lehnhardt, M., Eriksson, E., Steinau, H-U., Steinstraesser, L. (2006). Adenoviral Gene Delivery to Primary Human Cutaneous Cells and Burn Wounds. Mol. Med. 12: 199-207.

46. Popowicz, G., Dubin, G., Stec-Niemczyk, J., Czarna, A., Dubin, A., Potempa, J., Holak T.A. (2006). Functional and Structural Characterization of Spl proteases from Staphylococcus aureus. J. Mol. Biol. 358: 270-279.

47. Dubin, G. (2005). Proteinaceous cysteine protease inhibitors. Cell. Mol. Life. Sci. 62:653-669.

48. Dubin, A., Mak, P., Dubin, G., Rzychon, M., Stec-Niemczyk, J., Wladyka, B., Maziarka, K., Chmiel, D. (2005). New generation of peptide antibiotics. Acta Biochim. Pol. 52:633-638.

49. Dubin, G., Stec-Niemczyk, J., Dylag, T., Silberring, J., Dubin, A., Potempa, J. (2004) Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family. Biol. Chem. 385:543-546.

50. Dubin, G., Popowicz, G., Krajewski, M., Potempa, J., Dubin, A., Holak, T.A., (2004). Letter to the editor: 1H, 15N and 13C NMR resonance assignments of staphostatin A, a specific Staphylococcus aureus cysteine proteinase inhibitor. J. Biomol. NMR 28:295-96.

51. Dubin, G., Krajewski, M., Popowicz, G., Stec-Niemczyk, J., Bochtler, M., Potempa, J., Dubin, A., Holak, TA. (2003). A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus. Biochemistry. 42:13449-13456.

52. Dubin, G., (2003). Defense against own arms: staphylococcal cysteine proteases and their inhibitors. Acta Biochim. Pol. 50:715-724.

53. Dubin, G., (2002). Extracellular proteases of Staphylococcus spp. Biol. Chem. 383, 1075-1086.

54. Dubin, G., Chmiel, D., Mak, P., Rakwalska, M., Rzychon, M., Dubin, A. (2001). Molecular cloning and biochemical characterization of proteases form Staphylococcus epidermidis. Biol. Chem. 382, 1575-1582.

Patent applications

Dubin, G., Potempa, J., BioCentrum sp. z o. o., Uniwersytet Jagielloński. Proteinaza SplA i peptydy przez nią rozpoznawane oraz ich zastosowania. (nr. PL382770), rok 2007

Dubin, G., Potempa, J., BioCentrum sp. z o. o., Uniwersytet Jagielloński. A protease from Staphylococcus aureus, particularly SplA or SplB, peptides it recognises and their use. Zgłoszenie PCT (nr. WO 2008/153429), rok: 2008

Dubin, G., Popowicz, G., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Znakowane peptydy, sposoby wykrywania substancji modulujących oddziaływanie mdm2-p53 i/lub mdmx-p53 oraz zestaw przeznaczony do wykrywania substancji modulujących. (nr. P387416) rok: 2009

Wladyka, B., Dubin, G., Uniwersytet Jagielloński. Wysokowydajny promotor ekspresji w komórce bakteryjnej, konstrukt genowy, wektor, transformant, systemy ekspresji i sekrecji heterologicznego białka oraz ich zastosowania. (nr. P.389523), rok: 2009

Feder, M., Dubin, G., Bulkowska, U., Jaszczewska, J.A., Burchard, E., Kalinowska, I., Lewandowski, W., Adamed sp. z o.o. 1,5-dihydropyrrol-2-one derivatives as inhibitors of p53-MDM2/MDM4 protein-protein interaction. (zgłoszenie PCT nr. WO/2015/004610 na podstawie zgłoszenia nr. PL404651 (A1))

Dubin, G., Pecak, A., Malicki, S., Majewski, P., Uniwersytet Jagielloński. Aptamery posiadające powinowactwo do białka Mdm2, ich kompozycje oraz zastosowanie. (nr. PL405115 (A1))

Obtained patents

Dubin, G., Potempa, J., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Polipeptyd wykazujący powinowactwo do centrum aktywnego proteinazy SplB, białko, sekwencja nukleotydowa kodująca polipeptyd i białko, zastosowanie sekwencji polipeptydu, sposób otrzymywania białka oraz zastosowania proteinazy SplB. (nr. P.382638)

Dubin, G., Potempa, J., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Mutant proteinazy SplB i sposób otrzymywania mutanta proteinazy SplB. (nr. P.394914).

Dubin, G., Potempa, J., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Proteinaza posiadająca aktywność proteinazy SplB. (nr. P.394913)

Dubin, G., Potempa, J., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Wariant proteinazy SplA i sposób otrzymywania proteinazy SplA. (nr. P.394912)

Dubin, G., Potempa, J., Uniwersytet Jagielloński, BioCentrum sp. z o.o. Proteinaza posiadająca aktywność proteinazy SplA. (nr. P.394911)

Commercial implementation

CleanCut - protease with high substrate specificity for use in the removal of fusion tags. Developed on the basis of the studies of the author, claimed in the patent application (WO 2008/153429) and granted patents (PL214451, PL214123, and PL213955) and commercialized with the participation of BioCentrum Ltd. on the basis of appropriate agreements with the Jagiellonian University. It is currently offered on the international market by following companies: Sigma-Aldrich (with the brand name CleanCut) and ThermoScientific (with the brand name WELQut).

Job opportunity

  1. Doctoral and Postdoctoral positions for biological and related field scientists.