Post Doc Fellowships
Romanian Society of Biochemistry
Romanian Journal of Biochemistry

 
                
Romana
 
 
      Department of Enzymology

Department of Enzymology

The structure & role of signaling enzymes

Current Work, Future Projects, Selected Publications, Lectures


Stefan Eugen Szedlacsek, PhD - Head of Department
Mihaela Balasu, PhD
Georgiana Petrareanu, PhD
Mihaela Pascaru Mentel, PhD Student
Rodica Badea, PhD Student
Sujay Turuwekere Mallikarjuna, PhD Student
Ingrid Cristiana Vreja, Student
Ionut Dumitru, Student
Luminita Barca, Secretary
Adina Mariana Monea, technician
Maria Dragan, technician
 

Current Work
The central research theme of the group is the study of structure-function relationships of signaling enzymes with an emphasis on protein tyrosine phosphatases. We aim to contribute to the understanding of how their structural characteristics are correlated with specific signaling functions. To this purpose we analyze each signaling enzyme which we are investigating from multiple directions:

i.)    as a classical enzyme, trying to evaluate its stability under various conditions, pH-dependence of the activity, specific activity, steady-state kinetic parameters, substrate specificity and also to identify the specific inhibitors as well as the corresponding inhibition constants;

ii.) as a protein, trying to crystallize the purified enzyme preparation and then to determine its 3D-structure

iii.) as a signaling entity, trying to find its subcellular localization, physiological substrate(s), regulatory interactions, role played in signaling pathways, etc.

 

The combination of results thus obtained in this way is further used in shedding light on the signaling mechanism and on the overall functional role of the given enzyme.

The group has good experience and is currently involved in production, isolation and purification of recombinant proteins, expressed both in prokaryotic and eukaryotic systems. The research activity of the group is performed by tools of molecular biology (recombinant DNA, site-directed mutagenesis, (RT)-PCR, Western blotting, immunoprecipitation, etc.), spectroscopic analysis (UV-VIS and fluorescence spectrophotometry), cellular biology, protein crystallization and enzyme kinetic analysis.

Our team is involved in numerous national and international research projects. Thus, one of our current projects is financed by the European Commission within the Framework Program 6. Also, we are running projects within Romanian Research Programs, both as coordinators and as collaborators.

 

ONGOING RESEARCH PROJECTS

Marie Curie Research Training Networks: PTPNET
”Protein Tyrosine Phosphatases : Structure, Regulation, and Biological Function”
funded by the European Commission Research Directorate General
Lead investigator: Dr. Stefan Szedlacsek
Institute of Biochemistry - partner
http://www.ich.ucl.ac.uk/ich/academicunits/PTPNET/Homepage

Two main goals are set for our group within the objectives of this project. First: to determine the 3D-crystal structure of non-catalytic cytoplasmic subdomains of PTPs. Second: to evaluate the substrate specificities and determine the effects of specific modulators and mutations on the enzymatic activity of PTPs. It is also intended to perform correlation analysis between structural and activity data thus obtained. Modern experimental procedures will be applied, like recombinant DNA technologies, protein expression, protein purification and crystallization, spectroscopy, enzyme kinetics analysis.

FOSFOTIR
Evaluation of the Substrate Specificity of several Protein Tyrosine Phosphatases involved in Diseases.

Funded by: CNMP (Romania)
Project Director: Dr. Stefan Szedlacsek
Partners: Politechnica University. Bucharest; S.C.CORAX, Miercurea-Ciuc

We are interested in finding new physiological substrates of three protein tyrosine phosphatases with essential roles in signal transduction under normal and pathological conditions. The three PTPases are: PRL-3, which is massively overexpressed in metastasis of colorectal cancer; hEya1, proved to be implicated in the branchio-oto-renal or Melnick-Fraser syndrome and receptor PTPm (RPTPm) which recently has been demonstrated to be involved in human glioblastoma multiforme, the most common malignant primary brain tumor.

 

In order to reach our goal, we are using various experimental approaches like:  in vitro dephosphorylation of the potential substrate, GST-pulldown using inactive substrate-trapping mutants and modulation of substrate tyrosine phosphorylation level in a cellular contex. The starting point in finding potential substrates was represented by previous studies on synthetic phosphopeptides libraries. Using this approach, it was proved that some phosphopeptides were efficiently dephosphorylated by the catalytic domain of RPTPμ


XPEK
Determination of 3D Structure for some interesting proteins by X-ray diffraction.

Funded by: UEFISCSU (Romania)
Project Director: Dr. Stefan Szedlacsek

Selected Publications

1. “Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis”, Petrareanu, G., Balasu, M.C., Zander, U., Scheidig, A.J., Szedlacsek, S.E., Acta Cryst. F66, 805–807 (2010).

 

2. "Interface analysis of the complex between ERK2 and PTP-SL.", PLoS ONE.,4(5),e5432 (2009) Balasu MC, Spiridon LN, Miron S, Craescu CT, Scheidig AJ, Petrescu A-J, Szedlacsek SE.

 

3. "Analysis of Molecular Determinants of PRL-3.", J Cell Mol Med 13(9B), 3141-50. Epub 2008 Jun 28. (2009) Pascaru M, Tanase C, Vacaru AM, Boeti P, Neagu E, Popescu I, Szedlacsek SE.

 

4. "Protein tyrosine phosphatases: structure-function relationships.", FEBS J.,275(5), 867-882 (2008) Tabernero L, Aricescu AR, Jones EY, Szedlacsek SE.

 

5. "A microarray strategy for mapping the substrate specificity of protein tyrosine phosphatase.", Angew Chem Int Ed Engl.,46(40),7700-7003 (2007) Köhn M, Gutierrez-Rodriguez M, Jonkheijm P, Wetzel S, Wacker R, Schroeder H, Prinz H, Niemeyer CM, Breinbauer R, Szedlacsek SE, Waldmann H.

 

6. "Functional, fractal nonlinear response with application to rate processes with memory, allometry, and population genetics.", Proc Natl Acad Sci USA.,104(12),4798-803 (2007) Vlad MO, Moran F, Popa VT, Szedlacsek SE, Ross J.

 

7. "Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids.", J Am Chem Soc, 128(9), 2822-2835 (2006)
Dursina B, Reents R, Delon C, Wu Y, Kulharia M, Thutewohl M, Veligodsky A, Kalinin A, Evstifeev V, Ciobanu D, Szedlacsek SE, Waldmann H, Goody RS, Alexandrov K.

 

8. "Human TPST1 Transmembrane Domain Triggers Enzyme Dimerisation and Localisation to the Golgi Compartment", J.Mol.Biol, 361, 436-449 (2006)
S. Goettsch, R. A. Badea, J. W. Mueller, C. Wotzlaw, B. Schoelermann, L. Schulz, M. Rabiller, P. Bayer, C. Hartmann-Fatu.

 

9. "The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu", J.Biol.Chem., 279, 26922-26931 (2004) Cismasiu VB, Denes SA, Reilander H, Michel H, Szedlacsek SE.

 

10. "Intramolecular interactions in protein tyrosine phosphatase RPTPµ: kinetic evidence.", Biochem Biophys Res Commun. 280: 319-327 (2001) Aricescu, A. R., Fulga, T. A., Cismasiu, V., Goody, R. S. and Szedlacsek, S. E.

 

11. "Crystal structure of PTP-SL/PTPBR7 catalytic domain: implications for MAP kinase regulation.", J.Mol.Biol.; 311, 557-568. (2001) Szedlacsek, S. E., Aricescu, A. R., Fulga, T. A., Renault, L. and Scheidig, A J.