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
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
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
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
funded by the European Commission Research Directorate General
Lead investigator: Dr. Stefan Szedlacsek
Institute of Biochemistry - partner
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.
Evaluation of the Substrate Specificity of several Protein Tyrosine
Phosphatases involved in Diseases.
Funded by: CNMP (Romania)
Project Director: Dr. Stefan Szedlacsek
Partners: PolitechnicaUniversity. 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
Determination of 3D Structure for some interesting proteins by X-ray
Funded by: UEFISCSU (Romania)
Project Director: Dr. Stefan Szedlacsek
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).
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,
of Molecular Determinants of PRL-3.", J Cell Mol Med 13(9B), 3141-50. Epub 2008 Jun 28. (2009)
M, Tanase C, Vacaru AM, Boeti P, Neagu E, Popescu I, Szedlacsek SE.
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.
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.
and specificity profiling of protein prenyltransferase inhibitors using new
fluorescent phosphoisoprenoids.", J Am Chem Soc,128(9), 2822-2835
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,
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.
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,
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.