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Enzymology Department

Enzymology

Our central research topic is the study of structure-function relationships in signaling enzymes, with a focus on protein tyrosine phosphatases. The aim is to contribute to the understanding of how their structural features are correlated with specific signaling functions. To this end, signaling enzymes are studied from several directions:

  • as a classical enzyme, trying to evaluate enzymes stability under different conditions, the pH dependence of the activity, the specific activity, the kinetic parameters at the steady state, the substrate specificity and also the identification of the specific inhibitors 
  • as a protein, trying to crystallize the purified enzyme preparation and then determine its 3D structure
  • as a signaling entity, trying to find its subcellular location, substrate(s), regulatory interactions, role played in signaling pathways, etc.

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

We have good experience and we are currently involved in the production, isolation and purification of recombinant proteins, expressed in both prokaryotic and eukaryotic systems. Our research activity is carried out through tools of molecular biology (recombinant DNA, site-directed mutagenesis, (RT)-PCR, Western blot, immunoprecipitation, etc.), spectroscopic analysis (UV-VIS and fluorescent spectrophotometry), cell biology, protein crystallization and enzyme kinetics.

Our ongoing research projects are:

  • Design, preparation, characterization and testing of new molecular vectors targeting specific leukemic cells for targeted diagnosis and targeted therapy
  • Study of tau protein acetylation and its involvement in neurodegenerative diseases 

I. “Development of a cyclotron-driven neutron activator for radiopharmaceutical production; case study    of holmium-166 labelled affibody for targeted diagnostics and therapeutics” ; Acronym: NARAD; https://www.nipne.ro/proiecte/pn3/20-projects.html

Contracting Authority: UEFISCDI, code: PN-III-P2-2.1-PED/2019-4184; Number/Date of the contract:548/PED/02.11.2020, 

Partner 1: Institute of Biochemistry: starting date/finishing date: 02.11.2020-31.10.2022 

Project value P1: 250.000 RON

Project team P1:  Dr.Stefan Szedlacsek: Project manager P1; Dr.Horea Szedlacsek: Researcher; PhD Student: Adina Gabriela Puiu; PhD Student: Andrei Mihai Vasilescu; Technical assistant: Catalin Constantin Bica; Technical assistant: Otilia Cristina Donțu, Logistics and communication support:

Objectives:

The stages of the project and delivery dates:

1. The concept of a Neutron Activator in line with the cyclotron. 2. Design of a Neutron Activator in line with TR19 cyclotron for  radiopharmaceuticals production. 3. Neutron Activator testing. Case study of Holmium-166 labelled affibody, for targeted diagnostics and therapeutics,

Objectives achieved by P1 IBAR within the partnership:

(a) Large-scale expression and affinity purification of anti-HER2 Affibody; (b) Development of analytical methods for monitoring the monomer-dimer composition of affibody; (c) Control coupling of affibody with the MMA derivative of a fluorescent dye; (d) Coupling to affibody of selected chelators (MMA-DOTA, MMA-NODAGA, s.a.); (e) Development of analytical methods for monitoring the chelator-affibody  coupling reaction; (f) High purity purification of affibody-chelator conjugates

The technological maturity level (TRL) at the end of the project achieved by P1 IBAR is TRL 3-4, as was proposed in the project plan. 

This level was met because P1 demonstrated that the necessary compounds (the two anti-HER2 Affibody constructs) can be obtained for in vitro and in vivo functional studies and furthermore, the anti-HER2 affibody conjugated  with a lanthanide chelator was validated for mouses imaging by our collaborators.

II. "Genomic mapping of population from polluted area with radioactivity and heavy  metals to increase national security"; AcronymARTEMIS. http://www.umfcluj.ro/component/content/article/8-ro/532-genomic-mapping-of-population-from-polluted-area-with-radioactivity-and-heavy-metals-to-increase-national-security-artemis?Itemid=216

Contracting Authority: UEFISCDI, Code: PN-III-P1-1.2-PCCDI-2017-0737; Number/Date of the contract:35/PCCDI/03.04.2018

Partner 3: Institute of Biochemistry/IBAR: starting date/finishing date: 03.04.2018-31.12.2021 

Project value P3: 939.600 RON

Project team P3: Dr.Stefan Szedlacsek: Project manager P3Asc.Horea Szedlacsek; Dr.Rodica-Aura Badea, PhD Student  Aura Elena Ionescu; Asc.Simona Călăraș; Asc.Adina Gabriela Puiu; Asc.Andrei Mihai Vasilescu; Technical assistant: Otilia Cristina Donțu, Logistics and communication support: 

Project summary: The ARTEMIS project represented a complex approach to major environmental issues. ARTEMIS set out to pursue new ways of controlling the population's exposure to polluting agents (present in water and soil), with a major role in the occurrence of cancer, following the evolution of genomic changes, in the population that inhabits NW ROMANIA. 

The proposed consortium was composed of 5 well-known members of the Romanian research community, who intended to develop innovative technologies for the prevention of oncological pathologies, following environmental pollution. 

National Research-Development Institutes, Research Institutes belonging to the  Academy of Medical Sciences and Universities have created collaborations at the  national level in the study of molecular profiles among the affected population  versus the incidence of cancer in areas with a clean environment.

For each of the three types of cancer (lung/thyroid/colorectal), the Institute of Biochemistry of the Romanian Academy, P3 partner,  carried out the selection of protein tumor markers, followed by the selection of peptides derived from them. 

Mass spectrometry of these markers was used from human blood plasma  samples, collected both from cancer patients (lung/thyroid/colorectal)  and from healthy patients (used as control).

The following results were obtained:

(1) enrichment of samples in tumor markers, (2) quantification of unique peptides, (3) improving data on lung, thyroid and colorectal cancers.

Partner 3 succeeded in obtaining new information about the relative abundance of the selected markers in these types of cancer and the applicability of the new method developed in their diagnosis.

III. „The development in oncology of novel radiopharmaceuticals and nuclear techniques for diagnostic imaging and personalized treatment at molecular level”; Acronym: ONCORAD  https://www.nipne.ro/proiecte/pn3/9-projects.html

Contracting Authority: UEFISCDI, Code: PN-III-P1-1.2-PCCDI-2017-0769; Number/Date of the contract:64/PCCDI/31.03.2018

Partner 1 : Institute of Biochemistry/IBAR: starting date/finishing date: 31.03.2018-30.09.2021 

Project value P1: 404.475 RON

Project team P1: Dr.Stefan Szedlacsek: Project manager P1; Dr.Rodica Badea, Asc.Alexandra Maria Bănică, Technical assistant: Catalin Constantin Bica, Technical assistant: Otilia Cristina Donțu, Logistics and communication support.

Objectives: The main objective of ONCORAD consortium is the development of innovative targeted radiopharmaceuticals and nuclear medicine techniques for diagnostic imaging and improved radiotherapy in cancer. 

Results obtained by IBAR, Partner 1:

(1) Production of Sca1, Ly6C Cell Marker Antigens for finalizing the immunization of rabbits producing immune sera reactive to the markers of interest. Coordinator: IFIN-HH together with partner IBAR, (2) Obtaining pure Ly6C and ScaI proteins in sufficient quantities and characterized biochemically to complete the immunizations, but also to characterize the titers of the immune sera

 

Current research directions:

(a) expression, purification and study of the mechanism of action of some proteins involved in synaptic transmission;

(b) creation of new conjugates based on afibodies with a potential therapeutic role in different forms of leukemia;

(c) biochemical study of tau protein acetylation with applicability in neurodegenerative diseases;

(d) study of signaling mechanisms involved in various cancer forms

 

 

Stefan Szedlacsek, Dr.
Stefan Szedlacsek, Dr.

Head of Department

Professor Dr. Stefan Eugen Szedlacsek is the Head of the Enzymology Department at the Institute of Biochemistry of the Romanian Academy. He holds a PhD degree in Biotechnology from Polytechnic University of Bucharest as well as a MSc in Organic Synthesis (Polytechnic University- Bucharest) and MSc in Mathematics (Bucharest University). As a visiting scientist, he performed research in the field of cholesterol metabolism University of Illinois at Urbana-Champaign (USA), where he succeeded to evidence a new pathway in the metabolism of oxysterols. He is an “Alexander von Humboldt“ fellow and worked in Germany, in the Institute of Biochemistry (Kiel University) More...

Adina-Gabriela Puiu, PhD Student
Adina-Gabriela Puiu, PhD Student

Research Assistant

Adina-Gabriela Puiu is a researcher in the Institute of Biochemistry of the Romanian Academy. Adina-Gabriela is currently working in Enzymology in the Enzymology.

Andra-Elena Cosoreanu
Andra-Elena Cosoreanu

Andra-Elena Cosoreanu is a researcher in the Institute of Biochemistry of the Romanian Academy. Andra-Elena is currently working in Enzymology in the Enzymology.

Andrei-Mihai Vasilescu, PhD Student
Andrei-Mihai Vasilescu, PhD Student

Research Assistant

Andrei-Mihai Vasilescu is a researcher in the Institute of Biochemistry of the Romanian Academy. Andrei-Mihai is currently working in Enzymology in the Enzymology.

Bica Constantin Catalin, MSc
Bica Constantin Catalin, MSc

Asistent

Bica Constantin Catalin is a researcher in the Institute of Biochemistry of the Romanian Academy. Bica is currently working in Enzymology in the Enzymology.

Horea Szedlacsek
Horea Szedlacsek

Research Assistant

Horea Szedlacsek is a researcher in the Institute of Biochemistry of the Romanian Academy. Horea is currently working in Enzymology in the Enzymology.

Otilia-Cristina Donțu, Ms.
Otilia-Cristina Donțu, Ms.

Assistant

2008-2012: Bachelor's degree in economics, specialization in accounting and management informatics, Faculty of Economics and Business Administration, "Dunarea de Jos" University of Galati, Romania. 2021 - present: Master' More...

Viorel Enache, MSc.
Viorel Enache, MSc.

Research Assistant

Viorel Enache is a researcher in the Institute of Biochemistry of the Romanian Academy. Viorel is currently working in Enzymology in the Enzymology.

RECENT PUBLICATIONS

 

1.

Synthesis and characterization of a novel [52Mn]Mn-labelled affibody based radiotracer for HER2+ targeting”:  Balázs Váradi,   Károly Brezovcsik,   Zoltán Garda,   Eniko Madarasi,   Horea Szedlacsek,   Rodica-Aura Badea,   Andrei-Mihai Vasilescu,   Adina-Gabriela Puiu,   Aura Ionescu,   Livia-Elena Sima,   Cristian V.A. Munteanu,   Simona Călăraș,   Adrienn Vagner,   Dezső Szikra,   Ngô Minh Toàn,   Tibor Nagy,   Zoltán Szűcs,   Stefan Eugen Szedlacsek,   Gabor Nagy  and  Gyula Tircsó, Inorganic Chemistry Frontiers, Accepted:29 May 2023; First published: 06 Jun 2023; DOI https://doi.org/10.1039/D3QI00356F,

 

2.

 

Designed Peptide Inhibitors of STEP Phosphatase-GluA2 AMPA Receptor Interaction Enhance the Cognitive Performance in Rats":  Szedlacsek HS, Bajusz D, Badea RA, Pop A, Bică CC, Ravasz L, Mittli D, Mátyás D, Necula-Petrăreanu G, Munteanu CVA, Papp I, Juhász G, Hritcu L, Keserű GM, Szedlacsek-SE.; J Med Chem. 2022 Jan;  https://pubmed.ncbi.nlm.nih.gov/34962802/

 

3.

 

Trojan horse treatment based on PEG-coated extracellular vesicles to deliver doxorubicin to melanoma in vitro and in vivo": Patras L, Ionescu AE, Munteanu C, Hajdu R, Kosa A, Porfire A, Licarete E, Rauca VF, Sesarman A, Luput L, Bulzu P, Chiroi P, Tranca RA, Meszaros MS, Negrea G, Barbu-Tudoran L, Potara M, Szedlacsek S, Banciu M; Cancer Biol Ther. 2021;  https://pubmed.ncbi.nlm.nih.gov/34964693/

 

4.

 

Comparative Analysis Between Cellular Oncogenes and Viral Oncogenes”: Puiu, AG; Grigoras, O; Preda, MI; Constantin, M; Vasilescu, AM; Biointerface Research in Applied Chemistry; Review; Volume11; Issue3; Page 9939-9951; DOI10.33263/BRIAC113.99399951; Published JUN 15 2021;  https://www.webofscience.com/wos/woscc/full-record/WOS:000591668100010.

 

5.

 

,,Regulation of TRPM8 Channel activity by Src-mediated Tyrosine Phosphorylation”: Alexandra Manolache, Tudor Selescu, G. Larisa Maier, Mihaela Mentel, Aura Elena Ionescu, Cristian Neacșu , Alexandru Babeș, Ștefan Eugen Szedlacsek. Journal of Cellular Physiology; 2020; https://pubmed.ncbi.nlm.nih.gov/31729029/

 

6.

 

Analysis of EYA3 phosphorylation by Src kinase identifies residues involved in cell Proliferation”:  Aura E. Ionescu, Mihaela Mentel, Cristian V.A. Munteanu, Livia E. Sima, Eliza  C. Martin, Georgiana Necula-Petrareanu; Ștefan E. Szedlacsek. International Journal of Molecular Sciences, Volume 20, Issue 24, 6307; 2019; https://pubmed.ncbi.nlm.nih.gov/31847183/

 

7.

 

Biological and molecular modifications induced by cadmium and arsenic during breast and prostate cancer development”:  Environmental Research; Zimta, AA; Schitcu, V; Gurzau, E; Stavaru, C ; Manda, G; Szedlacsek, S; Berindan-Neagoe, Volume: 178, Article Number: 108700, DOI: 10.1016/j.envres; 2019; https://pubmed.ncbi.nlm.nih.gov/31520827/

 

8.

 

Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate”:  Journal of Structural Biology:Scheidig, AJ; Horvath, D; Szedlacsek, SE , Volume 207, Issue 1, Page 85-102; 2019; https://pubmed.ncbi.nlm.nih.gov/31059775/

 

9.

 

Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR-377/VE-PTP axis”:  Rosca AM, Mitroi DN, Cismasiu V, Badea R, Necula-Petrareanu G, Preda MB, Niculite C, Tutuianu R, Szedlacsek S, Burlacu A. J Cell Mol Med. 22, 4700-4708; 2018; https://pubmed.ncbi.nlm.nih.gov/30044046/

 

10.

 

WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton”:  Mentel M, Ionescu AE, Puscalau-Girtu I, Helm MS, Badea RA, Rizzoli SO, Szedlacsek SE. Sci Rep. 8, 2910; 2018; https://pubmed.ncbi.nlm.nih.gov/29440662/

 

11.

 

Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics“, Petrareanu G, Balasu MC, Vacaru AM, Munteanu CV, Ionescu AE, Matei I, Szedlacsek SE.: Appl Microbiol Biotechnol. 98, 7855-67; 2014; https://pubmed.ncbi.nlm.nih.gov/24740691/ 

 

12.

 

Protein tyrosine phosphatase structure-function relationships in regulation and pathogenesis“: Böhmer F, Szedlacsek S, Tabernero L, Ostman A, den Hertog J., Febs J. 280, 413-31; 2013; https://pubmed.ncbi.nlm.nih.gov/22682070/

 

13.

 

Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis”: Petrareanu, G, Balasu, MC, Zander, U, Scheidig, AJ and Szedlacsek, S.E., Acta Cryst. F66, 805–807; 2010; https://pubmed.ncbi.nlm.nih.gov/20606278/

 

14.

 

Interface Analysis of the Complex between ERK2 and PTP-SL”: Balasu MC, Spiridon   LN, Miron S, Craescu CT, Scheidig AJ, Petrescu AJ, Szedlacsek SE. PLoS One. 4(5), e5432; 2009; https://pubmed.ncbi.nlm.nih.gov/19424502/

 

15.

 

Analysis of Molecular Determinants of PRL-3”:  Pascaru M, Tanase C, Vacaru AM, Boeti P, Neagu E, Popescu I, Szedlacsek SE., J Cell Mol Med. 13(9B), 3141-50; 2009;  https://pubmed.ncbi.nlm.nih.gov/19040419/

 

16.

 

Protein tyrosine phosphatases, structure-function relationships”:Tabernero L, Aricescu AR, Jones EY, Szedlacsek SE; Febs J. 275, 867-82; 2008; https://pubmed.ncbi.nlm.nih.gov/18298793/

 

17

 

Functional, fractal nonlinear response with application to rate processes with memory, allometry, and population genetics” : Vlad MO, Morán F, Popa VT, Szedlacsek SE, Ross J. Proc Natl Acad Sci USA., 104, 4798-803; 2007; https://pubmed.ncbi.nlm.nih.gov/17360340/

 

18

 

Identification and specificity profiling of protein prenyltransferase inhibitors using new fluorescent phosphoisoprenoids”:  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; . J Am Chem Soc. 128, 2822-35; 2006;  https://pubmed.ncbi.nlm.nih.gov/16506760/

 

19

 

Fisher's theorems for multivariable, time- and space-dependent systems, with applications in population genetics and chemical kinetics”: Vlad MO, Szedlacsek SE, Pourmand N, Cavalli- Sforza LL, Oefner P, Ross J;  Proc Natl Acad Sci USA. 102, 9848-53; 2005; https://pubmed.ncbi.nlm.nih.gov/15994224/

 

Compound for inhibition of certain signaling processes related to the evolution of the cognitive processes 2017-2018
Acronym: Ctr.327/27.03.2017/Company CRU SRL, Medical Services Company/ Dunakesz, Hungary
Project director: Stefan Szedlacsek

The main idea of this project was that by inhibiting at least one of these two interactions of GluA2, the internalization of AMPAR will be reduced and therefore the synapse resistance will be increased, thus leading to improved cognitive functions.

Compound for inhibition of certain signaling processes related to the evolution of the cognitive processes
Novel radiolabeled affibodies for targeted imaging and therapy 2019-2022
Acronym: Bilateral agreement no.3698/13.09.2018 Romanian Academy- Hungarian Academy of Sciences
Project director: Stefan Szedlacsek

The project is agreed as a bilateral collaboration between IBAR and ATOMKI, and the University of Debrecen and IFIN-HH participate in this project voluntarily.

Radiolabelling of affibody for tumor diagnostic and theranostic application in the nuclear medicine 2022-2024
Acronym: Bilateral agreement no.2886/15.09.2021 Romanian Academy- Hungarian Academy of Sciences
Project director: Stefan Szedlacsek

The project is agreed as a joint collaboration among IBAR, ATOMKI and UD, the latter being a cost free participant. There are two main directions envisaged by the proposed project: - receptors mapping and therapy, using an affibody against HER2 receptors, combined with an adequate radioisotope. In this respect, the specific objectives are: a) expression and purification of affibodies; b) establishing labelling procedures; c) ex vivo and/or in vivo testing of optimal compounds.

Biochemical and cellular investigations on the potential use of affibodies in leukemia therapy 2022 - 2023
Acronym: LEUKEMIA. Grant no.779/14.06.2022
Project director: Stefan Szedlacsek

Affibody molecules are highly promising therapeutic candidates due to their advantageous features like: small size, efficient delivery, straightforward engineering towards improved formats, site-directed conjugation of payloads, possibility of GMP production by chemical synthesis or inexpensive bacterial production leading to low product costs.

Biochemical and cellular investigations on the potential use of affibodies in leukemia therapy
Protein Tyrosine Phosphatases: Structure, regulation and biological function 2007 - 2011
Acronym: PTPNET
Project director: Stefan Szedlacsek

Project Funded under: Human resources and Mobility in the specific programme for research, technological development and demonstration "Structuring the European Research Area" under the Sixth Framework Programme 2002-2006. PTPNET is a training network for young scientists in the field of protein tyrosine phosphatase (PTP) research.

Reconstruction of Ancestor of Receptor Protein Tyrosine Phosphatase Catalytic 2011 - 2016
Acronym: PTPs
Project director: Stefan Szedlacsek

Proteins are essential players of all biological processes and they are involved in practically every function performed by a living cell.