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:
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:
1. Experiment-based design of lead compounds with potential cognitive enhancement effect
The decline of cognitive capacity is one of the most debilitating features of neurodegenerative diseases. To a great extent, this is due to changes in the molecular composition of postsynaptic membranes which in turn leads to reduced synaptic plasticity. Synaptic function depends on synaptic plasticity which can either potentiate or depress information transfer. As a rule, high-frequency stimulation potentiates synaptic activity leading to long-term potentiation (LTP) while low-frequency stimulation depresses synaptic activity leading to long-term depression (LTD). Long term changes in synaptic functions can be induced by activation of NMDA receptors which modify synaptic strength through regulating the number of postsynaptic AMPA receptors (AMPAR). NMDAR activation leads to Ca2+ influx through the receptor coupled ion channel which can initiate either LTP or LTD, depending on the spatiotemporal activation profile. Cognitive impairment and learning ability of the brain is directly linked to synaptic plasticity as measured in LTP changes in animal models of brain diseases.
AMPARs are glutamate-activated ion channels which mediate the fast excitatory ion current underlying information transmission in the brain. An increase in the number of postsynaptic AMPARs leads to increased synaptic strength during LTP, while a decrease in postsynaptic AMPAR number produces LTD. Increased number of AMPAR during LTP can be mediated by both exocytosis of AMPARs and/or lateral diffusion of AMPARs from the peri-synaptic membrane to the synapse. Conversely, LTD leads to AMPAR diffusion away from the synapse and receptor endocytosis.
Post-translational modifications of AMPAR cytoplasmic region like tyrosine phosphorylation were proved to play important role in receptor trafficking and other processes so that a specific phosphorylation pattern of this receptor might be associated with a physiological or pathological state. The main idea of this project is to modulate the phosphorylation state of AMPA receptors in such a manner to favor a physiological functionality of the receptor. Eventually, we aim at identifying lead compounds with potential cognitive enhancement effect.
2. Mapping of tyrosine phosphorylation sites and functional analysis of EYA 3
Eyes absent (eya) proteins are members of a regulatory network of evolutionary conserved transcription factors and cofactors, termed retinal determination gene network (RDGN) in Drosophila, along with twin of eyeless (toy), eyeless (ey), sine oculis (so) and dachshund (dac). From insects to humans, there are correspondent gene families - Pax (for toy and ey), Six (for so), Eya (for eya) and Dach (for dac) - referred to as the PSEDN (Pax-Six-Eya-Dach) network. This network holds important roles in the development and homeostasis of various tissues and organs - eyes, kidneys, nervous system, ears, muscles - as well as in the context of limb formation, gonadogenesis and neurogenesis. Loss of function mutations in the Eyes absent genes can lead to several congenital syndromes, for example cardiofacial syndrome, bronchio-oto-renal syndrome, oto-facio-cervical syndrome, congenital cataract, late onset of deafness. On the other hand, overexpression of Eyes absent has been detected in diverse types of cancers like epithelial ovarian cancer, Wilms’ tumors, lung adenocarcinoma, colorectal cancer, colon cancer, esophageal adenocarcinoma.
Post-translational modifications of EYA proteins may influence their implication in physiological and pathological events. Recently, we have demonstrated and reported that Src kinase phosphorylates human EYA1 and EYA3 and their nuclear and cytoskeletal localization are controlled by Src phosphorylation. In the same time, we have found that EYA1 and EYA3 are capable of autodephosphorylation. We have also shown that Src kinase has phosphorylation sites in both N-terminal and C-terminal domains of EYA3 protein. This data brings into discussion the implication of tyrosine phosphorylation in regulating the physiological activities of eyes absent proteins and potential interacting partners in mammalian cells. Thus, in this project we perform a detailed mass spectrometric analysis of human EYA3 phosphorylation by protein tyrosine kinase Src and analyze whether the phosphorylation sites can be autodephosphorylated. In terms of our future objectives we plan to identify the physiological impact of the phosphorylation of the detected tyrosine residues.
3. Identification of signaling mechanisms involved in tumorigenesis, as a result of EYA3 dephosphorylation of the specific substrate WDR1.
Eyes Absent protein 3 (EYA3), active c-Src, WDR1 as well as tyrosine phosphorylated form of WDR1 have been reported as playing important roles in breast cancer initiation, progression and metastasis. It has been also shown that protein tyrosine phosphatase activity of EYA3 mediates breast cancer cell migration, invasion, transformation and metastasis through an as yet unidentified mechanism. Our recently reported findings indicate that WDR1, a significant mediator of actin cytoskeleton reorganization i) is phosphorylated by active c-Src, ii) is an in vitro cytoplasmic substrate of EYA3 and iii) its dephosphorylation by EYA3 generates major changes to the cellular actin cytoskeleton. The central task of the present project is the in vivo validation of WDR1 dephosphorylation by EYA3 and identification of the downstream pathways. Initially, we will assess how the expression of EYA3, c-Src, WDR1 and the Tyr phosphorylation level of WDR1 correlate with the tumor-promoting effects in several representative breast cancer cell lines, what are WDR1/EYA3 interactors and what molecular mechanism(s) underly the identified correlations and interactions. Moreover, we wish to shed light on how the dephosphorylation of WDR1 by EYA3 is connected to Rho GTPase signaling pathways. Finally, we aim to elucidate the involvement of in vivo dephosphorylation of WDR1 by EYA3 in the tumorigenic properties of breast cancer cells using subcutaneous xenografted mice.
4. Design and laboratory experimentation of a molecular vector based on Holmium166 for re-diagnosis and targeted radiotherapy.
Our project is structured on three distinct panels of experimental development and applied research and has as final result the realization of a functional model of a neutron activator (NA) controlled by a cyclotron, to obtain a radiopharmaceutical based on an antibody coupled to an activated nanoparticle surface; the nanoparticles contain radionuclide that emits β Holmium-166 (t1 / 2 = 26.8 h), a type of radiation proven to be effective for targeted cancer therapy.
· Research Grant 548PED/2020-2022 from UEFISCDI for "Development of a functional model of a neutron activator (NA) controlled by a cyclotron to obtain a radiopharmaceutical based on an affibody coupled to a surface of activated nanoparticles", Partner (P) team, PN-III-P2-2.1-PED-2019-4184;
· Research Grant 35PCCDI/2018-2021 from UEFISCDI for „Genomic mapping of population in area contaminated with radioactivity and heavy metals”, Partner 3, PN-III-P1-1.2-PCCDI-2017-0737;
· Research Grant 64PCCDI/2018-2021 from UEFISCDI for „Development of radio-pharmaceuticals and nuclear technics in oncology for imaging and personalized therapy at molecular level”, Partner 1, PN-III-P1-1.2-PCCDI-2017-0769;
1. Trojan horse treatment based on PEG-coated extracellular vesicles to deliver doxorubicin to melanoma in vitro and in vivo.
Cancer Biol Ther. 2021 Dec 29:1-16. doi: 10.1080/15384047.2021.2003656. Online ahead of print. PMID: 34964693
Designed Peptide Inhibitors of STEP Phosphatase-GluA2 AMPA Receptor Interaction Enhance the Cognitive Performance in Rats.
J Med Chem. 2021 Dec 28. doi: 10.1021/acs.jmedchem.1c01303. Online ahead of print. PMID: 34962802
3.Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation.
Ionescu AE, Mentel M, Munteanu CVA, Sima LE, Martin EC, Necula-Petrareanu
G, Szedlacsek SE.
Int J Mol Sci. 2019 Dec 13;20(24). pii: E6307. doi: 10.3390/ijms20246307.
4. Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation.
Manolache A, Selescu T, Maier GL, Mentel M, Ionescu AE, Neacsu C, Babes
A, Szedlacsek SE.
J Cell Physiol. 2019 Nov 14. doi: 10.1002/jcp.29397. Published: June 2020
5. Biological and molecular modifications induced by cadmium and arsenic during breast and prostate cancer
Zimta AA, Schitcu V, Gurzau E, Stavaru C, Manda G, Szedlacsek S, Berindan-Neagoe I. Environ Res. 2019, Nov;178:108700. doi: 10.1016/j. Review.
6. Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate.
Scheidig AJ, Horvath D, Szedlacsek SE. J Struct Biol. 2019 Jul 1;207(1):85-102.
7. 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 SE, Burlacu A. J.Cell.Mol.Med., 2018, 22(10), 4700-4708
8. 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. 2018 Feb 13;8(1):2910.
9. 20-HETE promotes glucose-stimulated insulin secretion in an autocrine manner through FFAR1.
Tunaru S, Bonnavion R, Brandenburger I, Preussner J, Thomas D, Scholich K, Offermanns S. Nat Commun. 2018 Jan 12;9(1):177.
10. Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis.
Schmitz K, Brunkhorst R, de Bruin N, Mayer CA, Häussler A, Ferreiros N, Schiffmann S, Parnham MJ, Tunaru S, Chun J, Offermanns S, Foerch C, Scholich K, Vogt J, Wicker S, Lötsch J, Geisslinger G, Tegeder I. Acta Neuropathol Commun. 2017 Jun 2;5(1):42.
11. The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity.
Hohmann SW, Angioni C, Tunaru S, Lee S, Woolf CJ, Offermanns S, Geisslinger G, Scholich K, Sisignano M. Sci Rep. 2017 Mar 27;7(1):446.
12. The leukotriene B4 receptors BLT1 and BLT2 form an antagonistic sensitizing system in peripheral sensory neurons.
Zinn S, Sisignano M, Kern K, Pierre S, Tunaru S, Jordan H, Suo J, Treutlein EM, Angioni C, Ferreiros N, Leffler A, DeBruin N, Offermanns S, Geisslinger G, Scholich K. J Biol Chem. 2017 Apr 14;292(15):6123-6134.
13. Expression, Purification, and Kinetic Analysis of PTP Domains.
Mentel M, Badea RA, Necula-Petrareanu G, Mallikarjuna ST, Ionescu AE, Szedlacsek SE. Methods Mol Biol. 2016;1447:39-66.
14. Arachidonic Acid Metabolite 19(S)-HETE Induces Vasorelaxation and Platelet Inhibition by Activating Prostacyclin (IP) Receptor.
Tunaru S, Chennupati R, Nüsing RM, Offermanns S. PLoS One. 2016 Sep 23;11(9):e0163633.
15. Loss of FFA2 and FFA3 increases insulin secretion and improves glucose tolerance in type 2 diabetes.
Tang C, Ahmed K, Gille A, Lu S, Gröne HJ, Tunaru S, Offermanns S. Nat Med. 2015 Feb;21(2):173-7.
16. 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. 2014 Sep;98(18):7855-67.
17. A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera.
Preuss B, Tunaru S, Henes J, Offermanns S, Klein R. Clin Exp Immunol. 2014 Jul;177(1):179-89.
18. Conserved MIP receptor-ligand pair regulates Platynereis larval settlement.
Conzelmann M, Williams EA, Tunaru S, Randel N, Shahidi R, Asadulina A, Berger J, Offermanns S, Jékely G. Proc Natl Acad Sci U S A. 2013 May 14;110(20):8224-9.
19. Protein tyrosine phosphatase structure-function relationships in regulation and pathogenesis.
Böhmer F, Szedlacsek S, Tabernero L, Ostman A, den Hertog J. FEBS J. 2013 Jan;280(2):413-31.
20. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.
Tunaru S, Althoff TF, Nüsing RM, Diener M, Offermanns S. Proc Natl Acad Sci U S A. 2012 Jun 5;109(23):9179-84.
Molecular modeling of a set of peptides that can disrupt the GluA2-Cterm complex with STEP (Complex A) or BRAG2 (ComplexB).
The project is agreed as a joint collaboration among IBAR and ATOMKI, UD and IFIN-HH is a cost free participant. There are two main directions envisaged by the proposed project: receptors mapping and therapy, using an affibody against HER2 receptor, combined with an adequate radioisotope. In this respect, the specific objectives are: a) expression and purification of affibodies; b) establish labeling procedures; c) ex vivo and/or in vivo testing of optimal compounds.
Enzymatic characterization of both mEya3 and hEya1 to identify the similarities and differences between these two proteins with relatively low sequence homology.
Synthesis of the catalytic domain of ancestral PTP and its characterization both in vitro and in vivo.
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.