Enzymology external website

• Research Grant 64PCCDI/2018 from UEFISCDI for „Development of radio-pharmaceuticals and nuclear technics in oncology for imaging and personalized therapy at molecular level” (Partner No. 1)

• Research Grant 35PCCDI/2018 from UEFISCDI for „Genomic mapping of population in area contaminated with radioactivity and heavy metals” (Partner No. 3)

1. Experiment-based design of lead compounds with potential cognitive enhancement effect

• Dr Rodica Badea
• Dr Horea Szedlacsek

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

• PhD student Aura Ionescu

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 PTPs which dephosphorylates given substrates

• Dr Rodica Badea

Protein tyrosine phosphorylation represents only a tiny fraction (~ 0.05%) from the total protein phosphorylation of eukaryotic cells. However, it has emerged as a major regulatory mechanism in signal transduction. Many essential cellular processes such as cell growth, differentiation, adhesion and migration, cell cycle control, gene transcription, angiogenesis as well as regulation of ion channels in nerve transmission are modulated by tyrosine phosphorylation. The perturbation of the balance between tyrosine phosphorylation and dephosphorylation has severe pathological consequences leading to numerous human diseases including cancer, diabetes, immune and neuronal diseases. Therefore, maintaining of the adequate level of protein tyrosine phosphorylation in eukaryotic cells is a process which is tightly regulated by the concerted action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs).

At present, one of the most problematic drawbacks in understanding the involvement of PTPs in regulation of signaling processes remains the poor knowledge of their physiological substrates. Thus, for relatively few cases tyrosine-phosphorylated proteins (pY-proteins) having essential roles in different biological processes, the specific dephosphorylating PTPs are known. Identification of PTPs able to dephosphorylate physiologically important pY residues not only would help to enlighten the complex map of tyrosine phosphorylation-mediated pathways but also would offer new tools for diagnostic and therapy. Thus, the aim of the present project is to develop a new technique for direct identification of PTP(s) that specifically dephosphorylate pY-proteins and to apply this novel procedure to the identification of PTPs which dephosphorylate given tyrosine-phosphotylated proteins of high interest for cell signaling.

4. Molecular pharmacology of GPCRs and oGPCRs

• Dr. Sorin Tunaru
• PhD Student Alexandra Banica

G-protein coupled receptors (GPCRs) are the largest family of plasma membrane proteins in mammals encoded by 800 genes in humans and more than 1000 in rodents. A large number of them are olfactory receptors whereas the rest respond to a diverse spectrum of ligands regulating a plethora of physiological processes. Although intensive research performed by pharma industry and academia led to development of drugs acting on GPCRs, the human genome encodes more than 120 receptor members with unknown ligands and physiological functions, known as “orphan GPCRs” (oGPCRs).

By using a combination of reverse- and forward- pharmacology, complemented by advanced mouse genetic models we aim to understand the biological roles of oGPCRs highly expressed mainly in the nervous system, adipose tissue and pancreatic cells.

Critical for successful identification of the biological roles of oGPCRs are the core technologies we are currently employing and further expanding. They include, but are not limited to:

• Functional screening of ligands of interest/ligand libraries. It consists of several assays to determine the activation of heterologously expressed oGPCR after ligand stimulation and it uses the G-protein dependent/independent modulation of intracellular signaling pathways. The usual read-outs are intracellular calcium, cAMP or beta-arrestin translocation.
• Receptor characterization includes methods designed to define receptor’s pharmacological profile: agonist/antagonist identification, intracellular signaling pathway, desensitization mechanisms, potential therapeutic properties.
• Functional siRNA screening. This is a powerful technology originally developed by us at the Max Planck Institute of Heart and Lung Research (Tunaru et al 2012, 2016, 2018) with the aim to overcome limitations of reverse & forward pharmacology. It includes screening of ligand libraries for cellular effects followed by receptor identification with pools of siRNA against all known and putative GPCRs.

Mouse genetics. To understand the biological role of selected oGPCRs we are currently developing advances mouse genetics in collaboration with Max Planck Institute for Heart and Lung Research. These models include generation of mice lacking the receptor gene, in tissue- specific and -conditional manner. Also, to obtain valuable information regarding receptor expression, we plan to generate genetic reporter mouse models, in which fluorescent proteins are expressed under the endogenous promoter of the oGPCR.

5. Investigations on the structure-function relationship in TRPM8 channel (in collaboration with Professor A. Babes – University of Bucharest

• Viorel Enache
• PhD student Alexandra Manolache (Bucharest University and Institute of Biochemistry)

Transient receptor potential melastatin (TRPM) channels are a subfamily of transient receptor potential (TRP) family of ion channels with a high genetic and functional diversity. TRPM8 is a polymodal receptor-channel expressed in primary afferent sensory neurons and also in non-neuronal tissue. It is activated by cooling and plant-derived compounds (menthol, eucalyptol, camphor) and modulated in a voltage channel-dependent manner. Its main sensory role is to transduce innocuous cooling, being widely accepted to be a major sensor of environmental cold temperature. It is also involved in a variety of pathological pain states, including neuropathic and inflammatory hypersensitivity to cold.  Recent reports have shown that TRPM8 is necessary to the initiation and progression of tumors and the abnormal expression of TRPM8 was found in various tumor forms. Therefore, TRPM8 is considered one of the most promising novel therapeutic targets in cancer therapy. The activity of TRPM8 is regulated by various intracellular messenger molecules and signaling pathways. Thus, it is reasonable to assume that post-translational modifications could play a significant role in regulation of TRPM8 activity.

Our project intends to investigate the effect of post-translational modifications of TRPM8 on channel function, to identify the molecular determinants involved in this process and to evaluate the structural impact of this post-translational modification.

Financial resources:

1. Research Grant 64PCCDI/2018 from UEFISCDI for „Development of radio-pharmaceuticals and nuclear technics in oncology for imaging and personalized therapy at molecular level” (Partner No. 1)
2. Research Grant 35PCCDI/2018 from UEFISCDI for „Genomic mapping of population in area contaminated with radioactivity and heavy metals” (Partner No. 3)

RECENT PUBLICATIONS      

1.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.

2. 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. [Epub ahead of print]

3. Biological and molecular modifications induced by cadmium and arsenic during breast and prostate cancer

    development.

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.

4.    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.

5.    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

6.      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.

7.      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.

8.      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.

9.      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.

10.  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.

11.   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.

12.    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.

13.  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.

14.  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.

15.  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.

16.  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.

17.  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.

18.  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.

• Alexandra Manolache, Tudor Selescu, G. Larisa Maier, Mihaela Mentel, Aura Elena Ionescu, Cristian Neacsu, Alexandru Babes, Stefan Eugen SzedlacsekAlexandra Manolache et al . Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation. Journal of Cellular Physiology, 2020, 235(6): 5192-5203IF=4.52
• Ionescu AE, Mentel M, Munteanu CVA, Sima LE, Martin EC, Necula-Petrareanu G, Szedlacsek SEIonescu AE et al . Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation. International Journal of Molecular Sciences, 2019, 20(24): 6307IF=4.18
• Scheidig AJ, Horvath D, Szedlacsek SE.Scheidig AJ et al . Crystal structure of a xylulose 5-phosphate phosphoketolase. Insights into the substrate specificity for xylulose 5-phosphate. Journal of Structural Biology, 2019, 207(1): 85-102IF=3.75
• Rosca AM, Mitroi DN, Cismasiu V, Badea R, Necula-Petrareanu G, Preda MB, Niculite C, Tutuianu R, Szedlacsek S, Burlacu ARosca AM et al . Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR-377/VE-PTP axis. Journal of cellular and molecular medicine, 2018, 22(10): 4700-4708IF=4.66
• Mentel M, Ionescu AE, Puscalau-Girtu I, Helm MS, Badea RA, Rizzoli SO, Szedlacsek SEMentel M et al . WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton. Scientific reports, 2018, 8(1): 2910
• Mentel M, Badea RA, Necula-Petrareanu G, Mallikarjuna ST, Ionescu AE, Szedlacsek SEMentel M et al . Expression, Purification, and Kinetic Analysis of PTP Domains. Methods in molecular biology (Clifton, N.J.), 2016, 1447: 39-66
• Tanase CA. Histidine domain-protein tyrosine phosphatase interacts with Grb2 and GrpL. PloS one, 2010, 5(12): e14339IF=4.41
• Pascaru M, Tanase C, Vacaru AM, Boeti P, Neagu E, Popescu I, Szedlacsek SEPascaru M et al . Analysis of molecular determinants of PRL-3. Journal of cellular and molecular medicine, 2009, 13(9B): 3141-50
• Thummel R, Li L, Tanase C, Sarras MP, Godwin ARThummel R et al . Differences in expression pattern and function between zebrafish hoxc13 orthologs: recruitment of Hoxc13b into an early embryonic role. Developmental biology, 2004, 274(2): 318-33
• Zhang J, Bai S, Tanase C, Nagase H, Sarras MP JrZhang J et al . The expression of tissue inhibitor of metalloproteinase 2 (TIMP-2) is required for normal development of zebrafish embryos. Dev Genes, 2003, 8(213): 382-9