Enzymatic caracterization and identification of potential native substrates for Eyes absent, protein responsible for Branchio-Oto-Renal syndrome

Enzymatic caracterization and identification of potential native substrates for Eyes absent, protein responsible for Branchio-Oto-Renal syndrome 2006 - 2008
Acronym: BOR
Budget: 247.294 lei
Project director: Stefan Szedlacsek
CNCSIS766 - no.11GR/25.04.2006

The project focuses on characterizing the branchio-oto-renal (BOR) syndrome, an autosomal dominant genetic disorder caused by mutations in the EYA gene. The goal is to enzymatically analyze the conserved C-terminal domain of the Eya3 protein and identify physiological substrates to better understand the molecular mechanisms involved in this syndrome.

The research project focuses on branchio-oto-renal (BOR) syndrome, an autosomal dominant genetic condition first described in 1976, characterized by branchial defects (such as fistulas and cysts), ear abnormalities (resulting in hearing loss ) and various kidney problems (from hypoplasia to bilateral renal aplasia).

The gene responsible for this syndrome, EYA, located on chromosome 8q13.3, shares significant homology with the absent eye (eya) gene in Drosophila. In vertebrates, several isoforms of the EYA gene have been identified that play essential roles in eye development and other biological processes.

 The project aims to provide a detailed enzymatic characterization of the conserved C-terminal domain of the Eya3 protein and to investigate the possibility of dimerization to discover new regulatory elements of the enzyme activity and transcription factor capacity. In addition, the project aims to identify the physiological substrate for the ED domain of the Eya protein. Methods will include gene cloning, expression in prokaryotic and eukaryotic systems, enzyme kinetic characterization and the use of pull-down techniques to isolate substrates. The results will contribute to a deeper understanding of the complex mechanisms by which this enzyme-transcription factor combination leads to BOR syndrome.

Stefan Szedlacsek, Dr.
Stefan Szedlacsek, Dr.

Head of Department

Prof. Dr. Stefan 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 Polytechnical University of Bucharest as well as a MSc in Organic Synthesis (Polytechnical University, Bucharest) and MSc in Mathematics (University of Bucharest). As a visiting scientist, he performed research in the field of cholesterol metabolism at the 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 Biochem More...

Objective 1: Cloning of the gene, its expression in prokaryotic systems, purification to the C-terminal domain of the Eya3 homolog in mouse mEya3(ED). 

Objective 2: Enzyme characterization and determination of kinetic parameters for the mEya3(ED) domain.  

Objective 3. Gene cloning and expression for the integral form of the mEya3 protein in prokaryotic and eukaryotic systems.  

Objective 4: Study of the influence of the N-terminal domain upon the enzymatic activity of the C-terminal mEya3(ED). 

Objective 5: Identification of physiological substrates of mEya3(ED) in vitro.


Project Coordinator: Dr.Ștefan Szedlacsek

1. Bălașu Mihaela,                      Dr.

2. Mențel (Pascaru) Mihaela         PhD Student

3. Petrăreanu Georgiana             PhD Student

4. Badea Rodica                         PhD Student


In the first stage of the project, the optimization of the expression of the mEya3(ED) catalytic domain was achieved. Methods for purifying the fusion protein mEya3(ED) were developed, both with glutathione-S-transferase (GST-mEya3(ED)) and with a sequence of 6 histidine residues (6xHis-mEya3(ED)). The purification of the 6xHis-mEya3(ED) fusion protein was successfully accomplished with a yield of 8mg/l, which allowed for the performance of enzymatic activity studies.

The enzymatic activity of the catalytic domain was tested on two substrates: pNPP, with activity measured in the visible spectrum at 405 nm, at 37°C, for 10 minutes, and DiFMUP, with activity measured by fluorescence with excitation at 360 nm and emission at 450 nm, at 25°C, for 10 minutes. The catalytic constants calculated for the phosphatase activity of the mEya3(ED) domain on pNPP are Km = 11.55 mM, Kcat = 64.42 s⁻¹.

Inhibition tests of the phosphatase activity of the mEya3(ED) domain were performed using 2 inhibitors: sodium orthovanadate and EDTA. Concentrations higher than 7 mM of sodium orthovanadate make the detection of phosphatase activity impossible, while at concentrations lower than 7 mM, a progressive decrease in phosphatase activity is observed with increasing inhibitor concentration.

Inhibition studies showed that EDTA competitively inhibits the enzymatic activity of the mEya3(ED) domain, with a calculated inhibition constant of 89.09 mM.

In the second phase of the project, the hEya3 gene and its C-terminal catalytic domain were cloned from a complementary DNA library.

Expression assays were performed on the newly cloned proteins, with optimal expression achieved in the E. coli BL21(DE)3RIL strain at 30°C with 0.1mM IPTG. The proteins were purified to over 85% purity to allow enzymatic activity studies. This stage included tests to determine the self-dephosphorylation capacity of the full length hEya3 protein or only its catalytic domain, hEya3(ED), using phosphorylated peptides corresponding to amino acids around tyrosines predicted to be phosphorylated. The spectra obtained at 280nm UV and fluorescence between Em 285-345nm did not show self-dephosphorylation of hEya3.

The enzymatic activity of hEya3 and hEya3(ED) was tested on two substrates: pNPP, a classical substrate for protein tyrosine phosphatases, and DiFMUP, a fluorescent substrate.

The kinetic constants calculated for the phosphatase activity of hEya3 and its hEya3(ED) domain showed a 1.5-fold higher catalytic efficiency on pNPP for the full-length hEya3, suggesting that the enzymatic activity on this substrate is not influenced by the N-terminal domain of the enzyme. In contrast, for the fluorescent substrate DiFMUP, there is a significant difference between the two forms of the enzyme. The catalytic domain hEya3(ED) shows no enzymatic activity on this substrate, whereas the full-length hEya3 shows approximately 15 times higher specificity for the fluorescent substrate compared to pNPP. A possible explanation for this finding could be the lack of native conformation of the separately expressed catalytic domain.

In the final stage of the project, inactive mutants (D/A and D/N) of the Eya3 protein were obtained by site-directed mutagenesis.

The inactive mutants were successfully expressed and purified. Tests on 6207 tyrosine-phosphorylated peptides showed that 68 of them are dephosphorylated by Eya proteins with a yield of more than 70%. We therefore have 68 potential substrates for the protein of interest.

Statistical analysis of the data revealed the consensus sequence for Eya substrates: [Ser/Tyr] - [Tyr]- pTyr - Xaa1-2 - [Ser] - [Arg] - [Arg, Phe], where Xaa – can be any amino acid, and in brackets are the preferred amino acids for each position.

International/National Conferences

(1) 24th-28th; july; EUROPHOSPHATASE- Aveiro, Portugal; Poster: „Enzymatic characterization of Eyes absent protein-a non-cysteine dependent protein tyrosine phosphatases"; Authors: Pascaru M., Szedlacsek S. book,  pp 46, ISBN: 9789899550100; 2007.

(2) Annals of West University of Timisoara, Romania; Poster: "Purification and Characterization of mEya3 C terminal region"; Authors:  Pascaru M., Szedlacsek S.;  Series of Chemistry 16(2); 1-266, pag.66, ISSN 1224-9513; 2007


(1) "Enzymatic Characterization of a New Isoform of Human Eyes Absent Homolog 3"; Authors: Pascaru M., Szedlacsek. ROM. J. Biochem., 45,1, P60;  2008