Reconstruction of Ancestor of Receptor Protein Tyrosine Phosphatase Catalytic

Reconstruction of Ancestor of Receptor Protein Tyrosine Phosphatase Catalytic 2011 - 2016
Acronym: PTPs
Budget: 1.499.990 lei
Project director: Stefan Szedlacsek
PN-II-ID-PCE-2011-3-0743

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

The present project aims to synthesize the ancestral catalytic domain of PTPs as corresponding to the derived sequence, to characterize it by a multitude of in vivo and in vitro procedures and to offer in this way new information regarding the evolution of the PTPs and their role in the ancient world.

We expect that the sequence of the ancestor PTP together with its enzyme kinetic characteristics, in vitro and in vivo substrate specificity profiles will have a major impact on understanding how PTPs evolved from an ancestral form to the existing representatives.

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...

  1. Synthesis of an ancestor of receptor protein tyrosine phosphatase catalytic domain (PTPAD1). Preparation of mutant and truncated forms of PTP-AD1;
  2. Enzyme kinetic characterization of PTP-AD1 and derived forms on a synthetic PTP substrate;
  3. Evaluation of substrate specificity of PTP-AD1 and its derived forms using microarray profiling on pTyr-containing peptides;
  4. Evaluation of catalytic efficiency and substrate specificity of PTP-AD1 and its  derived forms on cellular pTyr-containing proteins.

Dr. Stefan Szedlacsek, Romanian Head of Project

Postdoctoral researcher: Mihaela Mențel

Postdoctoral researcher: Georgiana Petrăreanu

PhD Student: Rodica-Aura Badea

Msc: Aura-Elena Ionescu

Student: Ioana Iancu

1. The sequence of the catalytic domain of the ancestral receptor-type protein tyrosine phosphatase (PTP-AD1) has been deduced by phylogenetic analysis. It shows all the conserved motifs characteristic of PTPs and the vast majority of the secondary structure elements of this class of enzymes. The nucleotide sequence was synthesised and cloned into a prokaryotic expression vector.

2. Three truncated constructs and seven constructs with point mutations for PTP-AD1 were obtained and the corresponding proteins were purified to a high degree of purity. The proteins were then used to study phosphatase activity and to assess substrate specificity.

3. Kinetic analysis indicated that the ancestral protein and derived forms have phosphatase activity. PTP-AD1 and truncated constructs show a pH profile similar to most classical PTPases, with the highest catalytic activity at pH 5

4. The catalytic activity of all the AD1 variants studied is lower than that of PTP-AD1 WT, with the exception of the AD1-TK mutant, whose kcat/Km value is 2 times higher than that of the native protein.

5. Tests performed on a library of about 6000 phosphorylated peptides showed that the ancestral AD1 catalytic domain has a very high catalytic activity and a broad substrate specificity.

6. Point mutations in the substrate specificity region (KNRY motif) as well as C-terminal truncations lead to altered substrate specificity and catalytic efficiency. The AD1 proteins most active on phosphopeptides were also the most active on cellular phosphoproteins. Our results suggest that the evolution of PTPs from a common ancestral form to current forms has occurred through point mutations and truncations of the ancestral form sequence.

7. Overexpression of PTP-AD1 decreases the viability (at 72h) and proliferation (72 and 96h) of breast cancer cells. Overexpression of the ancestral PTPase reduces the degree of phosphorylation of phosphorylated proteins in human melanoma cells.  

 

 

Articles:

(1) Böhmer F, Szedlacsek S, Tabernero L, Ostman A, den Hertog J., “Protein tyrosine phosphatase structure-function relationships in regulation and pathogenesis”. FEBS J. 2013 Jan; 280(2):413-31. doi: 10.1111/j.1742-4658.2012.08655.x.; (2012) 

(2) Petrareanu G.,The molecular and cellular characterization of a non-receptor protein tyrosine phosphatase”, Rom. J. Biochem., 50, (2013); 

(3) Mentel M., Iancu I., Kibedi Szabo C. Z., Munteanu C.V.A., Szedlacsek S., “Co-expression of human wdr1 gene with a chaperone increases its protein solubility”, Rom. J. Biochem., 50, 1, 39–51 (2013)

Conference: 

Mentel M., Iancu I., Ionescu A.E., Kibedi Szabo C. Z., Munteanu C.V.A., Szedlacsek S.; "Identification of highly efficient substrate-trapping mutant of eyes absent tyrosine phosphatase”, Oral presentation at International Conference of RSBMB, June 5-6, 2014, Baile Felix, Oradea, Romania (2014)

Poster presentations:

(1) “Effects of site-directed mutagenesis on the enzymatic activity of non-receptor protein tyrosine phosphatase PTPD1” (Meeting abstracts-poster); FEBS Journal Special Issue: 22nd IUBMB & 37th FEBS Congress, Seville, 2012; ISSN/ISBN 1742-464X

(2) “Receptor-like protein tyrosine phosphatase mu dephosphorylates and interacts with phospholipase C gamma 2” (Meeting abstract-poster); FEBS Journal Special Issue: 22nd IUBMB & 37th FEBS Congress, Seville, 2012; ISSN/ISBN 1742-464X

(3) “Identification of potential phosphorylation sites of actin-interacting protein 1” (Meeting abstract-poster) FEBS Journal Special Issue: 22nd IUBMB & 37th FEBS Congress, Seville, 2012; ISSN/ISBN 1742-464X;

(4)The presentation of a poster entitled „Mass Spectrometric investigations regarding phosphorylation of human Eya3 by Src kinase”  (Meeting abstract-poster); Europhosphatase; Phosphorylation switches and cellular homeostasis; Authors: Aura Elena Ionescu, Mihaela Menţel, Cristian V. A. Munteanu, Cristian Marian Butnaru, Rodica Aura Badea, Ioana Iancu, Ştefan Eugen Szedlacsek), at the EMBO Conference - Turku, Finland; 2015