Protein Tyrosine Phosphatases: Structure, regulation and biological function

The overarching, scientific objective of the Network Programme is to provide fundamental advances in our understanding of the molecular, cellular and tissue functions of protein tyrosine phosphatase (PTP) enzymes, under normal cellular conditions and in diseased states.  A diverse range of PTPs will be studied, including receptor-like and cytoplasmic enzymes, and multidisciplinary approaches will be brought to bear. Our ultimate goal is to bring the field to the point where there is a real possibility of progressing into translational research on a broad front, applying knowledge to PTP-based therapeutics.

There are four main scientific objectives of the Consortium:

Objective 1 - Understanding the Structural Basis of PTP signalling

Objective 2 - Understanding the Mechanisms and Dynamics of PTP Regulation

Objective 3 - Understanding the Role of PTPs in Development and Disease

Objective 4 - Development of Novel Bioinformatics Resources for the PTP Research Community

 4.  Other additional information

Ø  Teams in Sweden and Germany studied how chemicals known as reactive oxygen species (ROS) can control PTPs. ROS are produced under many disease conditions including cancer and neurodegeneration;

Ø  French team discovered the role for a PTP in cells that make myelin - the "insulator" of nerve fibres. Myelin is damaged in multiple sclerosis and there is great interest in developing ways to repair it; 

Ø  Swiss industrial team also researched multiple sclerosis and documented several new PTPs as potential therapeutic targets. This work thus provided important new knowledge with translational potential;

Ø  Other teams discovered new roles for PTPs in both brain tumours and leukaemia, and a Dutch team completed the first ever screen of PTP gene function in zebrafish;

Ø  Finally, Manchester team successfully delivered the web resource PhosphaBase, a publically accessible database and research platform for studying PTPs across hundreds of organisms. 

This is a major success for the Network,leaving a positive and lasting legacy.

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 I.   Experimental

• Both the full-length hEya3 gene and the C-terminal domain of hEya3(ED) were cloned using a cDNA library;
• The experiments using the phosphorylated peptides could not demonstrate that hEya3 protein can autodephosphorilate at the sites we proposed;
• The enzymatic activity measured for both hEya3 full-length and hEya3ED on pNPP revealed that N-terminal domain of hEya3 has no influences on its phosphates activity;
• A big difference was observed when the enzymatic activity was measured on DiFMUP. The hEya3(ED) didn’t exhibit any activity, possible because of the lack of the native conformation;
• We report for the first time that a full-length Eye-absent protein exhibits enzymatic activity. hEya1 activity appears to be, in preliminary assays, inhibited by high concentration of pNPP;
• Investigating PLCγ2 as a potential substrate of RPTPμ, protein pull-down experiments with a series of mutant forms of RPTPm confirmed that these two proteins interact and can be efficiently co-precipitated from cells;
• The catalytic domain of RPTPμ has been shown to dephosphorylate both the pY753 and pY759 sites of PLCγ2, which may have physiological significance because these residues are involved in activation of PLCγ2. The data strongly suggest that PLCγ2 is a bona fide substrate of RPTPμ.

II.  Organization of workshops/courses: Institute of Biochemistry (IBI) organized the FEBS Advanced Course "Recombinant DNA Technology and Protein Expression" held in Bucharest between September 2008.

·         The ESR from the Institute of Biochemistry (Partner #4) and a member of the team from Jena University Hospital (Partner #4) attended this practical and theoretical course.

III.    Relevant training for the ESR trainee (Sujay Turuvekere Mallikarjuna) 

·        Participation and presentation of research results in the PTPNET meeting held in Manchester, between June 13-18, 2008

·   Participation in training course “Bioinformatics and Crystallography Workshop” held in Manchester between June 13-18, 2008 and in the FEBS Advanced Course "Recombinant DNA Technology and Protein Expression" held in Bucharest, bewteen June 13-18, 2008

·        Secondment in University of Oxford (Partner #10), between July 1- July, 31 (2008)

Published papers:

1. Protein tyrosine phosphatases: structure-function relatuionships; authors: Tabernero, L., A. R. Aricescu, E. Y. Jones, and S. E. Szedlacsek; Volume 275; Issue 5; Page 867-882; DOI10.1111/j.1742-4658.2008.06251.x; Febs Journal; 2008; https://www.webofscience.com/wos/woscc/full-record/WOS:000253041600005; 

2. Interface analysis of the complex between ERK2 and PTP-SL; authors: Balasu MC, Spiridon LN, Miron S, Craescu CT, Scheidig AJ, Petrescu AJ, Szedlacsek, SE; Volume 4; Issue 5; DOI10.1371/journal.pone.0005432; Plos One; 2009; https://www.webofscience.com/wos/woscc/full-record/WOS:000265933800007; 

3. Large scale  mammalian  expression and purification of protein tyrosine phosphatase PTPBR7; authors: Sujay Turuvekere Mallikarjuna, Stefan E. Szedlacsek; ROM.J.BIOCHEM., 49, 1, 13–20; 2012 ; http://journal.biochim.ro/archive/n49-1/pdfs_49-1/rjb49-1_02.pdf;.

4. Identificationof protein tyrosine phosphatase substrates; authors: Mallikarjuna, S.T.; Szedlacsek, S.E.; ROM.J.BIOCHEM., 49, 1/2012; https://www.cabidigitallibrary.org/doi/pdf/10.5555/20123292962

5. Protein tyrosine phosphatase structure-function relation ships in regulation and pathogenesis; authors: Bohmer, F., Szedlacsek, S., Tabernero, L., Ostman, A., den Hertog J., Volume 280, Issue 2, Page: 413-431; DOI10.1111/j.1742-4658.2012.08655.x1; Febs Journal; 2013; https://www.webofscience.com/wos/woscc/full-record/WOS:000313906000007;

Book Chapter:

1. Expression, Purification, and Kinetic Analysis of PTP Domains; authors: Mentel, M; Badea, RA; Necula-Petrareanu, G; Mallikarjuna, ST; Ionescu, AE; Szedlacsek S.E,; Volume1447; Page39-66; DOI10.1007/978-1-4939-3746-2_3; 2016; Book Series Methods in Molecular Biology;  https://www.webofscience.com/wos/woscc/full-record/WOS:000399045600004;

Poster presentations

29-31 may 2008: Bucharest, Romania; The Annual International Conference of RSBMB; „Enzymatic Characterization of a New Isoform of Human Eyes Absent Homolog 3”; Authors: Mihaela Pascaru; Stefan E. Szedlacsek.

23-31 august 2008: Osaka, Japan; Congress of the International Union of Ctrystallography; „Structure-function analysis of Eyes absent proteins – aspartate dependent protein tyrosine phosphatases”; Authors: Mihaela Pascaru, Stefan E. Szedlacsek.

14-18 July 2009: Egmond aan Zee; Netherland; The Europhosphatase Conference; „New evidence for PLC γ2 as a potential substrate of RPTP μ”; Authors: Rodica Badea; Mihaela Mentel Pascaru; Stefan Szedlacsek.

14-18 July 2009: Egmond aan Zee; Netherland; The Europhosphatase Conference; „Expression and crystallization trials of FERM and KIND domain of PTPL1/BL”; Authors: Sujay Mallikarjuna, Mihaela Mentel, Stefan Szedlacsek.

Oral presentations:

13-18 june 2008: Manchaster; UK; Seminar/Workshop; Oral presentation: Stefan Szedlacsek: „Purification and crystallization of proteins during the PTPNET-Marie-Curie training network: „Bioinformatics and Crystallography”.

8-14 september 2008: Bucharest, Romania; „Prokariotyc expression, purification and crystallization trials of FERM domain of PTP-BL/PTP-L1” at FEBS Advanced Theoretical and Practical Course: RECOMBINANT DNA TECHNOLOGY & PROTEIN EXPRESSION: Oral presentation : Sujay Turuvekere Mallikarjuna and Stefan Szedlacsek

10 october 2008: Bucharest, Romania; Institute of Biochemistry: „Prokariotyc expression, purification and crystallization trials of KIND and FERM domain of PTP-BL/PTP-L1”; Oral presentation: Sujay Turuvekere Mallikarjuna and Stefan Szedlacsek.

21-24 September 2010: Bucharest, Romania; Institute of Biochemstry; The Diaspora Conference; „Trends in medical genomics and proteomics”; Oral presentation; „Back in time to the origins of protein tyrosine phosphatases”; Stefan Szedlacsek