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Bacterial tyrosine kinases: a new class of enzymes as innovative therapeutic targets – BACTYRKIN

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Submission summary

Protein phosphorylation-dephosphorylation plays a dominant role in signal transduction networks. However, for many years, protein phosphorylation-dephosphorylation was considered a cell's regulatory arsenal that was exclusive to eukaryotes and it was only after a long period of controversy that the existence of this modification was documented in bacteria. More precisely, concerning tyrosine-kinases, bacteria were thought to be devoid of these enzymes and this persistent tenet was only ruled out in 1997. Since then, a number of bacterial tyrosine-kinases have been identified and characterized in various bacteria, leading thus to the emerging picture that protein phosphorylation on tyrosine is an important regulatory arsenal of bacterial physiology. However, most of bacterial tyrosine-kinases known to date are unrelated to their eukaryotic counterparts and their catalytic mechanisms differ considerably, rendering thus these enzymes hardly predictable. Therefore, it seems that bacteria have developed their own idiosyncratic type of authentic tyrosine-kinases. By doing so, searching for new bacterial tyrosine-kinases and phosphorylation targets should contribute not only to understand better this type of enzyme, but also to access their importance in the life cycle of bacteria, and especially pathogens. One promising venue in this respect is the search for specific inhibitors of this type of enzymes. They present ideal targets for drug development to interfere, slow down and stop the growth of certain bacteria. Hence, it is interesting to develop an integrative approach combining biochemistry, molecular biology, bioinformatics and molecular modelling firstly, toward the characterization of bacterial tyrosine-kinases and their role in the bacterial cell and then, toward the design of drugs to modulate their activity. This is the central theme of this project that can be divided in two main work packages. The first one will consist in extending our initial studies to identify and characterize novel bacterial tyrosine-kinases and their substrates or partners in E. coli grown in several environmental conditions. For this, we propose to perform a systematic approach combining the use of radioactive phosphate, 2D-electophoresis, phosphoaminoacides analysis and mass spectrometry. In addition, bioinformatic analyses will be simultaneously performed to corroborate the experimental data. Then, once identified, and depending on the nature and the function of each phosphoprotein, specific tests will be carried out to measure the effect of phosphorylation on phosphorylated proteins activity. This will notably require the identification of the phosphorylation sites of the targeted substrates. Finally, genomic mutants and protein derivatives will be generated to check how identified tyrosine-kinases and their substrates affect bacterial life cycle. The second work package will concern processing of our biological data to bring new hypothesis with regard to the functioning of tyrosine-kinases and to design drugs. For this, we will develop bioinformatic expert tools to establish common features important for tyrosine-kinases activity. Then, we will perform tyrosine-kinases modelling to design drugs in order to target and affect their activity. This will be based on the work done for eukaryotic protein kinases in which inhibitors are modified peptides with a moiety that can mimic the ATP molecule, the peptide part having a great affinity and specificity for the protein kinase. Then, we will access the potential of the designed drugs to influence the development of E. coli and other pathogenic bacteria. Drugs will be first used to analyze in vitro their effect on tyrosine-kinases, and, depending on the metabolic function of the tyrosine-kinases and substrates, in vivo experiments will be carried out to check the drugs effect on the growth of certain bacteria.

Project coordination

Christophe Grangeasse (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 200,000 euros
Beginning and duration of the scientific project: - 48 Months

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