Protein biochemistry and proteomics

Leader of the research program: Marek Šebela and the respective department

The research program includes four basic directions of research activities, which are based on new instrumentation and established methodologies. The research on proteomes of plant organelles, parasitic fungi and oomycetes will mainly utilize mass spectrometry. There is very limited information available about the majority of plant nuclear proteins, although they are related to DNA function. In order to discover and characterize new plant nuclear proteins and their possible interactors, we will employ multidisciplinary approaches including proteomics, biochemistry and cell biology. Planned investigations on fungi and oomycetes will involve MALDI mass spectrometric profiling of surface proteins and their identification. This should allow identification of new protein biomarkers that might become useful for an early diagnosis of infection in cultivated plants. Research in structural biology will be focused on structure-function analyses of plant nucleoside hydrolases, which catalyze the cleavage of the N-glycosidic bond in nucleosides. It has been suggested that they are involved in the metabolism of nucleic acid bases and cytokinins. Another point of our interest is represented by plant aldehyde dehydrogenases acting on aldehyde substrates. Our major interest resides in solving the crystal structures, understanding the processes of catalysis and analyzing the expression profiles of the coding genes. Phytochemistry research will be focused on aromatic cytokinins, a group of important phytohormones. Feeding experiments with radiolabeled precursors and hypothetical intermediates followed by immunoaffinity extraction, liquid chromatography and mass spectrometry may uncover their biosynthetic pathways in plants. Histidine kinases from poplar will be analyzed using several approaches including cloning, expression and computational chemistry. Research in plant proteomics will include affinity purification of proteins with immobilized derivatives of plant hormones and their analogues (chemical proteomics) and development of analytical methods for the study of protein phosphorylation induced by selected phytohormones. Initial experiments in chemical proteomics will be conducted with immobilized ligands derived from enzyme inhibitors. To search for specific protein interactors of the compounds, protein extracts of plant origin will be separated on affinity sorbents, divided by fractioned elution and analyzed by proteomic approaches. Protein phosphorylation in plants will be studied by the means of standardized protocols for isolation, identification and quantification of proteins and peptides.