Venturing into terra incognita of protein small molecule interactions using combination of novel and classical approaches
“The charm of fishing is that it is the pursuit of what is elusive but attainable, a perpetual series of occasions for hope”. —John Buchan
Arabidopsis (plant and cell cultures), tobacco and yeast are used as starting material for the biochemical and –omics analysis resulting in protein-metabolite interaction networks and identifications of the ligand-receptor pairs. Yeast photo was reproduced from WIKIPEDIA (taken by Mogana Das Murtey and Patchamuthu Ramasamy)
For us, the most interesting interactions are between ligand and receptor, the triggers of signaling events. While the number of small molecules with proven or proposed regulatory roles is rapidly growing, the lion's share of their protein receptors remains unknown. Conversely, there are numerous signalling proteins with predicted ligand-binding domains, the identity of the small-molecule counterparts of which remains elusive. The identification of such regulatory PMIs stands at the core of this project. In this research we use Arabidopsis (cell cultures and plants), tobacco and yeast; our technological platform, however, is applicable to other organisms.
Recently we developed simple yet effective in vivo method for global detection of small molecules bound to proteins guided by co-fractionation. Through combination of size filtration with size exclusion chromatography (SEC) our protocol facilitates the unbiased prediction of protein-metabolite interactions in a cell-wide manner without the need of protein or metabolite baits. A map of the protein‒metabolite complexes obtained from SEC experiments assisted in the selection of novel PMIs, now being examined by targeted approaches.
Team and Scientific Interest
Dr. Aleksandra Skirycz (PI) leads the project.
Dr. Monika Kosmacz (Post-Doc) is interested in 3’ 5’-cAMP and 2’ 3’-cAMP signaling.
Dr. Ewelina Sokolowska (Post-Doc) looks into pipecolic acid signaling/regulation.
Dr. Juan Camilo Moreno Beltran (Post-Doc) is interested in dipeptides signaling/regulation.
Dr. Michal Gorka (Post-Doc) is in charge of the proteomics platform and data analysis.
Izabela Kasprzyk (PhD) is interested in dipeptides signaling/regulation.
Marcin Luzarowski (PhD) expands SEC approach to Arabidopsis ecotypes and yeast.
Si Wu (PhD) uses GWAS to decipher dipeptide metabolism.
Techniques
• Mass spectrometry based metabolomics
• Mass spectrometry based proteomics
• Size exclusion chromatography for the cell wide predictions of protein-metabolite complexes
• Affinity chromatography for fishing out protein partners of the small molecules immobilized on the agarose resins
• Tandem affinity chromatography for fishing out small molecules partners of tagged proteins
• CETSA/Thermal Proteome Profiling
• Micro-scale thermophoresis
Possible applications
The identification of heretofore unknown PMIs carries the potential to revolutionize the existing knowledge of small-molecule signaling. In a manner analogous to biomedical research, this knowledge could be used to modulate signaling pathways, and hence plant physiology, using chemicals designed to target protein receptors. Technological pipeline developed in the lab is directly transferable to drug and herbicide research (see e.g. Skirycz et al., 2016; Trends in Biotechnology)
Publications
Affinity purification with metabolomic and proteomic analysis unravels diverse roles of nucleoside diphosphate kinases. Journal of Experimental Botany 68 (13), pp. 3487 - 3499 https://doi.org/10.1093/jxb/erx183
Veyel D, Kierszniowska S, Kosmacz M, Sokolowska EM, Michaelis A, Luzarowski M, Szlachetko J, Willmitzer L, Skirycz A. (2017) System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants. Sci Rep. 7:42387. doi: 10.1038/srep42387.
Skirycz A, Kierszniowska S, Méret M, Willmitzer L, Tzotzos G. (2016) Medicinal Bioprospecting of the Amazon Rainforest: A Modern Eldorado? Trends Biotechnol. 34:781-90. doi: 10.1016/j.tibtech.2016.03.006.