摘要详情

ID / 提交时间

565 / 2019-03-20 18:01:42

标题

Identification of novel downstream effectors of Arabidopsis thaliana SHAGGY-like kinases by means of thermal proteome profiling

关键字

SHAKKY-like kinase,Thermal Proteome Profilling

主题及专题

全体主题 > Omics& New Technology

状态

摘要录用

作者

QING LU / 1Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; 2Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium

Yonghong Zhang / 1Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; 2Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium

Wim Dejonghe / 1Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; 2Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium

Daniel Martinez Molina / 3Pelago Bioscience AB, 171 48 Solna, Sweden

Jarne Pauwels / 4Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium; 5Department of Biochemistry and Microbiology, Ghent University, 9052 Ghent, Belgium

Francis Impens / 4Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium; 5Department of Biochemistry and Microbiology, Ghent University, 9052 Ghent, Belgium; 6VIB Proteomics Core, Ghent, Belgium.

Kris Gevaert / 4Center for Medical Biotechnology, VIB, 9052 Ghent, Belgium; 5Department of Biochemistry and Microbiology, Ghent University, 9052 Ghent, Belgium

Eugenia Russinova / 1Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; 2Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium

摘要

Chemical genetics is a powerful approach for basic research that takes advantage of the complexity of small-molecule-protein interactions to explore various biological processes. However, the identification of small-molecule targets remains a major challenge. Cellular thermal shift assay (CETSA) is a label-free biophysical assay, that facilitates the direct assessment of small-molecule target engagement in intact cells. CETSA combined with quantitative mass spectrometry, termed thermal proteome profiling (TPP), enables the direct monitoring of small-molecule-target protein interactions and the mapping of components of signalling pathways at the proteome-wide scale. Here, we adapted the TPP to intact plant cells by using the known Arabidopsis thaliana SHAGGY-like kinase (AtSK) inhibitor bikinin. First, we found that bikinin stabilizes almost all of its direct targets, demonstrating the potential of CETSA for target identification in Arabidopsis. Second, we detected 62 proteins, including known AtSK interactors, that showed significant thermal stability shifts. The protein-protein interaction network (https://string-db.org) analysis revealed putative novel AtSK interactors that are now being validated. Collectively, the data suggest that TPP is a powerful method for the identification of small-molecule targets and for the discovery of new signalling pathway components.