A new tool to map the conformational proteome in cells to reveal the structural plasticity of the glucocorticoid receptor
Chloé Van Leene1,2, Annick Verhee1,2, Tijs Merckaert1,2, Sven Eyckerman1,2, Karolien De Bosscher1,2, Kris Gevaert1,2
1 VIB-UGent Center for Medical Biotechnology, Ghent, Belgium
2 UGent Department of Biomolecular Medicine, Ghent, Belgium
Introduction: The glucocorticoid receptor (GR) is a multi-domain transcription factor with high conformational plasticity. GR is triggered by glucocorticoids, which are steroidal hormones involved in the regulation of cellular stress, immune responses, cellular differentiation and metabolism. Synthetic variants of these glucocorticoids have been derived to combat inflammation and cancer. However, chronic usage hereof leads to side effects and therapy resistance. Structural insights on GR are currently only derived from in vitro analysis of either its ligand- and/or DNA-binding domain. Studying GR’s conformational flexibility in a cellular environment is essential to better understand its function and to identify targeted strategies that favour GR transcriptional programs with beneficial therapeutic effects.
Methods: Limited proteolysis coupled to mass spectrometry (LiP-MS) detects changes in protein conformation. The current LiP-MS technology works on cell or tissue lysates that undergo a first short incubation with proteinase K to introduce cuts in surface-accessible regions. Further and complete digestion with trypsin, comparison with a trypsin-only treated sample and mass spectrometric analysis leads to the identification of protein conformation-specific cuts via semi-tryptic peptides, termed conformotypic peptides. This workflow comes with several limitations such as not working in a native endogenous environment and experimental artefacts. Hence, we aim to develop a tightly controllable LiP workflow in living cells based on a rapamycin-controlled split-protease system.
Preliminary data: Three proteases were chosen as LiP proteases and their respective split fragments were generated. Full protease activity was evaluated in an activity assay. Importantly, no cell death was observed after 48 hours of LiP protease expression, allowing a comfortably large time window to set up the in cellulo LiP system. The reassembly of the protease fragments was validated in vitro and in cellulo. Spontaneous assembly of some split couples indicates the need for an iterative process to identify those split fragments that only reassemble and thereby form an active protease upon adding rapamycin. In future experiments, the activity of the in cellulo reconstituted protease will be tested. Once a controllable LiP protease is established, analysis of in cellulo ligand-induced GR conformotypic peptides via LC-MS/MS will follow. Such peptide patterns will be integrated with corresponding data of GR ligand-specific gene regulatory profiles aimed at finding those structural fingerprints resulting in a desired signalling pathway.
Novel aspect: Studying differences in GR conformation directly in live cells in a reproducible and quantifiable manner.
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