Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets

Moortel, Laura Van; Thommis, Jonathan; Maertens, Bert; Staes, An; Clarisse, Dorien; Sutter, Delphine De; Libert, Claude; Meijer, Onno C.; Eyckerman, Sven; Gevaert, Kris; Bosscher, Karolien De

doi:10.1016/j.biopha.2022.113218

Cited by 11 publications

(11 citation statements)

References 44 publications

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“…S2b). At these near-physiological expression levels, we also observed a similar upregulation of well-known anti-inflammatory GR target genes ( TSC22D3 and DUSP1 ) 22 upon dexamethasone supplementation, a potent GR agonist, indicating both cell lines still trigger activation of the same downstream targets and show a comparable GR-dependent transcriptional transactivation (Fig. S2c).…”

Section: Resultssupporting

confidence: 63%

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Delhaye,

Moschonas,

Fijalkowska

et al. 2023

Preprint

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SummaryProtein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods, and has quickly become the current state-of-the-art in the field. Nevertheless, tight control of proximity labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein-of-interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split-link design, we applied it to identify protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field.MotivationIn proximity labeling proteomics protein-protein interactions are identified byin vivobiotinylation. However, the current lack of a universally applicable negative control for differential analysis affects accurate mapping of the interactome. To bridge this gap, we conceptualized a system based on the T2A self-cleaving peptide to match expression levels between control and bait protein setups while using the same bait protein. In addition, we implemented a versatile modular cloning system to build mammalian expression vectors for, but not limited to, proximity labeling assays.

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Section: Resultssupporting

confidence: 63%

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Delhaye,

Moschonas,

Fijalkowska

et al. 2023

Preprint

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“…However, despite extensive research, none of the lead SEGRAMs reached the clinic (reviewed in (28)). In a recent study, we characterized a panel of well-known SEGRAMs and found that most of them behaved as (weak) agonists, with reduced GR transcriptional activity (29). We also found that both SEGRAMs and GR antagonists reduced the levels of GR Ser211 phosphorylation, a PTM which has been reported to facilitate the AF-1-mediated interaction with coregulator proteins (30).…”

Section: Introductionmentioning

confidence: 77%

Antagonism and selective modulation of the human glucocorticoid receptor both reduce recruitment of p300/CBP and the Mediator complex

Moortel

Verhee

Melchers

et al. 2023

Preprint

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Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with agonists, antagonists, or lead selective GR agonists and modulators (SEGRAMs), we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that the GR antagonist RU486 and the SEGRAM Dagrocorat both reduced GR interaction with CREB-binding protein (CBP)/p300 and the Mediator complex when compared to the full GR agonist Dexamethasone. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.

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“…An alternative strategy is to make use of allosteric modulation of GR to change functions, perhaps even at a subcellular resolution. 86…”

Section: Discussionmentioning

confidence: 99%

“…Existing strategies for biased signaling rely on the presence of either interacting (proinflammatory) transcription factors, or on the presence of specific coregulators molecules. An alternative strategy is to make use of allosteric modulation of GR to change functions, perhaps even at a subcellular resolution 86 . Such a next generation of glucocorticoids should take advantage of GR signaling bias for tailoring safety and efficacy.…”

Section: Discussionmentioning

confidence: 99%

Structural basis of glucocorticoid receptor signaling bias

Jeanneteau

Meijer

Moisan

2022

J Neuroendocrinology

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Dissociation between the healthy and toxic effects of cortisol, a major stress‐responding hormone has been a widely used strategy to develop anti‐inflammatory glucocorticoids with fewer side effects. Such strategy falls short when treating brain disorders as timing and activity state within large‐scale neuronal networks determine the physiological and behavioral specificity of cortisol response. Advances in structural molecular dynamics posit the bases for engineering glucocorticoids with precision bias for select downstream signaling pathways. Design of allosteric and/or cooperative control for the glucocorticoid receptor could help promote the beneficial and reduce the deleterious effects of cortisol on brain and behavior in disease conditions.

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Novel assays monitoring direct glucocorticoid receptor protein activity exhibit high predictive power for ligand activity on endogenous gene targets

Cited by 11 publications

References 44 publications

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

Antagonism and selective modulation of the human glucocorticoid receptor both reduce recruitment of p300/CBP and the Mediator complex

Structural basis of glucocorticoid receptor signaling bias

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