Glucocorticoid receptor signaling: intricacies and therapeutic opportunities.

The glucocorticoid receptor (GR) is a major nuclear receptor (NR) drug target for the treatment of inflammatory disorders and several cancers. Despite the effectiveness of GR ligands, their systemic action triggers a plethora of side effects, limiting long-term use. Here, we discuss new concepts of and insights into GR mechanisms of action to assist in the identification of routes toward enhanced therapeutic benefits. We zoom in on the communication between different GR domains and how this is influenced by different ligands. We detail findings on the interaction between GR and chromatin, and highlight how condensate formation and coregulator confinement can perturb GR transcriptional responses. Last, we discuss the potential of novel ligands and the therapeutic exploitation of crosstalk with other NRs.

Selective Modulation of the Human Glucocorticoid Receptor Compromises GR Chromatin Occupancy and Recruitment of p300/CBP and the Mediator Complex.

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the 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 the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.

Immunopeptidomics mapping of Listeria monocytogenes T cell epitopes in mice.

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6,000 mouse self-peptides presented on MHC Class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

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

Protein-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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (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.

The Biologist's Guide to the Glucocorticoid Receptor's Structure.

The glucocorticoid receptor α (GRα) is a member of the nuclear receptor superfamily and functions as a glucocorticoid (GC)-responsive transcription factor. GR can halt inflammation and kill off cancer cells, thus explaining the widespread use of glucocorticoids in the clinic. However, side effects and therapy resistance limit GR's therapeutic potential, emphasizing the importance of resolving all of GR's context-specific action mechanisms. Fortunately, the understanding of GR structure, conformation, and stoichiometry in the different GR-controlled biological pathways is now gradually increasing. This information will be crucial to close knowledge gaps on GR function. In this review, we focus on the various domains and mechanisms of action of GR, all from a structural perspective.

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

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and auto-immune diseases. Unfortunately, their use is hampered by many side effects and therapy resistance. Efforts to find more selective glucocorticoid receptor (GR) agonists and modulators (called SEGRAMs) that are able to separate anti-inflammatory effects via gene repression from metabolic effects via gene activation, have been unsuccessful so far. In this study, we characterized a set of functionally diverse GR ligands in A549 cells, first using a panel of luciferase-based reporter gene assays evaluating GR-driven gene activation and gene repression. We expanded this minimal assay set with novel luciferase-based read-outs monitoring GR protein levels, GR dimerization and GR Serine 211 (Ser211) phosphorylation status and compared their outcomes with compound effects on the mRNA levels of known GR target genes in A549 cells and primary hepatocytes. We found that luciferase reporters evaluating GR-driven gene activation and gene repression were not always reliable predictors for effects on endogenous target genes. Remarkably, our novel assay monitoring GR Ser211 phosphorylation levels proved to be the most reliable predictor for compound effects on almost all tested endogenous GR targets, both driven by gene activation and repression. The integration of this novel assay in existing screening platforms running both in academia and industry may therefore boost chances to find novel GR ligands with an actual improved therapeutic benefit.

Improved Glucocorticoid Receptor Ligands: Fantastic Beasts, but How to Find Them?

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and hematological cancers. Unfortunately, their use is associated with debilitating side effects, including hyperglycemia, osteoporosis, mood swings, and weight gain. Despite the continued efforts of pharma as well as academia, the search for so-called selective glucocorticoid receptor modulators (SEGRMs), compounds with strong anti-inflammatory or anti-cancer properties but a reduced number or level of side effects, has had limited success so far. Although monoclonal antibody therapies have been successfully introduced for the treatment of certain disorders (such as anti-TNF for rheumatoid arthritis), glucocorticoids remain the first-in-line option for many other chronic diseases including asthma, multiple sclerosis, and multiple myeloma. This perspective offers our opinion on why a continued search for SEGRMs remains highly relevant in an era where small molecules are sometimes unrightfully considered old-fashioned. Besides a discussion on which bottlenecks and pitfalls might have been overlooked in the past, we elaborate on potential solutions and recent developments that may push future research in the right direction.

Endothelial Response to Glucocorticoids in Inflammatory Diseases.

The endothelium plays a crucial role in inflammation. A balanced control of inflammation requires the action of glucocorticoids (GCs), steroidal hormones with potent cell-specific anti-inflammatory properties. Besides the classic anti-inflammatory effects of GCs on leukocytes, recent studies confirm that endothelial cells also represent an important target for GCs. GCs regulate different aspects of endothelial physiology including expression of adhesion molecules, production of pro-inflammatory cytokines and chemokines, and maintenance of endothelial barrier integrity. However, the regulation of endothelial GC sensitivity remains incompletely understood. In this review, we specifically examine the endothelial response to GCs in various inflammatory diseases ranging from multiple sclerosis, stroke, sepsis, and vasculitis to atherosclerosis. Shedding more light on the cross talk between GCs and endothelium will help to improve existing therapeutic strategies and develop new therapies better tailored to the needs of patients.