NanoBiT ‐ and NanoBiT/BRET ‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model

Background and Purpose Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD$_{7}$, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD$_{7}$, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD$_{7}$ using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD$_{7}$ was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD$_{7}$ obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD$_{7}$ binding should be performed using receptors expressed under endogenous promotion

Immunomodulation—A Molecular Solution to Treating Patients with Severe Bladder Pain Syndrome?

Background: Patients with bladder pain syndrome experience debilitating pain and extreme frequency of urination. Numerous therapeutic approaches have been tested, but as the molecular basis of disease has remained unclear, specific therapies are not available. Objective: Recently, a systematic gene deletion strategy identified interleukin-1 (IL-1) hyperactivation as a cause of severe cystitis in a murine model. Treatment with an IL-1 receptor antagonist (IL-1RA) restored health in genetically susceptible mice, linking IL-1–dependent inflammation to pain and pathology in the bladder mucosa. The study objective was to investigate whether IL-1RA treatment might be beneficial in patients with bladder pain syndrome. Design, setting, and participants: Patients diagnosed with bladder pain syndrome were invited to participate and subjected to daily IL-1RA injections for 1 wk, followed by a treatment break. Patients with other urological disorders accompanied by pain were included as controls. Outcome measurements and statistical analysis: When symptoms returned, treatment was resumed and responding patients were maintained on treatment long term, with individualized dosing regimens. Symptom scores were recorded and molecular effects were quantified by neuropeptide and gene expression analysis. DNA samples were subjected to exome genotyping. Results and limitations: IL-1RA treatment reduced bladder pain and the frequency of urination in 13/17 patients (p < 0.001). Substance P levels in urine were lowered, and responders returned to a more normal lifestyle. Neuroinflammatory-dependent and IL-1–dependent gene networks were inhibited, as well as regulators of innate immunity. Genotyping revealed disease-associated IL1R1, NLRP3, and IL1RN DNA sequence variants in the responders. Controls did not benefit from IL-1RA treatment, except for one patent with cystitis cystica. Conclusions: In this clinical study, IL-1RA treatment is proposed to reduce chronic bladder pain, immediately and in the long term. Despite the limited number of study patients, the potent acute effect and lasting symptom relief indicate that this therapeutic approach may be worth exploring in controlled clinical trials. Patient summary: Treatment with an interleukin-1 (IL-1) receptor antagonist is proposed for treating bladder pain syndrome, as it can result in symptom relief and increase quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects. Take Home Message: Interleukin-1 (IL-1) receptor antagonist immunotherapy is proposed as a new approach to treating bladder pain syndrome, a debilitating disorder. Treated patients experienced symptom relief and increased quality of life. Reduced neuroinflammation and IL-1 signaling provided molecular evidence of the treatment effects

Structural basis of frizzled 7 activation and allosteric regulation

Frizzleds (ten paralogs: FZD1-10) belong to the class F of G protein-coupled receptors (GPCRs), which remains poorly understood despite its crucial role in multiple key biological functions including embryonic development, stem cell regulation, and homeostasis in the adult. FZD7, one of the most studied members of the family, is more specifically involved in the migration of mesendoderm cells during the development and renewal of intestinal stem cells in adults. Moreover, FZD7 has been highlighted for its involvement in tumor development predominantly in the gastrointestinal tract. This study reports the structure of inactive FZD7, without any stabilizing mutations, determined by cryo-electron microscopy (cryo-EM) at 1.9 Å resolution. We characterize a fluctuating water pocket in the core of the receptor important for FZD7 dynamics. Molecular dynamics simulations are used to investigate the temporal distribution of those water molecules and their importance for potential conformational changes in FZD7. Moreover, we identify lipids interacting with the receptor core and a conserved cholesterol-binding site, which displays a key role in FZD7 association with a transducer protein, Disheveled (DVL), and initiation of downstream signaling and signalosome formation

Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7

Summary: The G protein-coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live-cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA-approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore-forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding

NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model

Background and Purpose: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach: SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results: With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion