Search Results (55)

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), continues to pose significant public health challenges due to the toxicity, poor tolerability, and limited efficacy of current treatments. Targeted protein degradation (TPD) using proteolysis-targeting chimeras (PROTACs) represents a novel therapeutic avenue by leveraging the ubiquitin–proteasome system to selectively degrade essential parasite proteins. This review introduces the conceptual framework of “TrypPROTACs” as a prospective strategy for T. cruzi, integrating a comprehensive analysis of druggable targets across critical biological pathways, including ergosterol biosynthesis, redox metabolism, glycolysis, nucleotide synthesis, protein kinases, molecular chaperones such as heat shock protein 90 (Hsp90), and epigenetic regulators such as T. cruzi bromodomain factor 3 (TcBDF3). It is important to note that no TrypPROTAC compound has yet been synthesized or experimentally validated in T. cruzi; the approach discussed herein remains theoretical and forward-looking. Representative inhibitors for each target class are compiled, highlighting potency, selectivity, and structural features relevant to ligand design. We also examine the parasite’s ubiquitination machinery and compare it to the human system to identify putative E3 ubiquitin ligases. Key aspects of linker engineering and ternary complex stabilization are discussed, alongside potential validation techniques such as the cellular thermal shift assay (CETSA) and bioluminescence resonance energy transfer (NanoBRET). Collectively, these insights outline a roadmap for the rational design of TrypPROTACs and support the feasibility of expanding targeted protein degradation strategies to neglected tropical diseases. Full article

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects. Full article

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously. Full article

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR–D2R, GABAA–D5R, and FGFR1–5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor–receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor–receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor–receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000–2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor–receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor–receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson’s disease. Full article

One of the hallmarks of Parkinson’s disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB₁R). The presence of CB₁R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB₁R in CB₁R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB₁R-NMDAR complex in transfected HEK-293T cells. Moreover, α-synuclein treatment induced a decrease in the cAMP and MAP kinase (MAPK) signaling of both CB₁R and NMDAR not only in transfected cells but also in neuronal primary cultures. Finally, the interaction between CB₁R and NMDAR was studied by Proximity Ligation Assay (PLA) in neuronal primary cultures, where it was observed that the expression of CB₁R-NMDAR complexes was decreased upon α-synuclein treatment. These results point to a role of CB₁R-NMDAR complexes as a new therapeutic target in Parkinson’s disease. Full article

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABA_B receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5’s interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER. Full article

Potassium Channel Tetramerization Domain 5 (KCTD5) regulates diverse aspects of physiology, ranging from neuronal signaling to colorectal cancer. A key feature of KCTD5 is its self-assembly into multi-subunit oligomers that seemingly enables participation in an array of protein–protein interactions. KCTD5 has recently been reported to form hetero-oligomeric complexes with two similar KCTDs (KCTD2 and KCTD17). However, it is not known if KCTD5 forms hetero-oligomeric complexes with the remaining KCTD protein family which contains over two dozen members. Here, we demonstrate that KCTD5 interacts with various KCTD proteins when assayed through co-immunoprecipitation in lysed cells. We reinforced this dataset by examining KCTD5 interactions in a live-cell bioluminescence resonance energy transfer (BRET)-based approach. Finally, we developed an IP-luminescence approach to map regions on KCTD5 required for interaction with a selection of KCTD that have established roles in neuronal signaling. We report that different regions on KCTD5 are responsible for uniquely contributing to interactions with other KCTD proteins. While our results help unravel additional interaction partners for KCTD5, they also reveal additional complexities in KCTDs’ biology. Moreover, our findings also suggest that KCTD hetero-oligomeric interactions may occur throughout the KCTD family. Full article

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn²⁺ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permitted BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn²⁺ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn²⁺ in serum samples and achieved Zn²⁺ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes. Full article

Despite G protein-coupled receptors (GPCRs) being important theapeutic targets, the signaling properties of many GPCRs remain poorly characterized. GPCR activation primarily initiates heterotrimeric G protein signaling. To detect ligand-induced G protein activation, Bioluminescence Resonance Energy Transfer (BRET)-based biosensors were previously developed. Here, we designed a novel set of Nanoluciferase (NLuc) BRET-based biosensors (REGA-SIGN) that covers all Gα protein families (i.e., Gα_i/o, Gα_Ss/L, Gα_12/13 and Gα_q/15). REGA-SIGN uses NLuc as a bioluminescent donor and LSS-mKATE2, a red-shifted fluorophore, as an acceptor. Due to the enhanced spectral separation between donor and acceptor emission and the availability of a stable substrate for NLuc, this donor–acceptor pair enables sensitive kinetic assessment of G protein activity. After optimization, the NLuc integration sites into the Gα subunit largely corresponded with previously reported integration sites, except for Gα_Ss/L for which we describe an alternative NLuc insertion site. G protein rescue experiments validated the biological activity of these Gα donor proteins. Direct comparison between EGFP and LSS-mKATE2 as acceptor fluorophores revealed improved sensitivity for nearly all G protein subtypes when using the latter one. Hence, REGA-SIGN can be used as a panel of kinetic G protein biosensors with high sensitivity. Full article

The selectivity of drugs for G protein-coupled receptor (GPCR) signaling pathways is crucial for their therapeutic efficacy. Different agonists can cause receptors to recruit effector proteins at varying levels, thus inducing different signaling responses, called signaling bias. Although several GPCR-biased drugs are currently being developed, only a limited number of biased ligands have been identified regarding their signaling bias for the M1 muscarinic acetylcholine receptor (M1mAChR), and the mechanism is not yet well understood. In this study, we utilized bioluminescence resonance energy transfer (BRET) assays to compare the efficacy of six agonists in inducing Gαq and β-arrestin2 binding to M1mAChR. Our findings reveal notable variations in agonist efficacy in the recruitment of Gαq and β-arrestin2. Pilocarpine preferentially promoted the recruitment of β-arrestin2 (∆∆RAi = −0.5), while McN-A-343 (∆∆RAi = 1.5), Xanomeline (∆∆RAi = 0.6), and Iperoxo (∆∆RAi = 0.3) exhibited a preference for the recruitment of Gαq. We also used commercial methods to verify the agonists and obtained consistent results. Molecular docking revealed that certain residues (e.g., Y404, located in TM7 of M1mAChR) could play crucial roles in Gαq signaling bias by interacting with McN-A-343, Xanomeline, and Iperoxo, whereas other residues (e.g., W378 and Y381, located in TM6) contributed to β-arrestin recruitment by interacting with Pilocarpine. The preference of activated M1mAChR for different effectors may be due to significant conformational changes induced by biased agonists. By characterizing bias towards Gαq and β-arrestin2 recruitment, our study provides insights into M1mAChR signaling bias. Full article

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL₁₄-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL₁₄ which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL₁₄ containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES. Full article

The renin-angiotensin system (RAS) is one of the main regulatory systems of cardiovascular homeostasis. It is mainly composed of angiotensin-converting enzyme (ACE) and angiotensin II receptors AT1 and AT2. ACE and AT1 are targets of choice for the treatment of hypertension, whereas the AT2 receptor is still not exploited due to the lack of knowledge of its physiological properties. Peptide toxins from venoms display multiple biological functions associated with varied chemical and structural properties. If Brazilian viper toxins have been described to inhibit ACE, no animal toxin is known to act on AT1/AT2 receptors. We screened a library of toxins on angiotensin II receptors with a radioligand competition binding assay. Functional characterization of the selected toxin was conducted by measuring second messenger production, G-protein activation and β-arrestin 2 recruitment using bioluminescence resonance energy transfer (BRET) based biosensors. We identified one original toxin, A-CTX-cMila, which is a 7-residues cyclic peptide from Conus miliaris with no homology sequence with known angiotensin peptides nor identified toxins, displaying a 100-fold selectivity for AT1 over AT2. This toxin shows a competitive antagonism mode of action on AT1, blocking Gαq, Gαi3, GαoA, β-arrestin 2 pathways and ERK_1/2 activation. These results describe the first animal toxin active on angiotensin II receptors. Full article

Bioluminescence (BL) is an excellent optical readout for bioassays and molecular imaging. Herein, we accomplished new near infrared bioluminescence resonance energy transfer (NIR-BRET) templates for monitoring molecular events in cells with higher sensitivity. We first identified the best resonance energy donor for the NIR-BRET templates through the characterization of many coelenterazine (CTZ)–marine luciferase combinations. As a result, we found that NLuc–DBlueC and ALuc47–nCTZ combinations showed luminescence in the blue emission wavelength with excellent BL intensity and stability, for example, the NLuc–DBlueC and ALuc47–nCTZ combinations were 17-fold and 22-fold brighter than their second highest combinations, respectively, and were stably bright in living mammalian cells for at least 10 min. To harness the excellent BL properties to the NIR-BRET systems, NLuc and ALuc47 were genetically fused to fluorescent proteins (FPs), allowing large “blue-to-red” shifts, such as LSSmChe, LSSmKate2, and LSSmNep (where LSS means Large Stokes Shift). The excellent LSSmNep–NLuc combination showed approximately 170 nm large resonance energy shift from blue to red. The established templates were further utilized in the development of new NIR-BRET systems for imaging steroid hormone activities by sandwiching the ligand-binding domain of a nuclear receptor (NR-LBD) between the luciferase and the FP of the template. The NIR-BRET systems showed a specific luminescence signal upon exposure to steroid hormones, such as androgen, estrogen, and cortisol. The present NIR-BRET templates are important additions for utilizing their advantageous imaging of various molecular events with high efficiency and brightness in physiological samples. Full article

Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase–Oplophorus luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of “free” zinc ion (also termed “mobile” or “labile”) using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo. Full article