Abstract Post-translational histone modifications are major regulators of gene expression. However, conventional immunoassays do not provide sufficient information regarding their spatial and temporal dynamic changes. Fluorescence/Förster resonance energy transfer (FRET)-based probes are capable of monitoring the dynamic changes associated with histone modifications in real-time by measuring the balance between histone-modifying enzyme activities. Recently, a genetically encoded histone-modification fluorescent probe using a single-chain variable region (scFv) fragment of a specific antibody was developed. The probe, modification-specific intracellular antibody, is capable of monitoring histone-acetylation levels in both cultured cells and living organisms based on the ratio of fluorescence intensities between the cell nucleus and cytoplasm. In this study, we constructed a FRET probe composed of yellow fluorescent protein attached at the N-terminus of an acetyl H3K9-specific scFv, tethered to a cyan fluorescent protein. When the FRET probe was expressed in human cells, both FRET efficiency and fluorescence intensity in the nucleus increased following histone-deacetylase inhibitor treatment. Using these two parameters, endogenous histone-acetylation levels were quantified over a high dynamic range. This probe provides a simple approach to quantify spatial and temporal dynamic changes in histone acetylation.
Abstract Transcription factor dynamics are used to selectively engage gene regulatory programs. Biomolecular condensates have emerged as an attractive signaling module in this process, but the underlying mechanisms are not well-understood. Here, we probed the molecular basis of YAP signal integration through transcriptional condensates. Leveraging light-sheet single-molecule imaging and synthetic condensates, we demonstrate charge-mediated co-condensation of the transcriptional regulators YAP and Mediator into transcriptionally active condensates in stem cells. IDR sequence analysis and YAP protein engineering demonstrate that the signaling specificity of YAP is established, in part, through complementary electrostatic interactions between negatively charged blocks within YAP and positively charged blocks within Mediator. YAP/Mediator co-condensation is counteracted by negative feedback from transcription, driving an adaptive transcriptional response that is well-suited for decoding dynamic inputs. Our work reveals a molecular framework for YAP condensate formation and sheds new light on the function of YAP condensates for emergent gene regulatory behavior.
Abstract Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton’s Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.
Fluorescence-based probes, especially those that utilize Förster resonance energy transfer (FRET) between fluorescent protein (FP) variants, are widely used to monitor various biological phenomena, most often detecting its ligand-induced conformational change through the receptor domain. While antibody provides a fertile resource of a specific receptor for various biomolecules, its potential has not been fully exploited. An exception is a pair of donor FP-fused VH and acceptor FP-fused VL fragments, which has been proven useful when their association increases in the presence of antigen (open sandwich fluoroimmunoassay, OS-FIA). However, probes for larger proteins such as serum albumin (SA) were difficult to produce, since the interaction between VH and VL of these antibodies is barely affected by the bound antigen. Here, we propose a novel strategy, called open flower fluoroimmunoassay (OF-FIA), using a probe composed of a donor-fused VH and an acceptor-fused VL linked by a disulfide bond between VH and VL (CyPet/YPet-dsFv). The probe gave high FRET efficiency due to the dimerization propensity of the FP pair, while the efficiency got lower as SA concentration increased, probably due to dimer disruption. The constructed probe could detect clinically relevant range of SA, showing its potential as a diagnostic reagent.
The kinase ataxia telangiectasia mutated (ATM) plays a key role in the DNA damage response (DDR). It is thus essential to visualize spatiotemporal dynamics of ATM activity during DDR. Here, we designed a robust ATM activity reporter based on phosphorylation-inducible green fluorescent protein phase separation, dubbed ATM-SPARK (separation of phases-based activity reporter of kinase). Upon ATM activation, it undergoes phase separation via multivalent interactions, forming intensely bright droplets. The reporter visualizes spatiotemporal dynamics of endogenous ATM activity in living cells, and its signal is proportional to the amount of DNA damage. ATM-SPARK also enables high-throughput screening of biological and small-molecule regulators. We identified the protein phosphatase 4 that blocks ATM activity. We also identified BGT226 as a potent ATM inhibitor with a median inhibitory concentration of ~3.8 nanomolars. Furthermore, BGT226 sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting that BGT226 might be combined with radiotherapeutic treatment. ATM-SPARK achieves large dynamic range, bright fluorescence, and simple signal pattern.
Abstract Dysregulation and enhanced expression of MYC transcription factors (TFs) including MYC and MYCN contribute to the majority of human cancers. For example, MYCN is amplified up to several hundred-fold in high-risk neuroblastoma. The resulting overexpression of N-myc aberrantly activates genes that are not activated at low N-myc levels and drives proliferation and cell survival. Whether increasing N-myc levels simply mediate binding to lower-affinity binding sites in the genome or fundamentally changes the activation process remains unclear. One such activation mechanism that could become important above threshold levels of N-myc is the formation of aberrant transcriptional condensates through phase separation. Phase separation has recently been linked to transcriptional regulation, but how strongly it contributes to gene activation remains unclear. Here we characterized the phase behavior of N-myc and showed that it can form dynamic condensates that bear the hallmarks of transcriptional activity. We tested the contribution of phase separation to N-myc-mediated gene expression by using a chemogenetic tool that allowed us to compare non-phase-separated and phase-separated conditions at identical N-myc levels, which both showed a strong impact on gene expression compared to no N-myc expression. However, we found that only a small fraction of <3% of N-myc-regulated genes is further affected by phase separation, but that these events include the activation of key oncogenes and the repression of tumor suppressors. Indeed, phase separation increases cell survival by ∼15% corroborating the biological effects of the transcriptional changes. However, our results also show that >97% of N-myc-regulated genes are not affected by N-myc phase separation, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery.
Homologous recombination catalyzed by the RAD51 recombinase eliminates deleterious DNA lesions from the genome. In the presence of ATP, RAD51 forms a nucleoprotein filament on single-stranded DNA, termed the presynaptic filament, to initiate homologous recombination-mediated DNA double-strand break repair. The SWI5-SFR1 complex stabilizes the presynaptic filament and enhances its ability to mediate the homologous DNA pairing reaction. Here we characterize the RAD51 presynaptic filament stabilization function of the SWI5-SFR1 complex using optical tweezers. Biochemical experiments reveal that SWI5-SFR1 enhances ATP hydrolysis by single-stranded DNA-bound RAD51. Importantly, we show that SWI5-SFR1 acts by facilitating the release of ADP from the presynaptic filament. Our results thus provide mechanistic understanding of the function of SWI5-SFR1 in RAD51-mediated DNA recombination.
Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton's Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.
Abstract Background Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo , in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. Results Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system. Conclusion Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro , which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.
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