eLife: latest articles by subject

Multiple modes of cholesterol translocation in the human Smoothened receptor

diwakar@illinois.edu (Diwakar Shukla) — Wed, 11 Mar 2026 00:00:00 +0000

Smoothened (SMO), a member of the G-protein-coupled receptor superfamily, mediates Hedgehog signaling and is linked to cancer and birth defects. SMO responds to accessible cholesterol in the ciliary membrane, translocating it via a longitudinal tunnel to its extracellular domain. Reaching a complete mechanistic understanding of the cholesterol translocation process would help in the development of cancer therapies. Experimental data suggest two modes of translocation to support entry of cholesterol from outer and inner membrane leaflets, but the exact mechanism of translocation remains unclear. Using atomistic molecular dynamics simulations (∼2 ms simulations) and biochemical assays of SMO mutants, we assess the energetic feasibilities of the two modes. We show that the highest energetic barrier for cholesterol translocation from the outer leaflet is lower than that from the inner leaflet. Mutagenesis experiments and complementary simulations of SMO mutants validate the role of critical amino acid residues along the translocation pathways. Our data suggests that cholesterol can take either pathway to enter SMO, thus explaining experimental observations in the literature. Thus, our results illuminate the energetics and provide a first molecular description of cholesterol translocation in SMO.

Concatenated modular BK channel constructs reveal divergent stoichiometry in gating control by LRRC26 (γ1), pore, and selectivity filter

jyan1@mdanderson.org (Guanxing Chen) — Thu, 05 Mar 2026 00:00:00 +0000

Big-conductance, Ca²⁺-activated K⁺ (BK) channels consist of Ca²⁺- and voltage-sensing, pore-forming α (BKα) subunits and regulatory auxiliary β or γ subunits. Concatenated subunit constructs are powerful tools for elucidating subunit stoichiometry in ion channel gating and regulation, allowing control over subunit arrangement, stoichiometry, and mutation. However, the additional S0 transmembrane segment in BKα places its N- and C-termini on opposite sides of the membrane, preventing tandem BK channel subunit construction by conventional methods. To investigate the atypical ‘all-or-none’ modulatory function of γ subunits and the subunit stoichiometry of BK channel gating, we developed concatenated constructs containing 2 or 4 BKα subunits by splicing them into modular forms that can be co-expressed to form functional channels. These constructs retained voltage and Ca²⁺ gating properties similar to intact BK channels. By fusing the LRRC26 (γ1) subunit to the N-terminus of tandem BKα constructs, we found that a single γ1 subunit per α subunit tetramer is sufficient to fully modulate the channel. Furthermore, the L312A mutation in the deep pore region exhibited a stoichiometrically graded effect on voltage-gated BK channel activation. In contrast, a V288A mutation at the selectivity filter induced channel inactivation only when present in all four BKα subunits. Thus, by engineering concatenated BKα constructs, we identified three distinct stoichiometric modes of BK channel gating control by LRRC26, the pore, and the selectivity filter. This study offers new molecular tools and advances our understanding of subunit stoichiometry in BK channel gating and modulation.

Intracellular expression of a fluorogenic DNA aptamer using retron Eco2

hannes.mutschler@tu-dortmund.de (Corbin Machatzke) — Tue, 03 Mar 2026 00:00:00 +0000

DNA aptamers are short, single-stranded DNA molecules that bind specifically to a range of targets such as proteins, cells, and small molecules. Typically, they are utilized in the development of therapeutic agents, diagnostics, drug delivery systems, and biosensors. Although aptamers perform well in controlled extracellular environments, their intracellular use has been less explored due to challenges of expressing them in vivo. In this study, we employed the bacterial retron system Eco2 to express a DNA light-up aptamer in Escherichia coli. Our data confirms that structure-guided insertion of the aptamer domain into the non-coding region of the retron enables reverse transcription and biosynthesis of functional aptamer constructs in bacteria. The purified DNA aptamer synthesized under intracellular conditions shows comparable activity to a chemically synthesized control. Our findings demonstrate that retrons can be used to express short DNA aptamers within living cells, potentially broadening and optimizing their application in intracellular settings.

Synthetic gene circuits that selectively target RAS-driven cancers

kobi.benenson@gmail.com (Gabriel Valentin Senn) — Tue, 24 Feb 2026 00:00:00 +0000

Therapies targeting mutated rat sarcoma (RAS), the most frequently mutated oncogene in human cancers, could benefit millions of patients. Recently approved RAS inhibitors represent a breakthrough but are limited to a specific KRAS^G12C mutation and prone to resistance. Synthetic gene circuits offer a promising alternative by sensing and integrating cancer-specific biomolecular inputs, including mutated RAS, to selectively express therapeutic proteins in cancer cells. A key challenge for these circuits is achieving high cancer selectivity to prevent toxicity in healthy cells. To address this challenge, we present a novel approach combining multiple RAS sensors into RAS-targeting gene circuits, which allowed us to express an output protein in cells with mutated RAS with unprecedented selectivity. We implemented a modular design strategy and modeled the impact of individual circuit components on output expression. This enabled cell-line-specific adaptation of the circuits to optimize selectivity and fine-tune expression. We further demonstrate the targeting capabilities of the circuits by employing them in different RAS-driven cancer cells and provide evidence for their therapeutic potential by linking them to the expression of a clinically relevant output protein, which induced robust killing of cancer cells with mutated RAS. This work highlights the potential of synthetic gene circuits as a novel therapeutic strategy for RAS-driven cancers, advancing the application of synthetic biology in oncology.

Citalopram exhibits immune-dependent anti-tumor effects by modulating C5aR1⁺ TAMs

zzhang@shsci.org (Chongyi Jiang) — Mon, 09 Feb 2026 00:00:00 +0000

Administration of selective serotonin reuptake inhibitors (SSRIs) is associated with a reduced cancer risk and shows significant anti-tumor effects across multiple tumor types, suggesting the potential for repurposing SSRIs in cancer therapy. Nonetheless, the specific molecular target and mechanism of action of SSRIs remain to be fully elucidated. Here, we reveal that citalopram exerts an immune-dependent anti-tumor effect in hepatocellular carcinoma (HCC). Interestingly, the anti-HCC effects of citalopram are not reliant on its conventional target, the serotonin transporter. Through various drug repurposing approaches, including global reverse gene expression profiling, drug affinity responsive target stability assay, and molecular docking, the complement component 5a receptor 1 (C5aR1) is identified as a new target of citalopram. C5aR1 is predominantly expressed by tumor-associated macrophages, and citalopram treatment enhances local macrophage phagocytosis and elicits CD8⁺ T anti-tumor immunity. C5aR1 deficiency or depletion of CD8⁺ T cells hinders the anti-HCC effects of citalopram. Collectively, our study reveals the immunomodulatory roles of citalopram in inducing anti-tumor immunity and provides a basis for considering the repurposing of SSRIs as promising anticancer agents for HCC treatment.

Aggregation-dependent epitope sequence and modification fingerprints of anti-Aβ antibodies

Hans.Maric@virchow.uni-wuerzburg.de (Annik Steiert) — Mon, 09 Feb 2026 00:00:00 +0000

A hallmark of Alzheimer’s disease (AD), the most common form of dementia, is the progressive accumulation of amyloid-beta (Aβ) peptides across distinct brain regions. Anti-Aβ antibodies (Aβ-Abs) targeting specific Aβ variants are essential tools for AD research, diagnostics, and therapy. The monoclonal antibodies Aducanumab, Lecanemab, and Donanemab have recently been approved as the first disease-modifying treatments for early AD, highlighting the clinical importance of their exact binding profiles. In this study, we systematically characterized the binding and modification requirements of 20 Aβ-Abs, including biosimilars of Aducanumab, Lecanemab, and Donanemab, across monomeric, oligomeric, and aggregated Aβ forms. Array-based analysis of 20,000 modified Aβ peptides defined binding epitopes at single-residue resolution and revealed the impact of sequence variation, including familial AD mutations, as well as diverse post-translational modifications (PTMs). Notably, genetic variants, such as H6R, impaired binding of therapeutic Aβ-Abs like Aducanumab. Donanemab showed strong preference for pyroglutamate-modified AβpE3–17, while Lecanemab and Aducanumab exhibited aggregation- and sequence-context-dependent binding requirements. Comparison of peptide binding profiles with binding of full-length and aggregated Aβ via immunoprecipitation-mass spectrometry, capillary immunoassays, Western blotting, and immunohistochemistry on AD brain tissue revealed distinct aggregation-dependent binding behaviours. The valency- and context-dependence of Aducanumab binding, together with its preference for Ser8-phosphorylated Aβ, supports a dimerization-mediated binding mechanism. For Lecanemab, our data suggest that additional structural contributions beyond the minimal N-terminal epitope are required for binding to aggregated Aβ, which remain to be fully resolved. Together, this work provides the most comprehensive dataset to date on aggregation-dependent sequence and modification selectivity of Aβ-Abs. By integrating mutational, PTM, and aggregation contexts in a unified experimental framework, we establish a resource that enables rational selection of antibodies for research and diagnostic applications and offers mechanistic insights that may inform the design and optimization of future therapeutic antibodies in AD.

Correction: Gq activity- and β-arrestin-1 scaffolding-mediated ADGRG2/CFTR coupling are required for male fertility

Thu, 05 Feb 2026 00:00:00 +0000

Gut microbe-derived trimethylamine shapes circadian rhythms through the host receptor TAAR5

brownm5@ccf.org (Adeline M Hajjar) — Thu, 29 Jan 2026 00:00:00 +0000

Elevated levels of the gut microbe-derived metabolite trimethylamine N-oxide (TMAO) are associated with cardiometabolic disease risk. However, the mechanism(s) linking TMAO production to human disease are incompletely understood. Initiation of the metaorganismal TMAO pathway begins when dietary choline and related metabolites are converted to trimethylamine (TMA) by gut bacteria. Gut microbe-derived TMA can then be further oxidized by host flavin-containing monooxygenases to generate TMAO. Previously, we showed that drugs lowering both TMA and TMAO protect mice against obesity via rewiring of host circadian rhythms (Schugar et al., 2022). Although most mechanistic studies in the literature have focused on the metabolic end product TMAO, here we have instead tested whether the primary metabolite TMA alters host metabolic homeostasis and circadian rhythms via trace amine-associated receptor 5 (TAAR5). Remarkably, mice lacking the host TMA receptor (Taar5^−/⁻) have altered circadian rhythms in gene expression, metabolic hormones, gut microbiome composition, and diverse behaviors. Also, mice genetically lacking bacterial TMA production or host TMA oxidation have altered circadian rhythms. These results provide new insights into diet–microbe–host interactions relevant to cardiometabolic disease and implicate gut bacterial production of TMA and the host receptor that senses TMA (TAAR5) in the physiologic regulation of circadian rhythms in mice.

Phenylhydrazone-based endoplasmic reticulum proteostasis regulator compounds with enhanced biological activity

jkelly@scripps.edu (Adrian Guerrero) — Mon, 26 Jan 2026 00:00:00 +0000

Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a molecule that promotes adaptive ER proteostasis remodeling through mechanisms including preferential activation of the ATF6 signaling arm of the unfolded protein response (Plate et al., 2016). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a potential molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated α1-antitrypsin (A1AT) variant and an epilepsy-associated GABA_A receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically diverse protein misfolding disorders.

Shifting the PPARγ conformational ensemble toward a transcriptionally repressive state improves covalent inhibitor efficacy

douglas.kojetin@vanderbilt.edu (Brian S MacTavish) — Wed, 21 Jan 2026 00:00:00 +0000

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662—extensively used as PPARγ inhibitors to assess off-target activity—weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang and Kojetin, 2024). These covalent inhibitors shift the LBD toward a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of—but also do not completely inhibit—ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPARγ dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPARγ LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

The interplay between biomolecular assembly and phase separation

bartolucci@ub.edu (Christoph A Weber) — Fri, 09 Jan 2026 00:00:00 +0000

Many biological functions and dysfunctions rely on two fundamental processes, molecular assembly and the formation of condensed phases such as biomolecular condensates. Condensed phases generally form via phase separation, while molecular assemblies are clusters of molecules of various sizes, shapes, and functionality. We developed a theory that relies on thermodynamic principles to understand the interplay between molecular assembly and phase separation. We propose two prototypical classes of protein interactions and characterize their different equilibrium states and relaxation dynamics. We obtain results consistent with recent in vitro experimental observations of reconstituted proteins, including anomalous size distribution of assemblies, the gelation of condensed phases, and the change in condensate volume during ageing. Our theory provides the framework to unravel the mechanisms underlying physiological assemblies essential for cellular function and aberrant assemblies which are associated with several neurodegenerative disorders.

Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during BV2 microglial activation

grutter@unistra.fr (Adeline Martz) — Fri, 09 Jan 2026 00:00:00 +0000

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP, an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively and covalently biotinylates endogenous P2X7 in BV2 cells, a murine microglial cell line, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1β release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level.

A green lifetime biosensor for calcium that remains bright over its full dynamic range

j.goedhart@uva.nl (Franka H van der Linden) — Fri, 19 Dec 2025 00:00:00 +0000

Fluorescent biosensors toggle between two states, and for the vast majority of biosensors, one state is bright and the other state is dim. As a consequence, there is a substantial difference in the signal-to-noise ratio (SNR) for the two states. The dim state has a low SNR, which is problematic when precise, quantitative measurements are needed. During the engineering of a red-shifted variant of an mTurquoise-based calcium sensor, we serendipitously generated a green-emitting sensor that shows high brightness in both the calcium-bound and -unbound state, while still showing a calcium-dependent lifetime change of >1 ns. This sensor, named G-Ca-FLITS, is comparable in brightness to the bright state of GCaMP3 and jGCaMP7c in mammalian cells. The calcium-induced loss in fluorescence intensity is only around 30% and therefore we observe little variation in the SNR when calcium levels change. G-Ca-FLITS shows negligible sensitivity to pH in the physiological range, like its turquoise parent. Using fluorescence lifetime imaging (FLIM), we measured the calcium concentration with G-Ca-FLITS in various organelles and observed in HeLa cells transient and spatially heterogeneous calcium elevations in mitochondria. Finally, we evaluated the use of G-Ca-FLITS and its turquoise predecessor for two-photon FLIM in Drosophila brains.

A systematic bi-genomic split-GFP assay illuminates the mitochondrial matrix proteome and protein targeting routes

yury.bykov@rptu.de (Bruno Senger) — Tue, 16 Dec 2025 00:00:00 +0000

The majority of mitochondrial proteins are encoded in the nuclear genome. Many of them lack clear targeting signals. Therefore, what constitutes the entire mitochondrial proteome is still unclear. We here build on our previously developed bi-genomic (BiG) split-GFP assay (Bader et al., 2020) to solidify the list of matrix and inner membrane mitochondrial proteins. The assay relies on one fragment (GFP_1-10) encoded in the mitochondrial DNA enabling specific visualization of only the proteins tagged with a smaller fragment, GFP₁₁, and localized to the mitochondrial matrix or the inner membrane. We used the SWAp-Tag (SWAT) strategy to tag every protein with GFP₁₁ and mated them with the BiG GFP strain. Imaging the collection in six different conditions allowed us to visualize almost 400 mitochondrial proteins, 50 of which were never visualized in mitochondria before, and many are poorly studied dually localized proteins. We use structure-function analysis to characterize the dually localized protein Gpp1, revealing an upstream start codon that generates a mitochondrial targeting signal and explore its unique function. We also show how this data can be applied to study mitochondrial inner membrane protein topology and sorting. This work brings us closer to finalizing the mitochondrial proteome and the freely distributed library of GFP₁₁-tagged strains will be a useful resource to study protein localization, biogenesis, and interactions.

General trends in the calnexin-dependent expression and pharmacological rescue of clinical CFTR variants

jschleba@purdue.edu (Andrew G McKee) — Thu, 11 Dec 2025 00:00:00 +0000

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Though most people with CF have one or two copies of the ΔF508 mutation, there are hundreds of other distinct CF mutations that vary in their mechanistic effects and response to therapeutics. Endogenous chaperones are known to have divergent effects on the druggability of CF variants. Nevertheless, it remains unclear how this proteostatic modulation is related to the underlying mechanistic effects of distinct classes of CF mutations. Here, we survey the effects of a previously discovered effector (calnexin, CANX) on the expression and pharmacological rescue of 232 CF variants using deep mutational scanning. We find that CANX is generally required for robust plasma membrane expression of the CFTR protein, particularly for CF variants that perturb its second nucleotide-binding domain. CANX also appears to be critical for the pharmacological rescue of CF variants with poor basal expression. Though corrector selectivity is generally dictated by the properties of mutations, we find that CANX enhances the sensitivity of CF variants within a domain-swapped region of membranes spanning domain 2 to the type III corrector VX-445. Overall, mutagenic trends suggest CANX modulates the later stages of CFTR assembly and disproportionately affects variants bearing mutations within the C-terminal domains. Interestingly, we find that the loss of CANX results in widespread perturbations of CF variant interactomes and that the proteostatic effects of CANX are generally decoupled from changes in CFTR activity. Together, our findings reveal how the proteostasis machinery may shape the variant-specific effects of corrector molecules.

Deep learning reveals endogenous sterols as allosteric modulators of the GPCR–Gα interface

deepaks@imtech.res.in (Aakash Gaur) — Mon, 08 Dec 2025 00:00:00 +0000

Endogenous intracellular allosteric modulators of GPCRs remain largely unexplored, with limited binding and phenotype data available. This gap arises from the lack of robust computational methods for unbiased cavity identification, cavity-specific ligand design, synthesis, and validation across GPCR topology. Here, we developed Gcoupler, an AI-driven generalized computational toolkit that leverages an integrative approach combining de novo ligand design, statistical methods, Graph Neural Networks, and bioactivity-based ligand prioritization for rationally predicting high-affinity ligands. Using Gcoupler, we interrogated intracellular metabolites that target and regulate the GPCR–Gα interface (Ste2p–Gpa1p), affecting pheromone-induced programmed cell death in yeast. Our computational analysis, complemented by experimental validations, including genetic screening, multi-omics, site-directed mutagenesis, biochemical assays, and physiological readouts, identified endogenous hydrophobic metabolites, notably sterols, as direct intracellular allosteric modulators of Ste2p. Molecular simulations coupled with biochemical signaling assessment in site-directed Ste2p mutants further confirmed that metabolites binding to GPCR–Gα obstruct downstream signaling, possibly via a cohesive effect. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed that elevated metabolite levels attenuate hypertrophic response, reinforcing the evolutionary relevance of this mechanism.

Mechanism of SK2 channel gating and its modulation by the bee toxin apamin and small molecules

jonathan.whicher@novartis.com (Jonathan R Whicher) — Thu, 04 Dec 2025 00:00:00 +0000

Small-conductance calcium-activated potassium channel 2 (SK2) serves a variety of biological functions by coupling intracellular calcium dynamics with membrane potential. SK2 modulators are in development for the treatment of neurological and cardiovascular diseases, though the mechanisms of pharmacological modulation remain incompletely understood. We determined structures of an SK2–4 chimeric channel in Ca²⁺-bound and Ca²⁺-free conformations and in complex with the bee toxin apamin, a small molecule inhibitor, and a small molecule activator. The structures revealed that the S3–S4 linker forms a hydrophobic constriction at the extracellular opening of the pore. Apamin binds to this extracellular constriction and blocks the exit of potassium ions. Furthermore, we identified a structurally related SK2 inhibitor and activator that bind to the transmembrane domains. The compounds exert opposing effects on gating by differentially modulating the conformation of the S6 helices. These results provide important mechanistic insights to facilitate the development of targeted SK2 channel therapeutics.

How one nutrient controls cell size

finleyl@mskcc.org (Angela Montero) — Wed, 19 Nov 2025 00:00:00 +0000

The metabolic fate of a nutrient called pyruvate determines how big cells become.

MCAK recognizes the nucleotide-dependent feature at growing microtubule ends

chenwill@mail.tsinghua.edu.cn (Jian-Feng He) — Wed, 19 Nov 2025 00:00:00 +0000

The growing plus-end is a key regulatory site for microtubule dynamics. MCAK (mitotic centromere-associated kinesin), a microtubule depolymerizing kinesin, is an end-binding regulator of catastrophe frequency. It is intriguing how MCAK specifically binds to growing microtubule ends. Here, we measure the end-binding kinetics of MCAK using single-molecule imaging and reveal that MCAK not only binds to the distalmost ends, but also to the proximal region of GTP cap where EB1 preferentially binds. Further analysis shows that MCAK strongly binds to GTPγS microtubules which mimic the GDP·Pi-tubulin-enriched region of GTP cap, and this binding preference is dependent on the nucleotide state of MCAK. This finding suggests that MCAK recognizes the nucleotide-dependent feature of microtubule ends. Moreover, we show that although MCAK and XMAP215 partly share binding regions at the distalmost ends, they act largely independently, influencing catastrophe frequency and growth rate, respectively. Overall, our findings provide new insights into how MCAK regulates microtubule end dynamics.

Growth inhibitory factor/metallothionein-3 is a sulfane sulfur-binding protein

yshinkai@toyaku.ac.jp (George Devitt) — Fri, 14 Nov 2025 00:00:00 +0000

Cysteine-bound sulfane sulfur atoms in proteins have received much attention as key factors in cellular redox homeostasis. However, the role of sulfane sulfur in zinc regulation has been underinvestigated. In this study, we identified growth inhibitory factor (GIF)/metallothionein-3 (MT-3) as a sulfane sulfur-binding protein from mouse brain. We also report here that cysteine-bound sulfane sulfur atoms serve as ligands to hold and release zinc ions in GIF/MT-3 with an unexpected C–S–S–Zn structure. Oxidation of such a zinc/persulfide cluster in Zn₇GIF/MT-3 results in the release of zinc ions, and intramolecular tetrasulfide bridges in apo-GIF/MT-3 efficiently undergo S–S bond cleavage by thioredoxin to regenerate Zn₇GIF/MT-3. Three-dimensional molecular modeling confirmed the critical role of the persulfide group in the thermostability and Zn-binding affinity of GIF/MT-3. The present discovery raises the fascinating possibility that the function of other Zn-binding proteins is controlled by sulfane sulfur.

Membrane-mimetic thermal proteome profiling (MM-TPP) toward mapping membrane protein–ligand dynamic interactions

fduong@mail.ubc.ca (Ashim Bhattacharya) — Wed, 12 Nov 2025 00:00:00 +0000

Integral membrane proteins (IMPs) are central targets for small-molecule therapeutics, yet robust, unbiased, and detergent-free approaches to assess their on- and off-target interactions remain limited. Previously, we introduced the Peptidisc membrane mimetic (MM) for water-soluble stabilization of the membrane proteome and interactome (Carlson et al., eLife, 2019). In this work, we combine the Peptidisc with thermal proteome profiling (TPP) to establish membrane-mimetic thermal proteome profiling (MM-TPP), a method that enables proteome-wide mapping of membrane protein–ligand interactions. Using a membrane protein library derived from mouse liver tissue, we detected the specific effects of ATP and orthovanadate on the thermal stability of ATP-binding cassette (ABC) transporters, as well as stability shifts driven by the hydrotropic effect of ATP and its by-products on G protein-coupled receptors (GPCRs). In contrast, detergent-based TPP (DB-TPP) with ATP–VO₄ failed to yield specific enrichment of ATP-binding proteins, underscoring the unique capacity of MM-TPP. To further validate the approach, we demonstrated the ability of MM-TPP to detect specific ligand-induced stabilization of cognate targets, exemplified by the selective thermal stabilization of the P2RY12 receptor by 2-methylthio-ADP. Together, these findings position MM-TPP as a robust platform for uncovering both on- and off-target effects of small molecules, providing insights into the druggable membrane proteome and its stability in consequence of changing dynamic ligands.

Engineering NIR-sighted bacteria

andreas.moeglich@uni-bayreuth.de (Andreas Möglich) — Mon, 03 Nov 2025 00:00:00 +0000

Spatially and temporally orchestrated gene expression underpins organismal development, physiology, and adaptation. In bacteria, two-component systems (TCS) translate environmental cues into inducible expression outputs. Inducible expression also serves as a versatile instrument in both basic and applied science. Here, we harness the photosensors of rhizobial bathy-phytochromes to construct synthetic TCSs for stringent activation of gene expression by near-infrared (NIR) light in laboratory and probiotic Escherichia coli strains, and in Agrobacterium tumefaciens. Orthogonal TCSs afford the multiplexed expression control of several genes by NIR and visible light. Notwithstanding substantial photochemical activation of bathy-phytochromes by visible radiation, the NIR-light-responsive systems hardly responded to red light. Evidently, light signals can be processed by TCSs into highly nonlinear responses at the physiologically relevant level of gene expression. These fundamental aspects likely extend to naturally occurring TCSs. Depending on their photosensor traits and environmental conditions, bathy-phytochromes may thus either be NIR-specific or function as colorblind receptors of light vs. darkness.

Characterization of binding kinetics and intracellular signaling of new psychoactive substances targeting cannabinoid receptor using transition-based reweighting method

diwakar@illinois.edu (Diwakar Shukla) — Mon, 03 Nov 2025 00:00:00 +0000

New psychoactive substances (NPS) targeting human cannabinoid receptor 1 pose a significant threat to society as recreational abusive drugs that have pronounced physiological side effects. These greater adverse effects compared to classical cannabinoids have been linked to the higher downstream β-arrestin signaling. Thus, understanding the mechanism of differential signaling will reveal an important structure-activity relationship essential for identifying and potentially regulating NPS molecules. In this study, we simulate the slow (un)binding process of NPS MDMB-Fubinaca and classical cannabinoid HU-210 from CB₁ using multi-ensemble simulation to decipher the effects of ligand binding dynamics on downstream signaling. The transition-based reweighing method is used for the estimation of transition rates and underlying thermodynamics of (un)binding processes of ligands with nanomolar affinities. Our analyses reveal major interaction differences with transmembrane TM7 between NPS and classical cannabinoids. A variational autoencoder-based approach, neural relational inference (NRI), is applied to assess the allosteric effects on intracellular regions attributable to variations in binding pocket interactions. NRI analysis indicates a heightened level of allosteric control of NPxxY motif for NPS-bound receptors, which contributes to the higher probability of formation of a crucial triad interaction (Y^7.53-Y^5.58-T^3.46) necessary for stronger β-arrestin signaling. Hence, in this work, MD simulation, data-driven statistical methods, and deep learning point out the structural basis for the heightened physiological side effects associated with NPS, contributing to efforts aimed at mitigating their public health impact.

Insight into the bioactivity and action mode of betulin, a candidate aphicide from plant metabolite, against aphids

zhouhong1990@swu.edu.cn (Danlong Jing) — Mon, 03 Nov 2025 00:00:00 +0000

Pest-resistant plants usually utilize secondary metabolites to cope with insect infestation. Betulin, a key bioactive compound in aphid-resistant wild peach, possesses promising applications in crop protection. Here, betulin, in both greenhouse and field experiments, displayed excellent control efficacy against Myzus persicae. RNA-seq, quantitative real-time PCR (qRT-PCR), and western blotting revealed that betulin significantly inhibited the expression of MpGABR (encoding a GABA_A receptor). Besides, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, microscale thermophoresis (MST) and voltage-clamp assays indicated that betulin bound to MpGABR (K_d = 2.24 µM) and acted as an inhibitor of MpGABR. Molecular docking, mutagenesis, and genome editing suggested that THR228 is a critical and highly conserved site in MpGABR that betulin binds to specifically, causing aphid death. Overall, the activity of betulin depends on specific targeting and inhibition of MpGABR. Elucidating the mechanism of action of this peach-derived insecticide may offer a sustainable green strategy for aphid control.

Brown adipose tissue and skeletal muscle coordinately contribute to thermogenesis in mice

hsakaue@tokushima-u.ac.jp (Hiroshi Sakaue) — Mon, 27 Oct 2025 00:00:00 +0000

Endotherms increase the rate of metabolism in metabolic organs as one strategy to cope with a decline in the temperature of the external environment. However, an additional major contributor to maintenance of body temperature in a cold environment is contraction-based thermogenesis in skeletal muscle. Here, we show that impairment of hind limb muscle contraction by cast immobilization induced a loss of function of skeletal muscle and activated brown adipose tissue (BAT) thermogenesis as a compensatory mechanism. BAT utilizes free branched-chain amino acids (BCAAs) derived from skeletal muscle as an energy substrate for thermogenesis, and interleukin-6 released by skeletal muscle stimulates BCAAs production in muscle for support of BAT thermogenesis. Additionally, this thermoregulatory system between BAT and skeletal muscle may also play an important role in response to cold temperatures or acute stress. Our findings suggest that BAT and skeletal muscle cooperate to maintain body temperature in endotherms.

Pleomorphic effects of three small-molecule inhibitors on transcription elongation by Mycobacterium tuberculosis RNA polymerase

carlosb@berkeley.edu (Alexander B Tong) — Fri, 03 Oct 2025 00:00:00 +0000

The Mycobacterium tuberculosis RNA polymerase (MtbRNAP) is the target of the first-line anti-tuberculosis inhibitor rifampin; however, the emergence of rifampin resistance necessitates the development of new antibiotics. Here, we communicate the first single-molecule characterization of MtbRNAP elongation and its inhibition by three diverse small-molecule inhibitors: N(α)-aroyl-N-aryl-phenylalaninamide (D-IX216), streptolydigin (Stl), and pseudouridimycin (PUM) using high-resolution optical tweezers. Compared to Escherichia coli RNA polymerase (EcoRNAP), MtbRNAP transcribes more slowly, has similar mechanical robustness, and only weakly recognizes E. coli pause sequences. The three small-molecule inhibitors of MtbRNAP exhibit strikingly different effects on transcription elongation. In the presence of D-IX216, which inhibits RNAP active-center bridge-helix motions required for nucleotide addition, the enzyme exhibits transitions between slowly and super-slowly elongating inhibited states. Stl, which inhibits the RNAP trigger-loop motions also required for nucleotide addition, inhibits RNAP primarily by inducing pausing and backtracking. PUM, a nucleoside analog of UTP, in addition to acting as a competitive inhibitor, induces the formation of slowly elongating RNAP-inhibited states. Our results indicate that the three classes of small-molecule inhibitors affect the enzyme in distinct ways and show that the combination of Stl and D-IX216, which both target the RNAP bridge helix, has a strong synergistic effect on the enzyme.

A drug repurposing approach reveals targetable epigenetic pathways in Plasmodium vivax hypnozoites

STEVEN.MAHER@uga.edu (Agnes Orban) — Tue, 30 Sep 2025 00:00:00 +0000

Radical cure of Plasmodium vivax malaria must include elimination of quiescent ‘hypnozoite’ forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets for hypnozoites, we screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library and a collection of epigenetic inhibitors against P. vivax liver stages. From both libraries, we identified inhibitors targeting epigenetics pathways as selectively active against P. vivax and P. cynomolgi hypnozoites. These include DNA methyltransferase inhibitors as well as several inhibitors targeting histone post-translational modifications. Immunofluorescence staining of Plasmodium liver forms showed strong nuclear 5-methylcystosine signal, indicating liver stage parasite DNA is methylated. Using bisulfite sequencing, we mapped genomic DNA methylation in sporozoites, revealing DNA methylation signals in most coding genes. We also demonstrated that methylation level in proximal promoter regions as well as in the first exon of the genes may affect, at least partially, gene expression in P. vivax. The importance of selective inhibitors targeting epigenetic features on hypnozoites was validated using MMV019721, an acetyl-CoA synthetase inhibitor that affects histone acetylation and was previously reported as active against P. falciparum blood stages. In summary, our data indicate that several epigenetic mechanisms are likely modulating hypnozoite formation or persistence and provide an avenue for the discovery and development of improved radical cure antimalarials.

Blue-shifted ancyromonad channelrhodopsins for multiplex optogenetics

John.L.Spudich@uth.tmc.edu (Alyssa Palmateer) — Fri, 19 Sep 2025 00:00:00 +0000

Light-gated ion channels from protists (channelrhodopsins or ChRs) are optogenetic tools widely used for controlling neurons and cardiomyocytes. Multiplex optogenetic applications require spectrally separated molecules, which are difficult to engineer without disrupting channel function. Scanning numerous sequence databases, we identified three naturally blue-shifted ChRs from ancyromonads. They form a separate branch on the phylogenetic tree and contain residue motifs characteristic of anion ChRs (ACRs). However, only two conduct chloride, whereas the closely related Nutomonas longa homolog generates inward cation currents in mammalian cells under physiological conditions, significantly exceeding those by previously known tools with similar spectral maxima (peak absorption at ~440 nm). Measurements of transient absorption changes and pH titration of purified proteins combined with mutant analysis revealed the roles of the residues in the photoactive site. Ancyromonad ChRs could be activated by near-infrared two-photon illumination, a technique that enables the deeper-tissue optogenetic activation of specific neurons in three dimensions. Both ancyromonad ACRs allowed optogenetic silencing of mouse cortical neurons in brain slices. Ancyromonas sigmoides ACR (AnsACR) expression in cholinergic neurons enabled photoinhibition of pharyngeal muscle contraction in live worms. Overall, our results deepen the mechanistic understanding of light-gated channel function and expand the optogenetic toolkit with potent, blue-shifted ChRs.