Biology subjects

Zhang, M.

Publications and source records attributed to Zhang, M..

At least 199 records · Page 11Linked to original sources

bioRxiv2025

Spliformer-v2 predicts multi-tissue RNA splicing and reveals functional genomic links with neurodegenerative diseases

Precise regulation of pre-mRNA splicing underlies transcriptomic diversity and is disrupted in aging and disease, yet tissue-specific splice-altering genetic variants remain poorly resolved. Here, we present Spliformer-V2, a SegmentNT-based deep learning model for predicting and interpreting variant effects on RNA splicing across human tissues. We generated a diploid sequence resolved RNA splice map from paired whole-genome-sequencing and RNA-seq data across 12 central nervous system (CNS) and 6 peripheral tissues for model development. Spliformer-V2 outperformed SpliceTransformer, Pangolin and AlphaGenome in predicting splice-site usage, identified tissue-specific splicing regulatory motifs, and revealed tissue vulnerability to pathogenic splice-altering variants. Analyses of loci associated with 8 neurological diseases prioritized CNS-specific mis-splice-vulnerable genes. In 1,405 amyotrophic lateral sclerosis (ALS) genomes, Spliformer-V2 nominated rare splice-altering variants enriched in PTPRN2, which showed reduced expression in TDP-43-depleted neurons. PTPRN2 overexpression rescued C9ORF72-patient derived motor neuron degeneration and modulated TDP-43 mislocalization, indicating it as a potential therapeutic modifier in ALS.

bioinformatics↗

bioRxiv2025

Cortex-Wide, Cellular-Resolution Volumetric Imaging with a Modular Two-Photon Imaging Platform

Mapping cortex-wide neuronal activity at single-cell resolution has been limited by the physical trade-off between numerical aperture and field-of-view (FOV) in two-photon microscopes. We present Meso2P, a modular two-photon platform that decouples excitation and detection by introducing a lateral paraboloid fluorescence collector. The design sustains an effective NA 0.87 over a contiguous 6 x 6 mm2 FOV at high speed (2,048 x 2,048 pixels at 7.67 Hz). The modular platform can be upgraded with optional modules for simultaneous multi-plane imaging (1-4 planes at full resolution and speed), volumetric imaging (6 x 6 x 0.5 mm3, 2,048 x 2,048 x 28 voxels at 1 Hz capturing> 210,000 neurons), and holographic two-photon optogenetic stimulation for targeted perturbations. To handle the resulting large-scale data, we provide an open-source deep-learning pipeline that automates motion correction, segmentation, and spike inference. We demonstrate cortex-wide sensory responses, layer-specific network synchrony during anaesthesia, and in-vivo tracking of micro and nanoplastic distribution. Meso2P therefore provides a reproducible route to high-throughput volumetric imaging across almost the entire cortex with high detection efficiency.

neuroscience↗

bioRxiv2025

The mechanism of oncogenic PI3K lipid kinase variants at the membrane and their cryptic pockets

Cancer-prone alleles exhibit single hotspot mutations. However, the combination of a cancer hotspot and a weak or moderate mutation (the one-two punch hypothesis) produces same-allele double variants with a significantly different and potentially graded clinical phenotypic spectrum. Oncogenic PI3K variants, which are also associated with benign tumors and neurodevelopmental disorders, offer statistical support for this model. Using atomistic molecular dynamics (MD) simulations, we revealed that PI3K variants with single and double mutations exhibit expanded conformational profiles. Double mutations significantly shift the conformational ensembles toward the active form--a more pronounced effect than a single mutation. These double mutants facilitate nSH2 release, iSH2 shift, and A-loop protrusion in solution, promoting PIP2 substrate recruitment at the membrane. We discovered that observable, potentially drug-targetable cryptic pockets are PI3K mutation-specific. Ab initio discovery of these pockets through simulations is essential for AI-aided virtual drug screening. A key challenge is that a single drug is often ineffective against PI3K variants due to their diverse conformational spectra. To address this, we propose a conformational selection strategy involving a combination of allosteric drugs for variants with graded conformational spectra, particularly those with strong double mutations; we identified such potentially targetable cryptic pockets in double mutants conformers.

biophysics↗

bioRxiv2025

Integrated analysis of crosslink-ligation data for the detection of RNA 2D/3D structures and interactions in vivo.

ii.SUMMARY/ABSTRACTThe latest advancements and implementation of crosslinking- and proximity-ligation based methods have transformed the study of RNA structure and interactions in living cells. Despite this progress, sequencing data must be meticulously processed to elucidate information on RNA. This presents distinct challenges, as noted in the Psoralen Analysis of RNA Interactions and Structures (PARIS) and Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC) studies. In this chapter, we detail the process used to analyze RNA sequencing data derived from crosslink-ligation based experiments. We detail the strategies used to pre-process raw data, map to a reference genome, classify reads, and assemble duplex groups. Further, we show an example of analyzing structural data from the ribosomal RNA.

bioinformatics↗

bioRxiv2025

Design principles of the common Gly-X6-Gly membrane protein building block

Protein behavior in lipid is poorly understood and inadequately represented in current computational models. Design and prediction abilities for bilayer-embedded molecular structures may be improved by characterizing membrane proteins most frequent, favored structural features to glean both context-specific and general principles. We used protein design to proactively interrogate the sequence-structure relationship and stabilizing atomic details of two highly prevalent antiparallel transmembrane (TM) motifs with Small-X6-Small consensus sequences. A fragment-based data-mining and sequence statistical inference method including cross-evolutionary structure-aligned covariance enabled engineering of de novo multi-span TM protein assemblies by successfully encoding Gly-X6-Gly and Ala-X6-Ala building blocks. A highly stable glycine-based designs X-ray structure hosts C-H{middle dot}{middle dot}{middle dot}O=C H-bonding alongside extensive backbone-directed van der Waals packing, idealizing features of this motif in Nature. Data-driven design navigates sequence space to directly inquiry upon how to encode and stabilize vital membrane protein structural elements, facilitating efficacious construction of lipid-embedded architectures of increasing complexity. SignificanceMembrane proteins comprised of -helices pack together within lipid bilayers, establishing stabilities and architectures that brace function and guide evolution. De novo design was used to clarify the consensus sequences and molecular features encoding of one exceedingly common TM helix packing architecture ([~]10% of those in membrane folds). Small-X6-Small residue patterns were proven to reliably drive minimal proteins into these antiparallel TM geometries, revealing glycine mainchain hydrogen bonding or via fully apolar interfaces can encode the motif. Optimizing steric packing was the most decisive feature amongst the synthetic proteins. New membrane-specific design methods and model molecules are validated in route to outlining this important sequence-structure relationship broadly impacting the membrane proteome and revealing generalized structure-energetic imperatives governing interactions in lipid.

biophysics↗

bioRxiv2025

Competing Programs Shape Cortical Sensorimotor-Association Axis Development

The neocortex is organized along a dominant sensorimotor-to-association (S-A) axis, anchored by modality-specific primary sensorimotor areas at one end and transmodal association areas that form distributed networks supporting abstract cognition at the other. The developmental mechanisms shaping this axis remain elusive. Here, we present converging multispecies evidence supporting the Multinodal Induction-Exclusion in Network Development (MIND) model, in which S-A patterning is governed by competing processes of induction and exclusion, driven by opposing transcriptomically-defined identity programs emerging from different nodes. Key molecular and connectional features of association cortices arise through pericentral programs, originating around fronto-temporal poles and partially regulated by retinoic acid. They progress inward toward central territories of the naive neocortex along fronto-temporally polarized trajectories. Central programs are induced through interactions between topographically separated first-order sensorimotor thalamocortical inputs and the neocortex, promoting the formation of primary areas while excluding pericentral programs. Influenced by SATB2 and ZBTB18, these evolutionarily conserved programs compete for the same territory and create spatial compartmentalization of axon guidance, cell-cell adhesion, retinoic acid signaling, synaptogenesis, Wnt signaling, and autism risk genes. Notably, PLXNC1 and SEMA7A exhibit anti-correlated expression and repulsive functions in shaping cortico-cortical connectivity along the S-A axis. These processes of induction and exclusion establish an S-A equilibrium and topography in which primary sensorimotor areas emerge as focal islands within the broader ocean of distributed associative networks. The MIND model provides a unifying framework for understanding experimental, evolutionary, and clinical phenomena, revealing induction and exclusion as antagonistic complementary principles shaping the S-A axis and processing hierarchies.

neuroscience↗

bioRxiv2025

The neural processes underpinning flexible semantic retrieval in visual and auditory modalities

Contemporary accounts of semantic cognition propose that conceptual knowledge is supported by a heteromodal conceptual store and controlled retrieval processes. However, it remains unclear how the neural basis of semantic control varies across modalities. Recent models of cortical organization suggest that control networks are distributed along a unimodal-to-heteromodal cortical gradient, with the semantic control network (SCN) located in more heteromodal cortex than the domain-general multiple demand network (MDN). We used fMRI to examine how these networks respond to semantic control demands in visual and auditory tasks. Participants judged the semantic relatedness of spoken and written word pairs. On half of the trials, a task cue specified the semantic feature to guide retrieval; on the remaining trials, no such cue was given. The SCN showed greater activation when task knowledge was available, consistent with a role in the top-down control of semantic retrieval across modalities. In contrast, the MDN showed greater activation for spoken words, likely reflecting increased demands in speech perception. These findings demonstrate a dissociation between control networks, with SCN involvement modulated by task structure and MDN activity influenced by input modality.

neuroscience↗

bioRxiv2025

Programmed downregulation of METTL3 is essential for decidualization in both humans and mice

N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in regulating gene expression. Our prior research, alongside that of others, demonstrated that conditional uterine knockout of methyltransferase-like 3 (METTL3), the enzyme responsible for m6A modification, led to complete failure of embryo implantation and decidualization. Intriguingly, METTL3 expression is downregulated rather than upregulated in human endometrial stromal cells (HESCs) during in vitro decidualization and in mouse decidual tissues during pregnancy. We hypothesized that this decline in METTL3 expression is indispensable for successful decidualization. To test this hypothesis, we overexpressed METTL3 in HESCs and observed impaired decidualization in vitro. Additionally, we generated genetically engineered mice with uterine-specific METTL3 overexpression using Pgr-Cre, which exhibited subfertility mainly due to impaired decidualization. Further investigation revealed a marked decrease in HAND2, a well-established regulator of decidualization, following METTL3 overexpression. Mechanistically, we uncovered that METTL3 destabilizes HAND2 mRNA via m6A modification at the 5'-UTR. In summary, our study underscores the critical role of programmed METTL3 downregulation in decidualization by sustaining HAND2 expression.

physiology↗

bioRxiv2025

ERM proteins regulate the shape and number of Endoplasmic Reticulum-Plasma Membrane Junctions in neurons

Endoplasmic Reticulum (ER) - Plasma Membrane (PM) Junctions (EPJs) are specialized contact sites between ER membrane and the inner leaflet of PM. These junctions are critical for lipid exchange and Ca2+ signaling. In excitable cells like neurons and muscle, EPJs further modulate membrane excitability by regulating Ca2+ homeostasis. The mechanisms controlling EPJ abundance and morphology remain poorly understood. Using in vivo fluorescence imaging and electron microscopy of C. elegans neurons, we showed that EPJs form discrete, patch-like structures distributed across the soma. Through a forward genetics screen, we identified two conserved ERM (Ezrin-Radixin-Moesin) proteins, FRM-4 and FRM-1, as key regulators of EPJ shape and abundance. Both proteins localize to EPJs and exhibit liquid-liquid phase separation properties (LLPS). in vitro, purified FRM-4 binds to FRM-1, and together bundle filamentous actin. However, their presence in LLPS condensates and actin-bundling activity are mutually exclusive. In vivo, F-actin cables surround--but do not penetrate--EPJs, where FRM proteins are enriched as phase-separated condensates. Loss of FRM-4, FRM-1, or disruption of F-actin led to increased mobility of EPJs that fused into fewer but enlarged junctions. Together, our findings demonstrate that FRM-4 and FRM-1 control EPJ morphology by organizing peri-junctional F-actin networks, thereby restricting EPJ mobility and fusion.

cell biology↗

bioRxiv2025

PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

Membraneless organelles (MLOs) formed by liquid-liquid phase separation contribute to intracellular compartmentalization of specific biological functions. The dynamic regulation of MLOs is critical for RNA metabolism, stress response, and signal transduction. While the number and size of MLOs serve as valuable indicators of cellular states, leveraging their spatial distribution and diverse quantitative characteristics to infer cellular conditions has posed significant challenges. Here, we present processing body (PB)-scope: an unsupervised deep learning-based framework designed for imaging-based phenotypic screening of PB, one representative MLO. The model was trained on a substantial dataset comprising over 400,000 single-cell confocal microscopy images from a colon cancer cell line treated with 280 compounds. PB-scope enabled precise drug classification based on their effects on PB characteristics, including number and size. This artificial intelligence framework offers insights previously obscured by the dynamic and subtle nature of PBs. Notably, PB-scope identified drugs with a shared function in the JAK signaling pathway, a previously unrecognized regulator of PB dynamics. PB-scope holds the potential for adapting to the study of other MLOs, offering broad applicability in this emerging field of cell biology.

cell biology↗

bioRxiv2025

A Thiopurine-like Mutagenic Process Defines TGCT Subtypes

Testicular germ cell tumors (TGCTs) are the most common malignancy in young men, exhibit a unique developmental origin and exceptional chemosensitivity. However, the molecular distinctions between TGCT subtypes remain poorly understood. Here we present a comprehensive genomic analysis of 252 treatment-naive primary TGCTs, integrating deep whole-genome sequencing with matched transcriptomic and epigenomic data. We identify new driver genes and uncover defining features of TGCTs, including pervasive chromosome X amplification with subtype-specific X chromosome inactivation, and a germ cell-like transcriptional program. Although previously reported, whole genome doubling (WGD) in TGCTs is further characterized here as ubiquitous, developmentally early, and associated with age at onset. Seminomas are enriched for early driver mutations, secondary WGD events, sustained XIST expression and replication stress-associated indel mutational signatures, while non-seminomas show greater structural complexity, subclonal diversity, relatively earlier-onset WGD, extended tumor latency, and telomere elongation. Moreover, we identify a mutational signature, SBS87, that is exceptionally rare across cancers with exception of thiopurine-treated leukemia, but strikingly prevalent in TGCT, especially non-seminomas. SBS87 is linked to extended tumor latency and telomere elongation, implicating possible environmental or endogenous processes that mimic thiopurine-induced DNA damage in TGCT pathogenesis. Collectively, our findings define TGCTs as molecularly distinct tumors shaped by early genomic instability and highlight SBS87 as a novel mutational footprint with potential etiologic and clinical relevance.

genomics↗

bioRxiv2025

ATP2B1 expression identifies human hematopoietic stem cells across ontogeny with superior repopulation and self-renewal capacity

Long-term hematopoietic stem cells (LT-HSC) maintain lifelong hematopoiesis while preserving the stem cell compartment through self-renewal. The human LT-HSC compartment is molecularly and functionally heterogeneous and also varies across ontogeny. Dissecting the molecular basis for this variation is impeded by the absence of immunophenotypic markers to resolve LT-HSC heterogeneity. Here, we identified ATPase plasma membrane Ca2+transporting 1 (ATP2B1/PMCA1) as a novel cell surface marker that is heterogeneously expressed by CD49f+ LT-HSC across ontogeny. ATP2B1 immunophenotypic expression stratified human CD49f+ LT-HSC from fetal liver (FL), neonatal cord blood (CB) and adult mobilized peripheral blood (mPB) sources into functionally distinct subpopulations in single-cell (sc) clonogenic assays. CD49f+ATP2B1+ LT-HSC exhibited superior long-term repopulation and self-renewal capacities in vivo compared to CD49f+ATP2B1- LT-HSC. Molecular profiling by scMultiome and immunofluorescence microscopy point to enrichment of an HSC self-renewal program that includes the TFEB-endolysosomal axis in CD49f+ATP2B1+ LT-HSC. Our study provides a new tool to dissect the heterogeneous molecular programs in LT-HSC across ontogeny.

cell biology↗

bioRxiv2025

Contrastive Learning-Enhanced Drug Metabolite Prediction Drives the Discovery of an Orally Bioavailable RSK4 Inhibitor for Esophageal Cancer

Drug metabolism liabilities are a major bottleneck in drug development, necessitating accurate metabolite prediction tools. While AI-driven methods have advanced, challenges remain in achieving high-precision, robustness, broad reaction coverage and seamless integration into the drug development workflow. To address this, we first constructed the most comprehensive human-specific drug metabolism database to date, encompassing 11,665 human metabolic reactions and 2,497 high-quality rules. Leveraging this resource, we developed ConMeter (Contrastive learning and feature interaction based Metabolic Reaction prediction), a novel model integrating chemical feature interaction and contrastive learning. ConMeter demonstrates significant improvements, achieving [~]80% accuracy in identifying at least one metabolite within the top 5 predictions and showing an average 25% relative enhancement in ranking true positives compared to state-of-the-art methods. The comprehensive database also expands the range of predictable metabolic reactions. To demonstrate the utility of ConMeter, we integrated it into a metabolite prediction-guided drug design strategy aimed at addressing the low oral bioavailability of a metabolically vulnerable RSK4 lead compound -- a pervasive issue among known RSK inhibitors. This strategy successfully yielded R636, a novel RSK4 inhibitor exhibiting remarkably improved bioavailability (a 63-fold increase, reaching 63% absolute bioavailability) while maintaining its potent activity. Notably, R636 showed favorable safety profile and significant anti-tumor efficacy against esophageal squamous cell carcinoma (ESCC) in vitro and in two distinct patient-derived xenograft (PDX) mouse models. This work introduces ConMeter as a powerful and practical tool for high-accuracy metabolite prediction, demonstrating its potential to accelerate lead optimization, exemplified by the discovery of R636 as a promising orally bioavailable candidate for ESCC.

cancer biology↗

bioRxiv2025

Lineage-Traced PTBP1 Depletion in Mature Astrocytes Reveals Distinct Splicing Alterations Without Neuronal Features

Astrocyte-to-neuron reprogramming via depletion of PTBP1, a potent repressor of neuronal splicing, has been proposed as a therapeutic strategy, but its efficacy remains debated. While some reported successful conversion, others disputed this, citing a lack of neuronal gene expression as evidence of failed reprogramming. This interpretation was further challenged, attributed to incomplete PTBP1 inactivation, fueling ongoing controversy. Mechanistic understanding of the conversion, or the lack thereof, requires investigating, in conjunction with lineage tracing, the effect of Ptbp1 loss of function in mature astrocytes on RNA splicing, which has not yet been examined. Here, we genetically ablated PTBP1 in adult Aldh1l1-Cre/ERT2 Ai14 mice to determine whether lineage traced Ptbp1 knockout astrocytes exhibited RNA splicing alterations congruent with neuronal differentiation. We found no widespread induction of neurons, despite a minuscule fraction of knockout cells showing neuron-like transcriptomic signatures. Importantly, PTBP1 loss in mature astrocytes induced splicing alterations unlike neuronal splicing patterns. These findings suggest that targeting PTBP1 alone is ineffective to drive neuronal reprogramming and highlight the need for combining splicing and lineage analyses. Loss of astrocytic PTBP1 is insufficient to induce neuronal splicing, contrasting with its well-known role in other non-neuronal cells, and instead affects a distinct astrocytic splicing program.

neuroscience↗

bioRxiv2025

An Antibody Cocktail-Based Immunoaffinity-LC-MS Method Enabled Ultra-Sensitive and Robust Quantification of Circulating Proinsulin Proteoforms and C-peptide

Accurately measuring circulating proinsulin proteoforms is crucial for clinical investigation of diabetes, but was previously not feasible owing to limited assay specificity/sensitivity. Here we devised a highly sensitive LC-MS-based strategy to quantify intact proinsulin, des-31,32 and des-64,65 proinsulin, and C-peptide in circulation. The method involves: i)quantitative, robust affinity capture using an optimized antibody cocktail, eliminating the severe quantitative bias across multiple proteomes typically introduced when using a single antibody; ii)Lys-C digestion producing unique signature peptides for each proteoform, and iii)trapping-nano-LC coupled with FAIMS/dCV-MS for an ultra-sensitive analysis. The selective trapping/delivery ensured sensitive/selective analysis of the targets while achieving excellent analytical robustness that is critical for clinical assays, and the FAIMS/dCV substantially reduces baseline noise/interferences, further enhancing S/N. The assay achieved exceptional sensitivity, with serum LOQs of 1.7, 2.3, and 3.6 pg/mL respectively for intact-proinsulin, des-31,32 and des-64,65, representing the first assay capable of sensitively quantifying these major circulating proinsulin proteoforms. We applied this assay to 78 subjects, including autoantibody positive(n=20) and new-onset type-1-diabetes(T1D, n=19) with respective age/sex/BMI-matched controls, enabling the first accurate profiling of proinsulin proteoforms in clinical groups. The assay results demonstrated a clear separation of control and new-onset T1D groups that a parallel total-proinsulin ELISA assay fails to capture. Furthermore, distinct expression patterns in relative abundance ratios among proteoforms were observed across clinical groups. This assay may provide valuable insights into the {beta}-cell functions and the onset/progression of diabetes and other associated conditions. Moreover, the strategy is broadly applicable to targeted measurement of other biomarker proteoforms.

biochemistry↗

bioRxiv2025

Engineering a mouse-adapted SADS-CoV and establishing a neonatal mouse model to study its infection

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging bat-origin alphacoronavirus causing severe disease in neonatal piglets, with significant economic losses to the swine industry as a consequence. The virus exhibits a broad species tropism, infecting cells derived from pigs, humans and mice, highlighting its potential for cross-species transmission. Due to drawbacks associated with the use of young piglets, there is a need for an appropriate small animal model to study SADS-CoV biology. Here we established a mouse infection model based on a murinized mutant of the virus, mSADS-CoV, in which the ectodomain of the viral spike protein was replaced by that of the murine coronavirus mouse hepatitis virus (MHV). This chimeric virus, generated through targeted RNA recombination, replicated efficiently in murine cell cultures and exhibited an age-dependent infection in neonatal mice that was lethal in 2-day-old BALB/c mice affecting various organs, notably the intestine. We validated our infection model by successfully verifying the efficacy of the RNA-dependent RNA polymerase inhibitor remdesivir (RDV). The model will serve as a valuable tool for studying SADS-CoV pathogenesis and for elucidating the roles of host factors in viral replication as well as for preclinical evaluation of vaccine candidates and antiviral compounds targeting the viral replication machinery. Author SummarySADS-CoV poses a dual threat to the swine industry and public health because of its broad species tropism and potential for cross-species transmission. The emergence of other bat-derived coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV), underscores the need for robust models to study these pathogens. The successful rescue of mSADS-CoV and the development of a mouse infection model represent significant advancements in SADS-CoV research. This model not only enables the evaluation of antiviral therapeutics such as RDV but also provides a powerful platform for investigating viral replication mechanisms and host-pathogen interactions, offering critical insights for pandemic preparedness.

microbiology↗

bioRxiv2025

Widespread distribution of the BpfA-carrying bacteria involved in the bisphenol F degradation in Pseudomonadota and Actinomycetota

The global use of bisphenol F (BPF) has led to widespread environmental contamination, posing significant threats to ecosystems and human health. However, the genes involved in BPF degradation remained unknown. In this study, a novel oxidase gene, bpfA, was obtained from strain Microbacterium sp. F2 through a four-step purification strategy. BpfA is classified as a member of the 4-phenol oxidizing (4PO) subfamily and contains a conserved FAD-binding domain (Asp152 and His393) as well as a Tyr-Tyr-Arg triad (Tyr93, Tyr474, Arg475). BpfA catalyzes the conversion of BPF to 4,4-dihydroxydibenzophenone (DHBP) through a series of three consecutive reactions. In addition, BpfA exhibits catalytic activity towards 4-alkylphenols, such as vanillyl alcohol (VA), 4-n-propylguaiacol (4PG), and 4-(methoxymethyl)phenol (4MOP). BpfA homologs are widely distributed in the environment, particularly in soil. Bioinformatics-based taxonomic profiling revealed that BpfA homologs are widely distributed in metagenomes from cultivated land and forests, mainly belonging to Pseudomonadota and Actinomycetota. This study enhances our understanding of the microbial degradation mechanism of BPF, elucidates the catalytic function of the novel oxidase gene bpfA and its distribution pattern in the environment, and provides important insights into the evolutionary origin of BPF degradation genes and the targeted mining of environmental microbial resources.

microbiology↗

bioRxiv2025

Programmed inhibition of an innate immune receptor via de novo designed transmembrane proteins

Transmembrane domains of immune complexes transmit precise signals across lipid bilayers. Probing their interactions has the potential to yield mechanistic insights relevant to therapeutic design. However, our capability to generate molecules directed to bind lipid-embedded sites is limited. Here, we demonstrate a computational strategy to design polypeptides targeting Toll-like receptor 4 (TLR4), a mediator of inflammatory signaling, directly within membranes. TLR4 poses a formidable molecular recognition challenge, as its transmembrane domain is largely apolar, lacks a defined sequence motif, and exhibits an underdetermined structure-function relationship. One synthetic protein binds TLR4s transmembrane domain and antagonizes NF{kappa}B signaling in human cells, proving that precise transmembrane domain interactions are essential for cross-membrane conformational coupling. This work refines design principles for encoding stable interactions in cellular membranes and expands the range of lipid-embedded mechanisms accessible to probe with computationally derived molecules.

biochemistry↗