Biology subjects

Yang, Z.

Publications and source records attributed to Yang, Z..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Targeting NAT10 suppresses TFH responses and alleviates allergic asthma via an ac4C-TOX2 axis

Chemical modifications of RNA, such as N4-acetylcytidine (acC), fine-tune gene expression, but their roles in cell fate determination within the immune system remain poorly understood. Here, we identify the RNA cytidine acetyltransferase NAT10 as a pivotal regulator of T follicular helper (TFH) cell differentiation and the germinal centre response. T cell-specific ablation of Nat10 in mice severely impaired TFH cell development, germinal centre formation, and antibody production following viral infection. Integrated epitranscriptomic and transcriptional profiling revealed that NAT10 deposits acC modifications on a cohort of mRNAs critical for immune function. We pinpointed the transcription factor TOX2 as a key downstream target, showing that acC modification within its mRNA coding sequence enhances both transcript stability and translation efficiency, thereby promoting TOX2 protein expression. Enforced TOX2 expression fully rescued the TFH differentiation defect in NAT10-deficient cells. This NAT10-acC-TOX2 axis is conserved in humans, where its activity correlates with the magnitude of vaccine-elicited TFH responses. Furthermore, we identified R428 as a potent NAT10 inhibitor and demonstrated that its administration could suppress pathogenic TFH responses and alleviate symptoms in a mouse model of allergic asthma. Our work elucidates a central epitranscriptomic mechanism controlling T helper cell fate and humoral immunity, and nominates NAT10 as a potential therapeutic target for TFH-driven diseases.

immunology↗

bioRxiv2026

Restoring Klf9 Expression with Pressure Overload Leads to Metabolic Maladaptation and Early Onset of Heart Failure

Klf9 is a cardiac-enriched transcription factor of the Kruppel-like factor (Klf) family. Klf9 levels decrease during cardiac hypertrophy; however, no studies have examined its transcriptional targets or role in the progression of hypertrophy. Here, we report genome-wide differential Klf9 occupancy during cardiac hypertrophy, with a predominant enrichment at the metabolic gene promoters. Further, using conditional Klf9 knock-in mice subjected to pressure overload for 1 or 2 weeks, we show that restoring Klf9 expression initially inhibits hypertrophy but later leads to early-onset heart failure. We conclude that a decrease in Klf9 is required for metabolic adaptations that support the development of compensatory hypertrophy.

cell biology↗

bioRxiv2026

Human antibody heavy chain variable region exon interacts with 3' regulatory region to form a stable somatic hypermutation center

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) of immunoglobulin heavy chain (HC) variable region exons in germinal center (GC) B cells, allowing selection of affinity-matured B cell receptors. The V(D)J exon location is privileged for SHM targeting of diverse sequences within it. In analogy to the related HC class switch recombination center, we proposed that cohesin-mediated loop extrusion juxtaposes widely separated enhancers, including 3 Igh regulatory region ("3RR"), with the V(D)J exon in GC B cells to establish a SHM-center (SHM-C) "privileged" for AID access. To test this hypothesis, we employed a human RAMOS GC B lymphoma cell SHM model with inducible AID expression in which we targeted an unmutated mouse HC V(D)J exon in place of its human counterpart. Activation of AID expression in this cycling RAMOS line for 10 days led to robust SHM accumulation in the mouse V(D)J exon with a pattern similar to that in mouse GC B cells. In this model, the mouse V(D)J exon was indeed juxtaposed to the two human 3RRs and other elements consistent with SHM-C formation. While deletion of individual 3RRs had little effect, deletion of both greatly reduced SHM, but left V(D)J exon transcription unabated. Moreover, the SHM-C and SHM accumulation remained intact over a 10-day period in viably G1-arrested and viably G1-arrested RAD21 (cohesin)-depleted versions of the model. We discuss implications for SHM-C structure and function, its long-term functional stability in the absence of the loop extrusion process proposed to assemble it, and for 3RR SHM functions beyond transcriptional activation. Significance StatementUpon encountering pathogens, B cells enter specialized lymphoid structures known as germinal centers (GCs). GC B cells express activation-induced cytidine deaminase (AID) that initiates somatic hypermutation (SHM) of the portion of antibody genes that encodes antigen recognition. B cells in which mutations increase antibody affinity for activating pathogen are selected to enhance antibody responses. We proposed SHM occurs in a chromosomal structure in the antibody gene locus termed a SHM-center (SHM-C). By studying SHM in a human B cell lymphoma model, we provide direct evidence for existence of a SHM-C assembled from different functional components normally separated by considerable distances. We also find that the SHM-C is surprisingly stable and continues to function for many days in non-dividing cells.

immunology↗

bioRxiv2026

A novel AR molecular glue degrader via bivalent engagement of the N-terminal domain and RNF213-UBE2J2 complex

Targeted protein degradation enables the elimination of disease-relevant proteins through induced proximity between E3 ligases and substrates. Current androgen receptor (AR) therapies act via the ligand-binding domain (LBD) but fail against constitutively active LBD mutants or splice variants such as AR-V7. Here, we identify GZL626, a small-molecule degrader that induces proteasome-dependent degradation of both full-length AR and AR-V7. GZL626 suppresses AR transcriptional activity, inhibits proliferation of AR-positive prostate cancer cells, and blocks SARS-CoV-2 infection. Mechanistically, GZL626 binds the intrinsically disordered AR N-terminal domain (NTD) and promotes formation of a noncanonical RNF213-UBE2J2-AR ternary complex that drives AR ubiquitination and degradation. The AR DNA-binding domain (DBD) acts as a structural hub within this assembly. Distinct from PROTACs or classical molecular glues, GZL626 engages both AR and RNF213 bivalently, remodeling protein interactions to stabilize a functional E3-E2-substrate complex. These findings establish GZL626 as a first-in-class NTD degrader with potential to overcome resistance in advanced prostate cancer.

biophysics↗

bioRxiv2026

Unearthing a fungal giant: Dianjunaceae fam. nov., a novel Paleocene lineage of Xylariales harbouring Dianjunus rex gen. et sp. nov.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene ([~]65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxons phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

microbiology↗

bioRxiv2026

A Sensory Pathway Controls Motile Microcolony Formation and Spatial Organization in Gliding Bacteria

How cells transition between distinct modes of collective organization is a fundamental question in biology. We uncover the regulatory landscape governing the choice between bacterial swarming and zorbing. In Flavobacterium johnsoniae, zorbs are spherical, biofilm-like microcolonies that migrate collectively, whereas swarms are multilayered and ordered populations that expand planarly. We identify RgzAB, an unorthodox sensor kinase-response regulator pair governing this transition; suppressor and knockout mutants establish the importance of RgzA phosphorelay and RgzB as a downstream component. Transcriptomic and perturbation analyses link this switch to iron availability, connecting environmental sensing to collective behavior. Remarkably, RgzA activity also governs spatial organization within mixed populations: activated cells encapsulate wildtype neighbors into segregated co-zorbs, within which wildtype cells assemble into networks undergoing an abundance-dependent, percolation-like transition. Together, these findings reveal that one sensory circuit governs bacterial collectives at nested scales, from motility choice to the internal architecture of the collective.

microbiology↗

bioRxiv2026

Restricting Dietary Isoleucine Promotes Foxo3-Dependent Mitochondrial Respiration by Reducing Caloric Intake

Our prior work demonstrated that lowering dietary branched-chain amino acids (BCAAs) improves cardiac outcomes during pressure overload-induced stress. Here, we identify isoleucine restriction (IleR) as the key driver of this effect. Dietary isoleucine restriction induces hypophagia and weight loss, recapitulating the effects of caloric restriction (CR). Although it does not prevent the initial development of left ventricular hypertrophy, it halts its progression and the decline in ejection fraction compared with controls. This is associated with preservation of electron transport chain (ETC) gene expression, cristae structure, NAD+/NADH levels, and mitochondrial respiratory capacity in cardiomyocytes, which is recapitulated by CR. Mechanistically, both IleR and CR diets increase Foxo3 expression, thereby blocking the decline in expression of its target ETC and mitochondrial genome-encoded genes. Consequently, this improves mitochondrial respiratory capacity and reduces cardiac fibrosis. We conclude that restricting dietary isoleucine improves cardiac health by increasing Foxo3 expression and mitochondrial function via a cell-autonomous mechanism and by reducing caloric intake.

molecular biology↗

bioRxiv2026

Pep2Mol: 3D Molecule Generation Targeting Protein-Protein Interfaces with Diffusion Models

Protein-protein interactions (PPIs) are central to biological processes. Designing small molecules that modulate dysregulated PPIs holds strong promise for targeting undruggable proteins. However, existing structure-based drug design approaches focus on well-defined small-molecule binding pockets and struggle to generalize to large, shallow, and chemically complex PPI interfaces. Here, we introduce Pep2Mol, a diffusion-based generative model for 3D molecule design that targets orthosteric PPI sites by explicitly incorporating binding peptides or proteins as structural guidance, moving beyond conventional pocket-conditioned generation. To enable model development and benchmarking, we curate a large-scale, high-quality dataset of 10,956 experimentally resolved protein complex structure pairs, each pairing an orthosteric competitive ligand with a protein binder at overlapping receptor interfaces. Pep2Mol integrates two SE(3)-equivariant graph neural networks that encode protein-ligand and protein-peptide interactions respectively, and fuses these representations via attention-based conditioning to jointly guide the diffusion trajectory. Extensive evaluations demonstrate that Pep2Mol generates chemically valid ligands with state-of-the-art binding affinities, providing a strong foundation for small-molecule inhibitor design against challenging PPI interfaces.

bioinformatics↗

bioRxiv2026

Ginsenoside Ro ameliorates diabetic cardiomyopathy by maintaining ciliary homeostasis and enhancing antioxidation

ObjectiveTo investigate and clarify the role of Ginsenoside Ro (GRo) in diabetic cardiomyopathy (DiaCM) and to elucidate the molecular mechanism by which GRo ameliorates DiaCM. Methods[circled1] The construct of type 2 diabetic mouse model. The bought C57BL/6 male mice were housed in a specific pathogen-free (SPF) animal facility and randomly divided into control, STZ (model), STZ + GRo, and control+GRo groups. The STZ (model) and STZ + GRo groups were fed a high-fat and high-glucose diet combined with intraperitoneal injection of streptozotocin (STZ). The control and control + GRo groups were fed a normal diet, while the control + GRo and STZ + GRo groups were treated with GRo via oral gavage. Then, all groups were evaluated for cardiac function and structure by small animal echocardiography and histological staining including hematoxylin and eosin (HE) and Massons trichrome staining to screen and confirm diabetic cardiomyopathy in mice. Finally, immunofluorescence staining of cilia in mouse heart tissue was performed to determine whether GRo inhibits abnormal ciliary growth. [circled2] The construct of cell models. First, the CCK-8 (Cell Counting Kit-8) assay was used to separately evaluate the cytotoxicity of GRo and the combination of TGF-{beta}1 and PA in myocardial fibroblasts and cardiomyocytes. Subsequently, mouse myocardial fibroblast lines (MCFs) were treated with transforming growth factor-beta 1 (TGF-{beta}1), and H9c2 cardiomyocytes were treated with palmitic acid (PA). Both cell types then received the GRo treatment. [circled3] Molecular and cellular testing. Firstly, we measured serum levels of cardiac injury markers (CK-MB, MYO, and TNNI3), glutathione (GSH), and malondialdehyde (MDA). Secondly, we examined the expression of myocardial fibrosis-related genes (Col1a1, etc.), myocardial hypertrophy markers (Nppa, etc.), cilia-specific genes (Pkd1, etc.), and oxidative stress-related genes (Nrf2, etc.) in both animal and cell samples by Western blotting and RT-qPCR. Finally, we used immunofluorescence staining of myocardial fibroblasts to detect cilia length and phalloidin staining of cardiomyocytes to measure their cross-sectional area. [circled4] The correlation mechanism. Firstly, the cilia-specific inhibitory drug HIP-4 was used to disrupt cilia homeostasis by inhibiting cilia growth. Secondly, small activating RNA (saRNA) was used to upregulate the Pkd1 gene to verify whether GRo exerts its anti-fibrotic effects through the inhibition of PC1. Results[circled1] Animal level. A diabetic cardiomyopathy mouse model was successfully established by combining STZ injection with a high-fat and high-glucose diet, and treatment with GRo significantly ameliorated the associated symptoms. [circled2] Cellular level. We successfully established a myocardial fibrosis model by treating myocardial fibroblasts with TGF-{beta}1, and a myocardial hypertrophy model by treating cardiomyocytes with PA. Immunofluorescence staining demonstrated that GRo significantly decreased cilia length in the fibrosis model, while phalloidin staining showed that GRo significantly attenuated the increase in cardiomyocyte cross-sectional area. [circled3] Molecular level. Compared with the model group, GRo treatment significantly reduced serum levels of cardiac injury markers (CK-MB, MYO and TNNI3), glutathione (GSH) and malondialdehyde (MDA). Western blotting and RT-qPCR analyses of both animal and cell samples revealed that GRo markedly alleviated indicators of myocardial fibrosis and hypertrophy, while also suppressing cilia-specific genes and oxidative stress-related genes. Overall, GRo significantly ameliorated the markers associated with myocardial fibrosis and hypertrophy, and inhibited cilia-specific protein expression as well as oxidative stress parameters. [circled4] The correlation mechanism. The cilia-specific drug hedgehog pathway inhibitor 4 (HPI-4) was used to revealed that cilia homeostasis is closely linked to myocardial fibrosis and shortened cilia inhibit the fibrosis progression. Furthermore, upregulation of the Pkd1 gene by small activating RNA demonstrated that PC1 overexpression abrogates the therapeutic effect of GRo. Finally, GRo can alleviate DiaCM.

Molecular Biology↗

bioRxiv2026

The HSV-1 immediate early protein ICP22 interacts with the human antisense function 1 protein to promote viral replication

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection.

microbiology↗

bioRxiv2026

A human intracranial map of consciousness returning from anesthesia

How consciousness arises from the brain remains a central yet unsolved question. Although substantial effort has been devoted to characterizing the neural correlates of conscious perception during wakefulness, how consciousness re-emerges from unconscious states remains poorly understood. Here, we took advantage of the rare opportunity to record human intracranial local field potentials from propofol-induced general anesthesia through the transition to behavioral engagement and subsequent full wakefulness, capturing both the stable states of unconsciousness and consciousness and the reconstituting neural dynamics that bridge them. Anesthesia cannot be defined by any single electrophysiological signature; rather, it is an organized low-frequency regime characterized by a constellation of coordinated neural phenomena, including aperiodic slow waves, alpha/beta periodicity, global alpha synchronization, and slow-wave-alpha/beta phase-amplitude coupling. Following anesthetic cessation, this regime progressively dissolved as neural excitability and dynamical complexity climbed. The appearance of conscious behavior coincided with a fast and dramatic transformation in high-gamma activity, from stochastic and unpredictable bursts to structured responses that were distributed, task-selective, and event-locked. These findings suggest that the recovery of consciousness is a multiscale reorganization in which distinct neural underpinnings rise and fall. Together, this work charts an electrophysiological map of how conscious cognition is extinguished, reconfigured, and restored in the human brain.

neuroscience↗

bioRxiv2026

Spatiotemporal Patterns and Structural Substrates of Individual Functional Variability in Youth

Youth is a period of emerging individuality and extensive neural remodeling, yet how functional brain individuality is organized across development remains unclear. Prior work has often conflated variability in functional topography with variability in functional connectivity, highlighting the need to examine them separately to better understand how functional individuality relates to brain structure and cognition. Here, using individualized functional parcellation in two large multimodal developmental cohorts (total N = 1211), we separately quantified variability in individualized functional parcellation (vIFP) and in functional connectivity (vFC). Both forms of variability were organized along the sensorimotor-association axis and were greatest in the association cortex. During development, vIFP increased significantly with age at both regional and whole-brain level, whereas vFC showed regionally specific age effects but no significant whole-brain increase. Both trajectories exhibited a common mid-adolescent inflection (14-16 years), marking a window of accelerated functional individualization. Despite shared spatial and temporal organization, vIFP and vFC showed dissociable associations with brain structure and cognition. vIFP was more strongly coupled to structural variability, whereas vFC was more strongly associated with cognitive variability. These findings reveal convergent and divergent developmental principles of topographic and connectional functional variability, highlighting their complementary roles in structural constraints and cognitive specialization.

neuroscience↗

bioRxiv2026

Systemic degradation of repressive transcription factors gates gene expression and cell fate specification

While proteasomes are best known for eliminating defective proteins or turning off signaling pathways, they also enable crucial cellular activities. Critical among these, proteasomes allow cells to initiate gene expression, but underlying targets and regulatory mechanisms remain poorly understood. Here, we report that proteasomes drive the systemic degradation of repressive trans-cription factors to eject TLE/Groucho-family co-repressors from chromatin and thereby constantly free transcription start sites for activator binding. This circuitry requires the E3 ligase SCFFBXL14, which modifies its targets dependent on presentation by TLEs, but independently of their identity. The continuous cycling of co-repressors off chromatin, as achieved by systemic turnover of a protein family, is essential for stem cells to translate developmental cues into lineage-specific gene expression, and it is disrupted by cancer mutations in TLE1 that impair SCFFBXL14-recruit-ment. We conclude that systemic degradation of repressive transcription factors establishes co-repressor dynamics required for genes expression and cell fate specification.

biochemistry↗

bioRxiv2026

Confidence-supported label-free metabolic imaging with FPhaS phase autofluorescence microscopy

Label-free optical redox imaging utilizes endogenous NAD(P)H and FAD autofluorescence to evaluate metabolism in living specimens. The conventional optical redox ratio collapses these two channels into a single value; however, it does not indicate whether a pixel has sufficient photon support or the cellular context necessary for quantitative aggregation. To address this limitation, we introduce FPhaS, a fixed-calibration phase- autofluorescence framework that integrates quantitative phase imaging (QPI) with simultaneous label-free autofluorescence multi-harmonic microscopy (SLAM), using fluorescence lifetime imaging (FLIM) solely for validation. Because QPI and SLAM are acquired with the same objective, a unified non-biological calibration aligns phase-derived structural data with the autofluorescence frame, yielding a residual error of 0.39 pixels. This calibration is maintained across all biological specimens. This shared geometric reference enables local evaluation of structural and metabolic information, rather than comparing approximately aligned images. FPhaS decomposes the data into cell presence, ratio credibility, and confidence-supported pooling. We validated FPhaS on A549 cells under high and low-photon conditions; the framework is designed to generalize to other cell and tissue types. Confidence-weighted intensity redox estimates were compared with lifetime-derived measurements within mask-locked cellular regions. Concordance improved exclusively when both the denominator photon support and an independent structural criterion were satisfied. The same reference layer generated cell-level descriptors of metabolic content, metabolic-structural organization, and measurement reliability, while also constraining the CombinedWLS reconstruction under diminished fluorescence acquisition. FPhaS redefines label-free metabolic imaging from producing comprehensive ratio maps to identifying regions where optical evidence substantiates quantitative inference.

bioengineering↗

bioRxiv2026

Genetic dependency of atrial fibrillation-associated risk genes across tissue types: Discovering novel therapeutic targets

BackgroundAddressing the underlying causes of atrial fibrillation (AFib) is critically important. While potential AFib-related genes have been recognized, the impact of modifying these genes in humans remains poorly understood. ObjectiveWe assessed the cellular dependencies of 309 genes previously associated with AFib through genome-wide association studies using data from the Cancer Dependency Map project, aiming to prioritize potential therapeutic targets with minimal off-target effects. MethodsWe analyzed CRISPR-Cas9 knockout (CHRONOS scores) and RNA interference (RNAi) knockdown (DEMETER2 scores) screening data from 1,927 human cell lines across 24 tissue types, focusing on tissues associated with AFib initiation, presentation, and progression: autonomic ganglia, central nervous system (CNS), and soft tissue. We examined the expression and dependency scores of the AFib-associated genes, identifying significant correlations between gene expression and cellular dependency within specific tissues using Pearson correlation coefficients and controlling the false discovery rate (FDR) at 5%. ResultsOut of the 309 AFib-associated genes, 206 genes (66.7%) had CHRONOS dependency scores and 229 (74.1%) had DEMETER2 dependency scores available. Several genes showed significant negative dependency scores (CHRONOS < -0.5) across multiple tissues, indicating potential off-target effects if inhibited. In contrast, we identified 12 genes with significant expression-driven dependencies within AFib-associated tissues. In CNS cell lines, HAND2 (R = -0.456, FDR = 0.002) and VGLL2 (R = -0.434, FDR = 0.005) showed significant negative correlations between gene expression and cellular dependency. In soft tissue cell lines, BEST3 (R = -0.679, FDR = 0.001) and PITX2 (R = -0.679, FDR = 0.001) also demonstrated strong negative correlations. Additionally, ERBB4 in CNS lines showed a significant negative correlation (R = -0.361, FDR = 0.048). These findings suggest that inhibiting these genes may selectively affect high-expressing cells in AFib-associated tissues while minimizing effects on other tissues. ConclusionOur analysis identified HAND2, VGLL2, BEST3, and ERBB4 as potential therapeutic targets for AFib, demonstrating significant expression-driven dependencies in AFib-associated tissues with no pan-tissue essentiality. These results provide a quantitative basis for developing targeted therapies with reduced off-target effects. CONDENSED ABSTRACTAtrial fibrillation (AFIB) is one of the most common cardiac arrhythmias with numerous known risk factors. Although many AFIB-associated genes have been identified, the impact of screening or the effects of modifying these genes in humans remain poorly understood. We examined CRISPR knockout and RNAi knockdown screen data from nearly 2,000 human cell lines to assess the cellular dependencies of 309 genes associated with AFIB, previously identified through genome-wide association studies. Some genes demonstrate broad cell dependencies across various tissue types, indicating potential off-target effects if inhibited. Conversely, HAND2, VGLL2, BEST3, and ERBB4 were identified as genes of interest because their genetic knockouts specifically impacted high-expressing cells from tissue lineages pertinent to AFIB and/or were not pan-dependent. Overall, analyses of genetic screen data identified AFIB-associated genes whose knockout or knockdown selectively affected cell lines of relevant tissue lineages, prioritizing targets for potential AFIB treatments.

genomics↗

bioRxiv2026

Enhanced Target Binding by Leritrelvir Restores Dimerization of Mpro Mutants and Mitigates Drug Resistance

The SARS-CoV-2 main protease (Mpro) has been a major target of antiviral drug development, leading to the development of inhibitors such as nirmatrelvir, the antiviral component of the COVID-19 drug Paxlovid. However, resistance-associated mutations that reduce the efficacy of current Mpro inhibitors, particularly nirmatrelvir, have emerged. Here, we evaluated the inhibitory activity of leritrelvir (RAY1216), an Mpro inhibitor approved in China for COVID-19 monotherapy, against a panel of Mpro variants carrying mutations at 12 resistance-associated residues distributed across four catalytic subsites. Using integrated biochemical, biophysical, structural, and cellular analyses, we demonstrate that leritrelvir retains stronger inhibitory activity against most tested resistant mutants compared with nirmatrelvir. Most of the tested mutations promote Mpro dimer dissociation, with E166V showing a particularly pronounced effect and markedly compromising nirmatrelvir binding. In contrast, thermal shift and size-exclusion chromatography assays demonstrate that leritrelvir binding restores dimerization of these Mpro mutants. Sixteen high-resolution crystal structures reveal that leritrelvir binding re-instates key dimer-interface interactions disrupted by resistance mutations. Mini-replicon assays further confirm leritrelvir to possess enhanced cellular antiviral efficacy compared with nirmatrelvir. Our findings indicate that tighter leritrelvir binding enables more effective inhibition of dissociation-prone Mpro mutants than nirmatrelvir, supporting its use as a more resilient antiviral agent for SARS-CoV-2 treatment.

molecular biology↗

bioRxiv2026

Dynamic Gating Mechanism of the b0,+AT-Mediated Arg Transport: Insights from ASMD Simulations

Heteromeric amino acid transporters (HATs) mediate essential amino acid flux across membranes, but the molecular dynamics of substrate translocation remain poorly defined for many family members. Here, using conventional and adaptive steered molecular dynamics (cMD and ASMD) simulations, we identify residue W230 in the b0,+AT transport channel as a dynamic gate that regulates arginine (Arg) influx through side chain flipping. By integrating dynamic network analysis with dynamical cross-correlation of residue motions, we show that regulatory signals propagate from the Arg binding site through transmembrane helix 5 (TM5), a connecting loop, and TM6 to reach W230. We propose a dynamic gating mechanism for b0,+AT - mediated amino acid transport. Arg binding at V186 triggers signal propagation that enhances cooperative interactions between W230 and Arg, driving the side chain flipping of W230. Our findings reveal a dynamic gating mechanism underlying b0,+AT - dependent Arg transport and suggest that residue-triggered side chain reorientation may represent a conserved and efficient strategy in transporter function. Author SummaryAmino acids are the essential building blocks of life, and their transport across cell membranes is vital for nutrition and cellular signaling. Heteromeric amino acid transporters (HATs) mediate this process, yet how they physically move substrates through the protein at the atomic level remains poorly understood. In this study, we used advanced computer simulations to observe, in unprecedented detail, how b0,+AT--a key HAT member--transports the amino acid arginine. Our simulations revealed that a single residue, tryptophan 230 (W230), functions as a molecular gate: its side chain flips open to allow arginine to pass and then closes behind it, ensuring one-way traffic into the cell. We further discovered that the initial binding of arginine sends a signal through specific structural elements (helices and loops) to trigger this gate opening. This work not only uncovers a dynamic gating mechanism for b0,+AT but also suggests that similar side-chain flipping events may represent a common and efficient strategy used by other transporters to control substrate movement. Our findings provide a new framework for understanding transporter function and could inform future drug design targeting these critical membrane proteins.

biophysics↗

bioRxiv2026

Multimodal physical evidence uncovers interpretable gene regulatory networks for perturbation prediction

Gene regulatory networks govern cell fate transitions through dynamic causal mechanisms1. Since exhaustively mapping this vast perturbation space experimentally is prohibitive2, scalable computational models are essential. Yet, current frameworks fall short because they infer statistical co-expression rather than physical mechanisms, remain blind to non-canonical regulators lacking classical DNA-binding motifs, and fail to generalize across unseen perturbation factors or cell lines. Here we show that a multimodal biophysical framework, VitaGRN, overcomes these barriers by constructing a biophysical regulatory scaffold from multimodal evidence and propagating interactions to capture non-canonical regulators. By leveraging structurally aligned protein embeddings, VitaGRN predicts zero-shot perturbation responses and uncovers non-canonical translational control programs. Notably, VitaGRN demonstrates robust generalization across unseen factors, cell lines, and developmental transitions. Ultimately, VitaGRN generates a confidence-calibrated virtual perturbation atlas spanning over a thousand factors. This resource reframes gene regulatory networks from static correlation graphs into dynamically generalizable and mechanistically trans-parent models, streamlining wet-lab candidate prioritization.

bioinformatics↗