Biology subjects

Yamashita, K.

Publications and source records attributed to Yamashita, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural mechanism of the type IX Retron-Kva2 anti-phage defense system

Retrons are prokaryotic genetic elements that protect bacterial cells from invading phages. The type IX retron comprises a non-coding RNA, a reverse transcriptase (RT), and dual effectors: a putative HEPN nuclease and a winged helix-turn-helix (WH) protein. Here, we show that the type IX retron system from Klebsiella variicola cleaves host rRNAs and tRNAs upon phage infection, mediating anti-phage defense via an abortive-infection mechanism. Cryo-electron microscopy analysis reveals that the RT, HEPN, and WH proteins, along with multicopy single-stranded DNA (msDNA), form a unique, sheet-like supermolecular complex. Notably, the HEPN active site is encircled by the WH and msDNA within the complex, suppressing the nuclease activity prior to phage infection. A phage-encoded exonuclease cleaves the msDNA, likely releasing the HEPN nuclease to cleave host RNAs and induce growth arrest in infected cells. Overall, these findings highlight the structural and functional diversity of prokaryotic anti-phage defense systems.

biochemistry↗

bioRxiv2026

Green genetically encoded IP3 biosensor for hierarchical analysis of its signaling network

Inositol 1,4,5-trisphosphate (IP3) is a key second messenger that regulates diverse physiological processes. Visualization of IP3 dynamics in living cells is therefore important for understanding its signaling processes. In this study, we developed genetically encoded green fluorescent IP3 biosensors named Green iPenguins with distinct half-maximal effective concentrations (EC50) for IP3, enabling detection of IP3 signals over a range of concentrations. The biosensors displayed more than a 4-fold increase in fluorescence intensity upon IP3 and showed high specificity for IP3 over structurally related molecules. When expressed in HEK293T cells, the biosensors enabled visualization of IP3 dynamics involved in different signaling pathways. They were also compatible with dual-color imaging, allowing simultaneous monitoring of IP3 together with cAMP or Ca2+ signals. In addition, the hierarchical relationship between IP3 and Ca2+ signaling was visualized, providing insight into the temporal relationship between these two second messengers. The biosensors are expected to facilitate future studies of physiological processes involving IP3 signaling networks.

bioengineering↗

bioRxiv2026

Coordinated synthesis of double-stranded DNA by a dual reverse transcriptase immune system

Defense-associated reverse transcriptase (DRT) systems mediate antiviral immunity through distinct modes of cDNA synthesis: Class 1 DRTs catalyze untemplated synthesis, whereas Class 2 DRTs polymerize noncoding RNA-templated products. However, how these distinct modes drive defense remains unclear. Here, we report that DRT3 immunity arises when Class 1 and Class 2 RT activities cooperate to produce self-complementary double-stranded DNA (dsDNA). DRT3a uses a 5'-ACACAC-3' RNA template to synthesize poly-(dTdG) repeats, whereas DRT3b synthesizes poly-(dCdA) repeats without any nucleic acid template. Cryo-electron microscopy reveals that DRT3b forms a hexamer and uses active-site-adjacent residues as deoxyadenosine and deoxycytidine gates to enforce alternating nucleotide addition, representing a unique example of amino acid-templated DNA polymerization. DRT3 is toxic in cells lacking RecBCD, implicating host recombination machinery in limiting dsDNA accumulation, and the phage-encoded RecBCD inhibitor Gam triggers DRT3-mediated abortive infection. These findings reveal how two polymerases with distinct templating strategies generate complementary DNA for defense.

microbiology↗

bioRxiv2026

Reduced cortico-accumbal excitatory input due to Nav1.2 haploinsufficiency impairs sociability independently of dopamine

Mutations in SCN2A, which encodes the voltage-gated sodium channel Nav1.2, are associated with a wide spectrum of neurodevelopmental and neuropsychiatric disorders, including epilepsy, autism spectrum disorder (ASD), and schizophrenia. Although dysfunction of SCN2A-dependent neural circuits has been implicated in these disorders, the circuit mechanisms underlying social behavioral abnormalities remain poorly understood. Here, we investigated the neural circuit basis of social behavioral deficits associated with Scn2a dysfunction, focusing on the nucleus accumbens (NAc), a key hub in cortico-limbic circuits that regulates emotional and motivational behaviors. Using conditional genetic and chemogenetic approaches in mice, we examined the roles of dorsal telencephalic excitatory neurons, including those in the cerebral cortex, hippocampus, and amygdala, as well as parvalbumin-positive fast-spiking interneurons (PV FSIs) in the NAc. Mice with Scn2a haploinsufficiency in dorsal telencephalic excitatory neurons (Scn2afl/+/Emx1-Cre) exhibited reduced sociability in the three-chamber social interaction test. Similarly, chemogenetic inhibition of NAc PV FSIs decreased sociability without affecting locomotor activity or anxiety-like behavior. Scn2afl/+/Emx1-Cre mice also showed a trend toward reduced prepulse inhibition of the acoustic startle response. Notably, dopamine release into the NAc in the Scn2afl/+/Emx1-Cre and systemic Scn2a heterozygous knockout (Scn2a+/-) mice was largely comparable to that in control mice. Together, these findings indicate that reduced activity of dorsal telencephalic excitatory neurons or NAc PV FSIs is sufficient to impair sociability independently of mesolimbic dopamine hypofunction. Our results highlight a potential role of cortico-accumbal circuits in social behavioral deficits associated with SCN2A dysfunction.

neuroscience↗

bioRxiv2026

Iron-dependent reprogramming of damage-associated peptide receptor signaling coordinates immunity and phosphate stress adaptation

Plant adaptation to inorganic phosphate (Pi) limitation entails extensive developmental and metabolic reprogramming, collectively termed the phosphate starvation response (PSR). How plants balance nutrient acquisition with immune competence remains unresolved. Here, we show that PSR selectively remodels pattern-recognition receptor (PRR) signaling in Arabidopsis thaliana. Membrane proteomics reveals increased abundance of the Pep receptor PEPR1 under Pi deficiency, whereas most receptor kinases, including the microbial PRRs FLS2 and CERK1, are reduced. Immunoblotting further shows increased PEPR2 abundance. Consistently, Pep-triggered responses are strongly potentiated under Pi depletion, while flagellin- and chitin-induced responses are not, indicating selective PEPR sensitization rather than global immune suppression. This sensitization requires the PSR-associated ferroxidases LPR1/LPR2, NADPH oxidases RBOHD/RBOHF, and transcription factor WRKY33, linking iron-dependent redox remodeling to immune amplification. Beyond inducible defense, PEPRs promote basal PSR transcriptional reprogramming and growth under Pi limitation. Loss of PEPR1/PEPR2 compromises bacterial resistance and alters root-associated microbiota assembly under nutrient deficiency. Together, Pi depletion selectively sensitizes specific PRR pathways. PEPRs exhibit context-dependent dual functionality, promoting either immunity or nutrient stress adaptation, potentially in response to distinct PROPEP-derived ligands, thereby supporting nutrient acquisition without compromising immune vigilance. These findings provide a framework for how nutritional status selectively remodels PRR signaling to coordinate immunity with nutrient stress adaptation.

plant biology↗

bioRxiv2026

Electrical Surface Polarization as a Functionalization Strategy to Improve Bone Regeneration of Apatite-Based Graft Materials

Apatite-based bone graft materials are widely used for bone regeneration; however, their limited bioactivity and slow remodeling often hinder complete replacement by newly formed bone. Electrical surface polarization has emerged as a promising non-chemical strategy to modify biomaterial surface properties without altering bulk characteristics. In this study, we investigated the effects of electrical surface polarization on apatite-based biomaterials using synthesized carbonate apatite (CA) for mechanistic in vitro evaluation and a clinically relevant xenograft material for in vivo validation. Material characterization confirmed the formation of B-type carbonate apatite with bone-like mineral composition. Thermally stimulated depolarization current measurements verified successful induction of surface charges, with polarization intensity dependent on treatment conditions. In vitro studies using human peripheral blood-derived osteoclast precursors demonstrated that electrically polarized CA surfaces significantly enhanced osteoclast differentiation and resorptive activity compared to non-polarized controls, with the strongest effects observed on positively polarized surfaces. Three-dimensional analysis revealed increased resorption pit depth and volume, indicating enhanced osteoclast functionality. In vivo implantation of polarized xenograft materials into rat femoral defects resulted in significantly increased new bone formation and improved implant-bone integration compared to non-polarized materials. Higher polarization conditions promoted more mature bone tissue formation and greater bone-material affinity. These results demonstrate that electrical surface polarization effectively modulates osteoclast-material interactions and enhances bone regeneration, highlighting its potential as a simple and translatable functionalization strategy for apatite-based bone graft materials.

bioengineering↗

bioRxiv2026

Identification of a pharmacokinetic interaction between teicoplanin and sulfo-butyl ether-β-cyclodextrin, an excipient in the intravenous posaconazole formulation

Patients undergoing hematopoietic stem cell transplantation (HSCT) often receive multiple antibiotics and antifungals concurrently, making it crucial to understand their potential pharmacokinetic interactions of these agents. We report here an interaction between the glycopeptide antibiotic teicoplanin (TEIC) and sulfo-butyl ether-{beta}-cyclodextrin (SBECD), a solubilizing excipient in the intravenous formulation of posaconazole (PSCZ). We performed a single-center retrospective analysis of patients who underwent HSCT and received oral and intravenous PSCZ during TEIC therapy. The associations between PSCZ administration and TEIC concentration-to-dose (C/D) ratios were evaluated using linear mixed-effects models. We also examined the effects of intravenous PSCZ and SBECD on TEIC pharmacokinetics in rats by assessing the area under the concentration-time curve (AUC) and urinary excretion of total TEIC and its components. In addition, molecular docking and in vitro protein-binding assays were conducted to investigate the interaction between TEIC and SBECD. In patients who underwent HSCT, TEIC C/D ratio was significantly lower following intravenous PSCZ administration than without administration. In contrast, the effect of oral PSCZ administration relative to non-administration was not statistically significant. In rats, intravenous PSCZ and SBECD decreased the AUC of TEIC and increased urinary excretion, particularly in the A2 group. Docking simulations indicated that the hydrophobic side chain of TEIC A2-2 fit within the SBECD cavity, and in vitro assays confirmed SBECD concentration-dependent increases in TEIC unbound fractions. The co-administration of intravenous PSCZ containing SBECD may reduce TEIC protein binding, thereby enhancing renal excretion.

pharmacology and toxicology↗

bioRxiv2026

Cryo-EM structure of pro-aggregant P301L/S320F double-mutant tau filaments formed in mouse brains following peripheral AAV delivery

Tauopathies are characterized by the accumulation of abnormally phosphorylated tau filaments, with disease-specific folds revealed by cryo-electron microscopy (cryo-EM). Here, we delivered a recombinant human tau carrying the P301L/S320F double mutation into AppNL-G-F/MAPT double knock-in mice using a blood-brain-barrier-permeable AAV (AAV-PHP.eB), enabling rapid induction of tau aggregation in vivo. The mutant tau was systemically administered via retro-orbital injection, providing a minimally invasive approach for widespread neuronal transduction. Phosphorylated tau aggregation, as well as seeding activity, were observed without the need for exogenous seeds. Our cryo-EM analysis resolved a novel filament fold incorporating residues 279-329, in which the P301L and S320F mutations introduced stabilizing interactions that reinforced filament assembly. This conformation was distinct from previously reported folds, including Alzheimer and Pick tau filaments. Moreover, the filaments adopted a more compact architecture than those observed in patient-derived samples or other model mice. This finding demonstrates that P301L/S320F double-mutant tau adopts a structurally unique, highly aggregation-prone fold, and that this system provides a rapid platform for modeling tau filament formation.

neuroscience↗

bioRxiv2026

Atomic resolution cryo-EM at 200 keV

Atomic resolution in cryo-electron microscopy was first demonstrated six years ago. This was accomplished using 300 kV electron microscopes equipped with new hardware that provided narrower energy spread, aberration correction, and energy filtering. Here, we report the achievement of 1.24 [A] atomic resolution on an upgraded 200 kV electron microscope featuring a cold field emission gun, a high-resolution objective lens polepiece, and an energy filter. These components transform the instrument into a cost-effective single particle cryo-EM platform with performance comparable to that of significantly more expensive 300 kV systems. The microscope can also be operated at 100 kV and by using a high-speed hybrid-pixel detector we were able to reach sub-2 [A] resolution.

molecular biology↗

bioRxiv2025

Measurement of atomic scattering factors by cryo-electron microscopy

Determination of specimen structure from cryo-electron microscopy (cryo-EM) experiments relies on an accurate model of the electrostatic potential of the specimen. For biological macromolecules, the potential is strongly influenced by the presence of chemical bonds between atoms, a fact unaccounted for by models of electron scattering that are currently standard in the field. We propose a Bayesian approach to the estimation of atomic scattering factors which incorporates the effect of the molecular environment while remaining fast, interpretable and transferable between molecules. Our algorithm infers atomic scattering factors directly from maps of the electrostatic potential determined by cryo-EM single particle analysis, bypassing the need for computationally-intensive theoretical calculations. The algorithm is used to infer empirical scattering factors from high-resolution reconstructions of catalase enzymes. To illustrate its broad applicability, the algorithm is also applied to a training set of publicly-available cryo-EM data. The empirical scattering factors show improved agreement with a test set of cryo-EM reconstructions. The predictions are further validated by comparison with magnetic susceptibility values of organic compounds, as well as by application to the refinement of atomic models. Significance StatementUnderstanding the structure of biomolecules is key to explaining their function. Cryo-electron microscopy is a method for reconstructing the electrostatic potential distribution of a biological macro-molecule, a quantity which contains information about atomic positions and the redistribution of charge due to chemical bonding. These factors should be modelled when inferring the structure of the molecule from its electrostatic potential. We develop an improved model of the potential that takes into account chemical bonding while remaining computationally tractable. The parameters of the model are inferred from a selection of cryo-electron microscopy datasets using Bayesian methods.

biophysics↗

bioRxiv2025

Computed Tomography-Derived Elastic Modulus as a Noninvasive Marker of Aortic Wall Integrity: Correlation with Histopathology in the Ascending Aorta

BackgroundAscending aortic aneurysms and dissections are life-threatening conditions often requiring prophylactic surgeries. Current guidelines rely primarily on aortic diameter for intervention; however, many dissections occur without severe dilation. Mechanical properties, such as elastic modulus have emerged as potential predictors of disease progression; nonetheless, noninvasive clinical applications remain limited. This study evaluated the relationship between the computed tomography (CT)-derived elastic modulus of the ascending aorta and the histopathological characteristics of the aortic media. MethodsThirty patients who underwent surgical ascending aorta replacement were included in this study. Preoperative CT was used to calculate the aortic elastic modulus based on geometric measurements and pulse pressure. Resected aortic specimens were subjected to histological and immunohistochemical analyses to assess elastin, collagen, vascular smooth muscle cells (VSMCs), and smoothelin expression. Correlation analyses between the CT-derived elastic modulus and aortic media composition were conducted after adjusting for age and aortic diameter. ResultsThe CT-derived elastic modulus exhibited a significant negative correlation with elastin area and a positive correlation with collagen area. Additionally, a moderate negative correlation was observed between the elastic modulus and elastin fiber waviness. A strong negative correlation was detected between the elastic modulus and the proportion of contractile-type (smoothelin-positive) VSMCs. These findings remained significant after adjusting for confounders. ConclusionsCT-derived elastic modulus of the ascending aorta reflects the underlying pathological changes, including extracellular matrix remodeling and VSMC phenotypic modulation. Noninvasive assessment of aortic mechanical properties may provide novel insights into aortic disease progression and therapeutic responses.

pathology↗

bioRxiv2025

Insights from aquaporin structures into drug-resistant sleeping sickness

Trypanosoma brucei is the causal agent of African trypanosomiasis in humans and animals, the latter resulting in significant negative economic impacts in afflicted areas of the world. Resistance has arisen to the trypanocidal drugs pentamidine and melarsoprol through mutations in the aquaglyceroporin TbAQP2 that prevent their uptake. Here we use cryogenic electron microscopy to determine the structure of TbAQP2 from Trypanosoma brucei, bound to either the substrate glycerol or to the sleeping sickness drugs, pentamidine or melarsoprol. The drugs bind within the AQP2 channel at a site completely overlapping that of glycerol. Mutations leading to a drug-resistant phenotype were found in the channel lining. Molecular dynamics simulations showed the channel can be traversed by pentamidine, with a low energy binding site at the centre of the channel, flanked by regions of high energy association at the extracellular and intracellular ends. Drug-resistant TbAQP2 mutants are still predicted to bind pentamidine, but the much weaker binding in the centre of the channel observed in the MD simulations would be insufficient to compensate for the high energy processes of ingress and egress, hence impairing transport at pharmacologically relevant concentrations. The structures of drug-bound TbAQP2 represent a novel paradigm for drug-transporter interactions that could provide new mechanisms for targeting drugs into pathogens and human cells.

molecular biology↗

bioRxiv2025

Improved Tumor Blood Flow Enhances the Abscopal Effect: Preclinical Assessment in Mice Treated with Combined Radiation and PD-1 Blockade Therapy

The abscopal effect, where localized radiation therapy induces regression of distant metastatic lesions through immune activation, shows promise for treating metastatic cancer but occurs inconsistently. Here we demonstrate that tumor perfusion critically influences systemic immune responses to combination therapy with radiation and PD-1 blockade. Using multimodal imaging including DCE-MRI, EPR oximetry, and hyperpolarized 13C-MRI, we show that successful abscopal responses in MC38 tumors are characterized by enhanced perfusion, reduced hypoxia, decreased cellularity, and lower glycolytic activity in remote tumors. Notably, pre-treatment perfusion metrics (AUC1min) and extracellular volume (AUC10min) in primary tumors predict subsequent growth of remote tumors, while the same measurements in remote tumors lack predictive value. Based on these findings, we enhanced the abscopal effect by exposing mice to carbogen (95% O2 + 5% CO2) during radiation therapy. Carbogen exposure increased tumor perfusion by 71% (AUC1min) and significantly improved systemic responses in the checkpoint blockade responsive MC38 model but not in the poorly responsive B16.F10 tumors. The enhanced response correlated with increased activation of CD8+ T cells in tumor-draining lymph nodes and elevated serum HMGB-1 levels. RNA sequencing revealed significant extracellular matrix remodeling in carbogen-treated tumors. These results establish tumor perfusion as both a predictive biomarker and a modifiable determinant of systemic immune responses, suggesting that perfusion-based patient stratification and vascular modification strategies could improve outcomes in combination immunotherapy and radiation treatment.

cancer biology↗

bioRxiv2025

Low Field Magnetic Resonance Imaging to Detect Acute Kidney Injury

Renal oxygenation is essential for maintaining kidney function. Disruptions in oxygen delivery can lead to renal hypoxia, which can exacerbate kidney injury through multiple pathways, including inflammation, oxidative stress, and ischemia-reperfusion injury. Despite the recognized importance of oxygenation in renal pathology, non-invasive and reliable methods for assessing kidney oxygen levels are limited. Current techniques either lack sensitivity or involve invasive procedures, restricting their use in routine monitoring. Therefore, there is a pressing need for innovative approaches to assess renal oxygenation, particularly in kidney injury. This study evaluated Electron Paramagnetic Resonance (EPR)-based oxygen imaging using the paramagnetic tracer Ox071 to assess kidney oxygen levels in mice with cyclophosphamide-induced kidney injury. Urine pO2 was also assessed as a potential surrogate marker. EPR oximetry accurately measured kidney oxygen distribution, revealing a temporary increase in pO2 post-injury. Urine oximetry, however, did not reliably reflect changes in kidney oxygenation. Furthermore, EPR oximetry provided high-resolution spatial mapping of oxygen levels within the kidney, allowing for a detailed understanding of the impact of hypoxia on renal tissue. EPR oximetry is a promising, non-invasive tool for monitoring renal oxygenation, offering high-resolution mapping and longitudinal assessment. Its ability to provide detailed information about oxygen distribution within the kidney makes it a valuable tool for studying the pathophysiology of renal diseases and for developing novel therapeutic strategies. Translational Statement: Quantitative spatially resolved measurement of renal oxygenation has the potential to guide clinical decision making in renal disorders such as acute kidney injury. In this study we demonstrate the utility of electron paramagnetic resonance imaging to provide non-invasive and quantitative high-resolution mapping of kidney oxygen concentrations.

physiology↗

bioRxiv2024

Mitochondrial Dysfunction and ER Stress in CB1 Receptor Antagonist-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

Cannabinoid receptor type 1 (CB1R) is the key modulator of neuronal viability. In particular, CB1R antagonists provide neuroprotective effects on neurotoxicity caused by e.g. neuronal injury. However, the underlying mechanisms and potential limitations of CB1R antagonism remain unclear. Here we investigated the impact of environmental conditions on CB1R antagonist effects. Unexpectedly, we have found that cell-permeable CB1R antagonists, rimonabant and AM251, induced significant cell death in human neuroblastoma SH-SY5Y cells under serum-free conditions. Mitochondrial morphological analysis revealed mitochondrial swelling characterized by their network fragmentation and cristae reduction. Additionally, phosphoproteomics analysis showed an upregulation of phosphorylated EIF2AK3 (also known as PERK), leading to the activation of the eIF2/ATF4/CHOP pathway, indicative of endoplasmic reticulum (ER) stress. Rimonabant and AM251 treatment also triggered caspase-dependent apoptosis, evidenced by the detection of cleaved caspase 3 and cleaved PARP. These results suggest that cell-permeable CB1R antagonists promote apoptosis via mitochondrial dysfunction and ER stress under serum-free conditions in SH-SY5Y cells. Our findings indicate that while CB1R antagonists may be neuroprotective in certain conditions, they may also pose a neurotoxic risk in environments characterized by cellular stress or nutrient deprivation.

pharmacology and toxicology↗

bioRxiv2024

Band-selective IR PRESS for brain tumor spectroscopy allows robust detection of lactate

Lactate plays a critical role in the tumor microenvironment, driving tumor progression, metastasis, and immune evasion. Despite its importance, in vivo quantification of lactate using magnetic resonance spectroscopy (MRS) has faced challenges, primarily due to the overlapping lipid signal at 1.3 ppm. Current clinical practice employs a long echo time to exploit differences in T2 relaxation between lactate and lipids; however, this approach significantly suppresses signals from other metabolites. Lipid has a notably different T1 relaxation time than lactate and other metabolites, which may be exploited by an inversion recovery sequence to better distinguish them. However, this method has not found wide use because of the loss of signal in other metabolites. Here we introduce a selective inversion pulse with a short echo time MRS method (SPIR-PRESS), which mitigates this issue. In phantom experiments, SPIR-PRESS successfully suppressed lipid signals that could be misinterpreted as lactate in short TE PRESS spectra, while maintaining sensitivity to the full metabolite profile. SPIR-PRESS demonstrated superior performance in quantifying lactate compared to long echo time PRESS, with [~] 60 % increase in sensitivity for lactate detection compared to conventional PRESS with a 288 ms TE. In a mouse glioma model, SPIR-PRESS clearly detected lactate and other key tumor metabolites (choline, creatine, NAA) in the tumor, which were not detectable in conventional long TE PRESS. These findings highlight SPIR-PRESS as a promising technique for improved lactate quantification and comprehensive metabolite profiling in tumor environments.

biochemistry↗

bioRxiv2024

MAP4K1 and MAP4K2 regulate ABA-induced and Ca2+-mediated stomatal closure in Arabidopsis

Abscisic acid (ABA)-induced stomatal closure limits water loss from plants under drought stress. To investigate signaling pathways involved in stomatal closure, we performed a phosphoproteomic analysis of ABA-treated Arabidopsis guard cell protoplasts (GCPs). From this analysis, we discovered that MITOGEN-ACTIVATED PROTEIN 4 KINASE 1 (MAP4K1) is phosphorylated in response to ABA. Subsequent biochemical assays showed that Ser-479 of MAP4K1 is directly phosphorylated by SRK2E (OST1/SnRK2.6), a protein kinase that plays a central role in ABA-induced responses. Mutational analyses of MAP4K1, as well as closely related MAP4K2, revealed that both kinases positively regulate ABA-induced stomatal closure, and that Ser-479 of MAP4K1 was required for this phenotype. In map4k1map4k2, stomatal closure was induced by exogenous Ca2+ but not H2O2. Furthermore, electrophysiology experiments showed that MAP4K1/2 are required for ABA-dependent activation of Ca2+-permeable channels in GCPs. Together, our results indicate that SnRK2 and MAP4K1/2 function as a signaling module to regulate Ca2+-mediated stomatal closure.

plant biology↗

bioRxiv2023

Structural basis of anticancer drug recognition and amino acid transport by LAT1

LAT1 (SLC7A5) transports large neutral amino acids and their derivatives across the plasma membrane and plays pivotal roles in cancer cell proliferation, immune response and drug delivery across the blood-brain barrier. Despite recent advances in structural understanding of LAT1, how it discriminates substrates and inhibitors including the clinically relevant anticancer drugs remains elusive. Here we report six structures of LAT1, captured in three different conformations and bound with diverse bioactive ligands, elucidating its substrate transport and inhibitory mechanisms. JPH203, also known as nanvuranlat or KYT-0353 and currently in clinical trials as an anticancer drug, binds to the wide-open substrate-binding pocket of LAT1. It adopts a U-shaped conformer, with its amino-phenylbenzoxazol moiety pushing against transmembrane helix 3 (TM3), bending TM10 and arresting the transporter in the outward-facing conformation. In contrast, the physiological substrate L-Phe does not exhibit such inhibitory interactions, whereas melphalan, a slow substrate, poses steric hindrance in the pocket, explaining its inhibitory activity. Unexpectedly, the "classical" system L inhibitor BCH induces an occluded state, a key structural intermediate required for substrate transport. Trans stimulation assays show that BCH facilitates transporter turnover and is therefore a transportable substrate. These findings provide a structural framework for the intricate mechanisms of substrate recognition and inhibition of LAT1, paving the way for developing more specific and effective drugs against it.

biochemistry↗