Activation of the helper NRC4 immune receptor forms a hexameric resistosome.

Innate immune responses to microbial pathogens are regulated by intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) in both the plant and animal kingdoms. Across plant innate immune systems, "helper" NLRs (hNLRs) work in coordination with "sensor" NLRs (sNLRs) to modulate disease resistance signaling pathways. Activation mechanisms of hNLRs based on structures are unknown. Our research reveals that the hNLR, known as NLR required for cell death 4 (NRC4), assembles into a hexameric resistosome upon activation by the sNLR Bs2 and the pathogenic effector AvrBs2. This conformational change triggers immune responses by facilitating the influx of calcium ions (Ca2+) into the cytosol. The activation mimic alleles of NRC2, NRC3, or NRC4 alone did not induce Ca2+ influx and cell death in animal cells, suggesting that unknown plant-specific factors regulate NRCs' activation in plants. These findings significantly advance our understanding of the regulatory mechanisms governing plant immune responses.

Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine.

Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels.

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

Structural biology and molecular pharmacology of voltage-gated ion channels.

Voltage-gated ion channels (VGICs), including those for Na+, Ca2+ and K+, selectively permeate ions across the cell membrane in response to changes in membrane potential, thus participating in physiological processes involving electrical signalling, such as neurotransmission, muscle contraction and hormone secretion. Aberrant function or dysregulation of VGICs is associated with a diversity of neurological, psychiatric, cardiovascular and muscular disorders, and approximately 10% of FDA-approved drugs directly target VGICs. Understanding the structure-function relationship of VGICs is crucial for our comprehension of their working mechanisms and role in diseases. In this Review, we discuss how advances in single-particle cryo-electron microscopy have afforded unprecedented structural insights into VGICs, especially on their interactions with clinical and investigational drugs. We present a comprehensive overview of the recent advances in the structural biology of VGICs, with a focus on how prototypical drugs and toxins modulate VGIC activities. We explore how these structures elucidate the molecular basis for drug actions, reveal novel pharmacological sites, and provide critical clues to future drug discovery.

Structural Basis for Blocking Sugar Uptake into the Malaria Parasite Plasmodium falciparum.

Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα.

DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron.

The Omicron (B.1.1.529) variant of SARS-CoV-2 emerged in November 2021 and is rapidly spreading among the human population1. Although recent reports reveal that the Omicron variant robustly escapes vaccine-associated and therapeutic neutralization antibodies2-10, the pathogenicity of the virus remains unknown. Here we show that the replication of Omicron is substantially attenuated in human Calu3 and Caco2 cells. Further mechanistic investigations reveal that Omicron is inefficient in its use of transmembrane serine protease 2 (TMPRSS2) compared with wild-type SARS-CoV-2 (HKU-001a) and previous variants, which may explain its reduced replication in Calu3 and Caco2 cells. The replication of Omicron is markedly attenuated in both the upper and lower respiratory tracts of infected K18-hACE2 mice compared with that of the wild-type strain and Delta (B.1.617.2) variant, resulting in its substantially ameliorated lung pathology. Compared with wild-type SARS-CoV-2 and the Alpha (B.1.1.7), Beta (1.351) and Delta variants, infection by Omicron causes the lowest reduction in body weight and the lowest mortality rate. Overall, our study demonstrates that the replication and pathogenicity of the Omicron variant of SARS-CoV-2 in mice is attenuated compared with the wild-type strain and other variants.

A predictive model for therapy failure in chronic myeloid leukemia patients receiving tyrosine kinase inhibitor therapy.

Although tyrosine kinase inhibitor (TKI) therapy has markedly improved the survival of people with chronic-phase chronic myeloid leukemia (CML), 20-30% of people still experienced therapy failure. Data from 1,955 consecutive subjects with chronic-phase CML diagnosed by the European LeukemiaNet (ELN) recommendations from 1 center receiving initial TKI imatinib or a second-generation (2G-) TKI therapy were interrogated to develop a clinical prediction model for TKI therapy failure. This model was subsequently validated in 3,454 subjects from 76 other centers. Using the predictive clinical co-variates associated with TKI therapy failure, we developed a model that stratified subjects into low-, intermediate- and high-risk subgroups with significantly different cumulative incidences of therapy failure (p < 0.001). There was good discrimination and calibration in the external validation dataset, and the performance was consistent with that of the training dataset. Our model had the better prediction discrimination than the Sokal and ELTS scores did, with the greater time-dependent area under the receiver-operator characteristic curve (AUROC) values and a better ability to re-defined the risk of therapy failure. Our model could help physicians estimate the likelihood of initial imatinib or 2G-TKI therapy failure in people with chronic-phase CML.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase.

Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

Ribocentre-switch: a database of riboswitches.

Riboswitches are regulatory elements found in the untranslated regions (UTRs) of certain mRNA molecules. They typically comprise two distinct domains: an aptamer domain that can bind to specific small molecules, and an expression platform that controls gene expression. Riboswitches work by undergoing a conformational change upon binding to their specific ligand, thus activating or repressing the genes downstream. This mechanism allows gene expression regulation in response to metabolites or small molecules. To systematically summarise riboswitch structures and their related ligand binding functions, we present Ribocentre-switch, a comprehensive database of riboswitches, including the information as follows: sequences, structures, functions, ligand binding pockets and biological applications. It encompasses 56 riboswitches and 26 orphan riboswitches from over 430 references, with a total of 89 591 sequences. It serves as a good resource for comparing different riboswitches and facilitating the identification of potential riboswitch candidates. Therefore, it may facilitate the understanding of RNA structural conformational changes in response to ligand signaling. The database is publicly available at https://riboswitch.ribocentre.org.

Cryo-EM structure of human voltage-gated sodium channel Nav1.6.

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits ß1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the ß1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.

Dual-pocket inhibition of Nav channels by the antiepileptic drug lamotrigine.

Voltage-gated sodium (Nav) channels govern membrane excitability, thus setting the foundation for various physiological and neuronal processes. Nav channels serve as the primary targets for several classes of widely used and investigational drugs, including local anesthetics, antiepileptic drugs, antiarrhythmics, and analgesics. In this study, we present cryogenic electron microscopy (cryo-EM) structures of human Nav1.7 bound to two clinical drugs, riluzole (RLZ) and lamotrigine (LTG), at resolutions of 2.9 Å and 2.7 Å, respectively. A 3D EM reconstruction of ligand-free Nav1.7 was also obtained at 2.1 Å resolution. RLZ resides in the central cavity of the pore domain and is coordinated by residues from repeats III and IV. Whereas one LTG molecule also binds to the central cavity, the other is found beneath the intracellular gate, known as site BIG. Therefore, LTG, similar to lacosamide and cannabidiol, blocks Nav channels via a dual-pocket mechanism. These structures, complemented with docking and mutational analyses, also explain the structure-activity relationships of the LTG-related linear 6,6 series that have been developed for improved efficacy and subtype specificity on different Nav channels. Our findings reveal the molecular basis for these drugs' mechanism of action and will aid the development of novel antiepileptic and pain-relieving drugs.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis.

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Angiotensin II receptor inhibition ameliorates liver fibrosis and enhances hepatocellular carcinoma infiltration by effector T cells.

Although viral hepatocellular carcinoma (HCC) is declining, nonviral HCC, which often is the end stage of nonalcoholic or alcoholic steatohepatitis (NASH, ASH), is on an upward trajectory. Immune checkpoint inhibitors (ICIs) that block the T cell inhibitory receptor PD-1 were approved for treatment of all HCC types. However, only a minority of HCC patients show a robust and sustained response to PD-1 blockade, calling for improved understanding of factors that negatively impact response rate and duration and the discovery of new adjuvant treatments that enhance ICI responsiveness. Using a mouse model of NASH-driven HCC, we identified peritumoral fibrosis as a potential obstacle to T cell-mediated tumor regression and postulated that antifibrotic medications may increase ICI responsiveness. We now show that the angiotensin II receptor inhibitor losartan, a commonly prescribed and safe antihypertensive drug, reduced liver and peritumoral fibrosis and substantially enhanced anti-PD-1-induced tumor regression. Although losartan did not potentiate T cell reinvigoration, it substantially enhanced HCC infiltration by effector CD8+ T cells compared to PD-1 blockade alone. The beneficial effects of losartan correlated with blunted TGF-ß receptor signaling, reduced collagen deposition, and depletion of immunosuppressive fibroblasts.

A Bayesian approach to estimate MHC-peptide binding threshold.

Major histocompatibility complex (MHC)-peptide binding is a critical step in enabling a peptide to serve as an antigen for T-cell recognition. Accurate prediction of this binding can facilitate various applications in immunotherapy. While many existing methods offer good predictive power for the binding affinity of a peptide to a specific MHC, few models attempt to infer the binding threshold that distinguishes binding sequences. These models often rely on experience-based ad hoc criteria, such as 500 or 1000nM. However, different MHCs may have different binding thresholds. As such, there is a need for an automatic, data-driven method to determine an accurate binding threshold. In this study, we proposed a Bayesian model that jointly infers core locations (binding sites), the binding affinity and the binding threshold. Our model provided the posterior distribution of the binding threshold, enabling accurate determination of an appropriate threshold for each MHC. To evaluate the performance of our method under different scenarios, we conducted simulation studies with varying dominant levels of motif distributions and proportions of random sequences. These simulation studies showed desirable estimation accuracy and robustness of our model. Additionally, when applied to real data, our results outperformed commonly used thresholds.

Hemorrhoidal disease and its genetic association with depression, bipolar disorder, anxiety disorders, and schizophrenia: a bidirectional mendelian randomization study.

BACKGROUND: Hemorrhoids and psychiatric disorders exhibit high prevalence rates and a tendency for relapse in epidemiological studies. Despite this, limited research has explored their correlation, and these studies are often subject to reverse causality and residual confounding. We conducted a Mendelian randomization (MR) analysis to comprehensively investigate the association between several mental illnesses and hemorrhoidal disease. METHODS: Genetic associations for four psychiatric disorders and hemorrhoidal disease were obtained from large consortia, the FinnGen study, and the UK Biobank. Genetic variants associated with depression, bipolar disorder, anxiety disorders, schizophrenia, and hemorrhoidal disease at the genome-wide significance level were selected as instrumental variables. Screening for potential confounders in genetic instrumental variables using PhenoScanner V2. Bidirectional MR estimates were employed to assess the effects of four psychiatric disorders on hemorrhoidal disease. RESULTS: Our analysis revealed a significant association between genetically predicted depression and the risk of hemorrhoidal disease (IVW, OR=1.20,95% CI=1.09 to 1.33, P <0.001). We found no evidence of associations between bipolar disorder, anxiety disorders, schizophrenia, and hemorrhoidal disease. Inverse MR analysis provided evidence for a significant association between genetically predicted hemorrhoidal disease and depression (IVW, OR=1.07,95% CI=1.04 to 1.11, P <0.001). CONCLUSIONS: This study offers MR evidence supporting a bidirectional causal relationship between depression and hemorrhoidal disease.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.