DdmDE eliminates plasmid invasion by DNA-guided DNA targeting.

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Structural biology of innate immunity.

Innate immune responses depend on timely recognition of pathogenic or danger signals by multiple cell surface or cytoplasmic receptors and transmission of signals for proper counteractions through adaptor and effector molecules. At the forefront of innate immunity are four major signaling pathways, including those elicited by Toll-like receptors, RIG-I-like receptors, inflammasomes, or cGAS, each with its own cellular localization, ligand specificity, and signal relay mechanism. They collectively engage a number of overlapping signaling outcomes, such as NF-κB activation, interferon response, cytokine maturation, and cell death. Several proteins often assemble into a supramolecular complex to enable signal transduction and amplification. In this article, we review the recent progress in mechanistic delineation of proteins in these pathways, their structural features, modes of ligand recognition, conformational changes, and homo- and hetero-oligomeric interactions within the supramolecular complexes. Regardless of seemingly distinct interactions and mechanisms, the recurring themes appear to consist of autoinhibited resting-state receptors, ligand-induced conformational changes, and higher-order assemblies of activated receptors, adaptors, and signaling enzymes through conserved protein-protein interactions.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling.

Receptor clustering on the cell membrane is critical in the signaling of many immunoreceptors, and this mechanism has previously been attributed to the extracellular and/or the intracellular interactions. Here, we report an unexpected finding that for death receptor 5 (DR5), a receptor in the tumor necrosis factor receptor superfamily, the transmembrane helix (TMH) alone in the receptor directly assembles a higher-order structure to drive signaling and that this structure is inhibited by the unliganded ectodomain. Nuclear magnetic resonance structure of the TMH in bicelles shows distinct trimerization and dimerization faces, allowing formation of dimer-trimer interaction networks. Single-TMH mutations that disrupt either trimerization or dimerization abolish ligand-induced receptor activation. Surprisingly, proteolytic removal of the DR5 ectodomain can fully activate downstream signaling in the absence of ligand. Our data suggest a receptor activation mechanism in which binding of ligand or antibodies to overcome the pre-ligand autoinhibition allows TMH clustering and thus signaling.

Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense.

SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.

Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.

Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.

Oligomerization-mediated activation of a short prokaryotic Argonaute.

Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P)+ to induce bacterial cell death1. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein2. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA-DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR-APAZ. After the binding of RNA-DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P)+. In addition, we observe unique features of interactions between SPARTA and RNA-DNA, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.

CCR5 closes the temporal window for memory linking.

Real-world memories are formed in a particular context and are often not acquired or recalled in isolation1-5. Time is a key variable in the organization of memories, as events that are experienced close in time are more likely to be meaningfully associated, whereas those that are experienced with a longer interval are not1-4. How the brain segregates events that are temporally distinct is unclear. Here we show that a delayed (12-24 h) increase in the expression of C-C chemokine receptor type 5 (CCR5)-an immune receptor that is well known as a co-receptor for HIV infection6,7-after the formation of a contextual memory determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed expression of CCR5 in mouse dorsal CA1 neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dorsal CA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, and therefore closes the temporal window for memory linking. Our findings also show that an age-related increase in the neuronal expression of CCR5 and its ligand CCL5 leads to impairments in memory linking in aged mice, which could be reversed with a Ccr5 knockout and a drug approved by the US Food and Drug Administration (FDA) that inhibits this receptor, a result with clinical implications. Altogether, the findings reported here provide insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.

Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly.

Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are ATP-driven proton pumps comprised of a cytoplasmic V1 complex for ATP hydrolysis and a membrane-embedded Vo complex for proton transfer. They play important roles in acidification of intracellular vesicles, organelles, and the extracellular milieu in eukaryotes. Here, we report cryoelectron microscopy structures of human V-ATPase in three rotational states at up to 2.9-Å resolution. Aided by mass spectrometry, we build all known protein subunits with associated N-linked glycans and identify glycolipids and phospholipids in the Vo complex. We define ATP6AP1 as a structural hub for Vo complex assembly because it connects to multiple Vo subunits and phospholipids in the c-ring. The glycolipids and the glycosylated Vo subunits form a luminal glycan coat critical for V-ATPase folding, localization, and stability. This study identifies mechanisms of V-ATPase assembly and biogenesis that rely on the integrated roles of ATP6AP1, glycans, and lipids.

Gasdermin D pore structure reveals preferential release of mature interleukin-1.

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Dostarlimab for Primary Advanced or Recurrent Endometrial Cancer.

BACKGROUND: Dostarlimab is an immune-checkpoint inhibitor that targets the programmed cell death 1 receptor. The combination of chemotherapy and immunotherapy may have synergistic effects in the treatment of endometrial cancer. METHODS: We conducted a phase 3, global, double-blind, randomized, placebo-controlled trial. Eligible patients with primary advanced stage III or IV or first recurrent endometrial cancer were randomly assigned in a 1:1 ratio to receive either dostarlimab (500 mg) or placebo, plus carboplatin (area under the concentration-time curve, 5 mg per milliliter per minute) and paclitaxel (175 mg per square meter of body-surface area), every 3 weeks (six cycles), followed by dostarlimab (1000 mg) or placebo every 6 weeks for up to 3 years. The primary end points were progression-free survival as assessed by the investigator according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, and overall survival. Safety was also assessed. RESULTS: Of the 494 patients who underwent randomization, 118 (23.9%) had mismatch repair-deficient (dMMR), microsatellite instability-high (MSI-H) tumors. In the dMMR-MSI-H population, estimated progression-free survival at 24 months was 61.4% (95% confidence interval [CI], 46.3 to 73.4) in the dostarlimab group and 15.7% (95% CI, 7.2 to 27.0) in the placebo group (hazard ratio for progression or death, 0.28; 95% CI, 0.16 to 0.50; P<0.001). In the overall population, progression-free survival at 24 months was 36.1% (95% CI, 29.3 to 42.9) in the dostarlimab group and 18.1% (95% CI, 13.0 to 23.9) in the placebo group (hazard ratio, 0.64; 95% CI, 0.51 to 0.80; P<0.001). Overall survival at 24 months was 71.3% (95% CI, 64.5 to 77.1) with dostarlimab and 56.0% (95% CI, 48.9 to 62.5) with placebo (hazard ratio for death, 0.64; 95% CI, 0.46 to 0.87). The most common adverse events that occurred or worsened during treatment were nausea (53.9% of the patients in the dostarlimab group and 45.9% of those in the placebo group), alopecia (53.5% and 50.0%), and fatigue (51.9% and 54.5%). Severe and serious adverse events were more frequent in the dostarlimab group than in the placebo group. CONCLUSIONS: Dostarlimab plus carboplatin-paclitaxel significantly increased progression-free survival among patients with primary advanced or recurrent endometrial cancer, with a substantial benefit in the dMMR-MSI-H population. (Funded by GSK; RUBY ClinicalTrials.gov number, NCT03981796.).

25-Hydroxycholesterol Protects Host against Zika Virus Infection and Its Associated Microcephaly in a Mouse Model.

Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection in vitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role of CH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.

Inflammation NODs to Antagonists of RIP2-XIAP Interaction.

While innate immunity is crucial for host defense, dysregulated signaling activation leads to pathological inflammation. In this issue of Molecular Cell, Goncharov et al. (2018) present a strategy to combat inflammatory diseases by disrupting RIP2-XIAP interaction in NOD2-mediated signaling.

GWAShug: a comprehensive platform for decoding the shared genetic basis between complex traits based on summary statistics.

The shared genetic basis offers very valuable insights into the etiology, diagnosis and therapy of complex traits. However, a comprehensive resource providing shared genetic basis using the accessible summary statistics is currently lacking. It is challenging to analyze the shared genetic basis due to the difficulty in selecting parameters and the complexity of pipeline implementation. To address these issues, we introduce GWAShug, a platform featuring a standardized best-practice pipeline with four trait level methods and three molecular level methods. Based on stringent quality control, the GWAShug resource module includes 539 high-quality GWAS summary statistics for European and East Asian populations, covering 54 945 pairs between a measurement-based and a disease-based trait and 43 902 pairs between two disease-based traits. Users can easily search for shared genetic basis information by trait name, MeSH term and category, and access detailed gene information across different trait pairs. The platform facilitates interactive visualization and analysis of shared genetic basic results, allowing users to explore data dynamically. Results can be conveniently downloaded via FTP links. Additionally, we offer an online analysis module that allows users to analyze their own summary statistics, providing comprehensive tables, figures and interactive visualization and analysis. GWAShug is freely accessible at http://www.gwashug.com.

PGS-Depot: a comprehensive resource for polygenic scores constructed by summary statistics based methods.

Polygenic score (PGS) is an important tool for the genetic prediction of complex traits. However, there are currently no resources providing comprehensive PGSs computed from published summary statistics, and it is difficult to implement and run different PGS methods due to the complexity of their pipelines and parameter settings. To address these issues, we introduce a new resource called PGS-Depot containing the most comprehensive set of publicly available disease-related GWAS summary statistics. PGS-Depot includes 5585 high quality summary statistics (1933 quantitative and 3652 binary trait statistics) curated from 1564 traits in European and East Asian populations. A standardized best-practice pipeline is used to implement 11 summary statistics-based PGS methods, each with different model assumptions and estimation procedures. The prediction performance of each method can be compared for both in- and cross-ancestry populations, and users can also submit their own summary statistics to obtain custom PGS with the available methods. Other features include searching for PGSs by trait name, publication, cohort information, population, or the MeSH ontology tree and searching for trait descriptions with the experimental factor ontology (EFO). All scores, SNP effect sizes and summary statistics can be downloaded via FTP. PGS-Depot is freely available at http://www.pgsdepot.net.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

Identification of mEAK-7 as a human V-ATPase regulator via cryo-EM data mining.

Vacuolar-type adenosine triphosphatases (V-ATPases) not only function as rotary proton pumps in cellular organelles but also serve as signaling hubs. To identify the endogenous binding partners of V-ATPase, we collected a large dataset of human V-ATPases and did extensive classification and focused refinement of human V-ATPases. Unexpectedly, about 17% of particles in state 2 of human V-ATPases display additional density with an overall resolution of 3.3 Å. Structural analysis combined with artificial intelligence modeling enables us to identify this additional density as mEAK-7, a protein involved in mechanistic target of rapamycin (mTOR) signaling in mammals. Our structure shows that mEAK-7 interacts with subunits A, B, D, and E of V-ATPases in state 2. Thus, we propose that mEAK-7 may regulate V-ATPase function through binding to V-ATPases in state 2 as well as mediate mTOR signaling.

Targeting stem-loop 1 of the SARS-CoV-2 5' UTR to suppress viral translation and Nsp1 evasion.

SARS-CoV-2 is a highly pathogenic virus that evades antiviral immunity by interfering with host protein synthesis, mRNA stability, and protein trafficking. The SARS-CoV-2 nonstructural protein 1 (Nsp1) uses its C-terminal domain to block the messenger RNA (mRNA) entry channel of the 40S ribosome to inhibit host protein synthesis. However, how SARS-CoV-2 circumvents Nsp1-mediated suppression for viral protein synthesis and if the mechanism can be targeted therapeutically remain unclear. Here, we show that N- and C-terminal domains of Nsp1 coordinate to drive a tuned ratio of viral to host translation, likely to maintain a certain level of host fitness while maximizing replication. We reveal that the stem-loop 1 (SL1) region of the SARS-CoV-2 5' untranslated region (5' UTR) is necessary and sufficient to evade Nsp1-mediated translational suppression. Targeting SL1 with locked nucleic acid antisense oligonucleotides inhibits viral translation and makes SARS-CoV-2 5' UTR vulnerable to Nsp1 suppression, hindering viral replication in vitro at a nanomolar concentration, as well as providing protection against SARS-CoV-2-induced lethality in transgenic mice expressing human ACE2. Thus, SL1 allows Nsp1 to switch infected cells from host to SARS-CoV-2 translation, presenting a therapeutic target against COVID-19 that is conserved among immune-evasive variants. This unique strategy of unleashing a virus' own virulence mechanism against itself could force a critical trade-off between drug resistance and pathogenicity.

Aberrant dynamic functional network connectivity in progressive supranuclear palsy.

BACKGROUND: The clinical symptoms of progressive supranuclear palsy (PSP) may be mediated by aberrant dynamic functional network connectivity (dFNC). While earlier research has found altered functional network connections in PSP patients, the majority of those studies have concentrated on static functional connectivity. Nevertheless, in this study, we sought to evaluate the modifications in dynamic characteristics and establish the correlation between these disease-related changes and clinical variables. METHODS: In our study, we conducted a study on 53 PSP patients and 65 normal controls. Initially, we employed a group independent component analysis (ICA) to derive resting-state networks (RSNs), while employing a sliding window correlation approach to produce dFNC matrices. The K-means algorithm was used to cluster these matrices into distinct dynamic states, and then state analysis was subsequently employed to analyze the dFNC and temporal metrics between the two groups. Finally, we made a correlation analysis. RESULTS: PSP patients showed increased connectivity strength between medulla oblongata (MO) and visual network (VN) /cerebellum network (CBN) and decreased connections were found between default mode network (DMN) and VN/CBN, subcortical cortex network (SCN) and CBN. In addition, PSP patients spend less fraction time and shorter dwell time in a diffused state, especially the MO and SCN. Finally, the fraction time and mean dwell time in the distributed connectivity state (state 2) is negatively correlated with duration, bulbar and oculomotor symptoms. DISCUSSION: Our findings were that the altered connectivity was mostly concentrated in the CBN and MO. In addition, PSP patients had different temporal dynamics, which were associated with bulbar and oculomotor symptoms in PSPRS. It suggest that variations in dynamic functional network connectivity properties may represent an essential neurological mechanism in PSP.

Structures and functions of short argonautes.

Argonaute proteins (Agos) represent a highly conserved family of proteins prevalent in all domains of life and have been implicated in various biological processes. Based on the domain architecture, Agos can be divided into long Agos and short Agos. While long Agos have been extensively studied over the past two decades, short Agos, found exclusively in prokaryotes, have recently gained attention for their roles in prokaryotic immune defence against mobile genetic elements, such as plasmids and phages. Notable functional and structural studies provide invaluable insights into the underlying molecular mechanisms of representative short Ago systems. Despite the diverse domain arrangements, short Agos generally form heterodimeric complexes with their associated effector proteins, activating the effector's enzymatic activities upon target detection. The activation of effector proteins in the short Ago systems leads to bacterial cell death, a mechanism of sacrificing individuals to protect the community.