ABSTRACT
Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.
Subject(s)
Herpesvirus 4, Human , Viral Matrix Proteins , Humans , Cryoelectron Microscopy , Epstein-Barr Virus Infections/virology , Epstein-Barr Virus Infections/metabolism , HEK293 Cells , Herpesvirus 4, Human/metabolism , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/physiology , Models, Molecular , Mutation , Protein Multimerization , Signal Transduction , Viral Matrix Proteins/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/geneticsABSTRACT
During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.
Subject(s)
Cell Membrane Structures , Myosins , Neural Tube , Signal Transduction , Animals , Mice , Biological Transport , Cell Membrane Structures/metabolism , Hedgehog Proteins/metabolism , Myosins/metabolism , Pseudopodia/metabolism , Neural Tube/cytology , Neural Tube/metabolismABSTRACT
Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.
Subject(s)
Glycoproteins , Molecular Dynamics Simulation , Humans , Cryoelectron Microscopy , Glycoproteins/chemistry , Glycosylation , Polysaccharides/chemistryABSTRACT
Hybrid sterility restricts the utilization of superior heterosis of indica-japonica inter-subspecific hybrids. In this study, we report the identification of RHS12, a major locus controlling male gamete sterility in indica-japonica hybrid rice. We show that RHS12 consists of two genes (iORF3/DUYAO and iORF4/JIEYAO) that confer preferential transmission of the RHS12-i type male gamete into the progeny, thereby forming a natural gene drive. DUYAO encodes a mitochondrion-targeted protein that interacts with OsCOX11 to trigger cytotoxicity and cell death, whereas JIEYAO encodes a protein that reroutes DUYAO to the autophagosome for degradation via direct physical interaction, thereby detoxifying DUYAO. Evolutionary trajectory analysis reveals that this system likely formed de novo in the AA genome Oryza clade and contributed to reproductive isolation (RI) between different lineages of rice. Our combined results provide mechanistic insights into the genetic basis of RI as well as insights for strategic designs of hybrid rice breeding.
Subject(s)
Gene Drive Technology , Oryza , Hybridization, Genetic , Oryza/genetics , Plant Breeding/methods , Reproductive Isolation , Plant InfertilityABSTRACT
How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.
Subject(s)
Amino Acids/metabolism , CD8-Positive T-Lymphocytes/cytology , Immunologic Memory , Signal Transduction , Amino Acid Transport Systems/metabolism , Animals , CD8-Positive T-Lymphocytes/immunology , CRISPR-Cas Systems , Cell Cycle , Cell Differentiation , Disease Models, Animal , Female , Gene Knock-In Techniques , Lymphocytic Choriomeningitis/immunology , Male , Mice , Mice, Transgenic , Precursor Cells, T-Lymphoid/cytologyABSTRACT
Pediatric-onset colitis and inflammatory bowel disease (IBD) have significant effects on the growth of infants and children, but the etiopathogenesis underlying disease subtypes remains incompletely understood. Here, we report single-cell clustering, immune phenotyping, and risk gene analysis for children with undifferentiated colitis, Crohn's disease, and ulcerative colitis. We demonstrate disease-specific characteristics, as well as common pathogenesis marked by impaired cyclic AMP (cAMP)-response signaling. Specifically, infiltration of PDE4B- and TNF-expressing macrophages, decreased abundance of CD39-expressing intraepithelial T cells, and platelet aggregation and release of 5-hydroxytryptamine at the colonic mucosae were common in colitis and IBD patients. Targeting these pathways by using the phosphodiesterase inhibitor dipyridamole restored immune homeostasis and improved colitis symptoms in a pilot study. In summary, comprehensive analysis of the colonic mucosae has uncovered common pathogenesis and therapeutic targets for children with colitis and IBD.
Subject(s)
Inflammatory Bowel Diseases/pathology , Inflammatory Bowel Diseases/therapy , Intestinal Mucosa/pathology , Antigens, CD/metabolism , Apyrase/metabolism , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Cell Death/drug effects , Cellular Microenvironment/drug effects , Child , Cohort Studies , Colon/pathology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dipyridamole/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation/drug effects , Genetic Predisposition to Disease , Homeostasis/drug effects , Humans , Immunoglobulin G/blood , Immunologic Memory , Inflammation/pathology , Inflammatory Bowel Diseases/blood , Inflammatory Bowel Diseases/genetics , Interferon Type I/metabolism , Macrophages/drug effects , Macrophages/metabolism , Methylprednisolone/pharmacology , Myeloid Cells/drug effects , Myeloid Cells/metabolismABSTRACT
Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.
Subject(s)
Bacterial Proteins/chemistry , Legionella pneumophila/metabolism , Phosphoric Diester Hydrolases/chemistry , Ubiquitin/chemistry , Bacterial Proteins/metabolism , Biocatalysis , Crystallography, X-Ray , Dimerization , Phosphoric Diester Hydrolases/metabolism , Protein Binding , Protein Domains , Protein Structure, Quaternary , Ubiquitin/metabolism , UbiquitinationABSTRACT
The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.
Subject(s)
Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Amino Acid Sequence , Binding Sites , Intracellular Membranes/metabolism , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Protein Binding , Protein Domains , Protein Precursors/chemistry , Protein Precursors/metabolism , Protein Structure, Secondary , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence AlignmentABSTRACT
MCL-1 is essential for promoting the survival of many normal cell lineages and confers survival and chemoresistance in cancer. Beyond apoptosis regulation, MCL-1 has been linked to modulating mitochondrial metabolism, but the mechanism(s) by which it does so are unclear. Here, we show in tissues and cells that MCL-1 supports essential steps in long-chain (but not short-chain) fatty acid ß-oxidation (FAO) through its binding to specific long-chain acyl-coenzyme A (CoA) synthetases of the ACSL family. ACSL1 binds to the BH3-binding hydrophobic groove of MCL-1 through a non-conventional BH3-domain. Perturbation of this interaction, via genetic loss of Mcl1, mutagenesis, or use of selective BH3-mimetic MCL-1 inhibitors, represses long-chain FAO in cells and in mouse livers and hearts. Our findings reveal how anti-apoptotic MCL-1 facilitates mitochondrial metabolism and indicate that disruption of this function may be associated with unanticipated cardiac toxicities of MCL-1 inhibitors in clinical trials.
Subject(s)
Fatty Acids , Mitochondria , Animals , Mice , Apoptosis , Coenzyme A Ligases/genetics , Fatty Acids/metabolism , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Oxidation-ReductionABSTRACT
Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.
Subject(s)
Insulin-Secreting Cells/metabolism , Obesity/etiology , Obesity/metabolism , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Animals , Cell Proliferation , Cyclin D2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Diet, High-Fat/adverse effects , Disease Models, Animal , Female , Humans , Insulin/blood , Insulin/metabolism , Insulin-Secreting Cells/ultrastructure , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , MAP Kinase Signaling System , Male , Mice , Mice, Knockout , Multiprotein Complexes/metabolism , Obesity/drug therapy , Parabiosis , Protein Binding , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
Evidence shows a continuing increase in the frequency and severity of global heatwaves1,2, raising concerns about the future impacts of climate change and the associated socioeconomic costs3,4. Here we develop a disaster footprint analytical framework by integrating climate, epidemiological and hybrid input-output and computable general equilibrium global trade models to estimate the midcentury socioeconomic impacts of heat stress. We consider health costs related to heat exposure, the value of heat-induced labour productivity loss and indirect losses due to economic disruptions cascading through supply chains. Here we show that the global annual incremental gross domestic product loss increases exponentially from 0.03 ± 0.01 (SSP 245)-0.05 ± 0.03 (SSP 585) percentage points during 2030-2040 to 0.05 ± 0.01-0.15 ± 0.04 percentage points during 2050-2060. By 2060, the expected global economic losses reach a total of 0.6-4.6% with losses attributed to health loss (37-45%), labour productivity loss (18-37%) and indirect loss (12-43%) under different shared socioeconomic pathways. Small- and medium-sized developing countries suffer disproportionately from higher health loss in South-Central Africa (2.1 to 4.0 times above global average) and labour productivity loss in West Africa and Southeast Asia (2.0-3.3 times above global average). The supply-chain disruption effects are much more widespread with strong hit to those manufacturing-heavy countries such as China and the USA, leading to soaring economic losses of 2.7 ± 0.7% and 1.8 ± 0.5%, respectively.
ABSTRACT
Single-atom catalysts1 make exceptionally efficient use of expensive noble metals and can bring out unique properties1-3. However, applications are usually compromised by limited catalyst stability, which is due to sintering3,4. Although sintering can be suppressed by anchoring the metal atoms to oxide supports1,5,6, strong metal-oxygen interactions often leave too few metal sites available for reactant binding and catalysis6,7, and when exposed to reducing conditions at sufficiently high temperatures, even oxide-anchored single-atom catalysts eventually sinter4,8,9. Here we show that the beneficial effects of anchoring can be enhanced by confining the atomically dispersed metal atoms on oxide nanoclusters or 'nanoglues', which themselves are dispersed and immobilized on a robust, high-surface-area support. We demonstrate the strategy by grafting isolated and defective CeOx nanoglue islands onto high-surface-area SiO2; the nanoglue islands then each host on average one Pt atom. We find that the Pt atoms remain dispersed under both oxidizing and reducing environments at high temperatures, and that the activated catalyst exhibits markedly increased activity for CO oxidation. We attribute the improved stability under reducing conditions to the support structure and the much stronger affinity of Pt atoms for CeOx than for SiO2, which ensures the Pt atoms can move but remain confined to their respective nanoglue islands. The strategy of using functional nanoglues to confine atomically dispersed metals and simultaneously enhance their reactivity is general, and we anticipate that it will take single-atom catalysts a step closer to practical applications.
ABSTRACT
Networks of optical clocks find applications in precise navigation1,2, in efforts to redefine the fundamental unit of the 'second'3-6 and in gravitational tests7. As the frequency instability for state-of-the-art optical clocks has reached the 10-19 level8,9, the vision of a global-scale optical network that achieves comparable performances requires the dissemination of time and frequency over a long-distance free-space link with a similar instability of 10-19. However, previous attempts at free-space dissemination of time and frequency at high precision did not extend beyond dozens of kilometres10,11. Here we report time-frequency dissemination with an offset of 6.3 × 10-20 ± 3.4 × 10-19 and an instability of less than 4 × 10-19 at 10,000 s through a free-space link of 113 km. Key technologies essential to this achievement include the deployment of high-power frequency combs, high-stability and high-efficiency optical transceiver systems and efficient linear optical sampling. We observe that the stability we have reached is retained for channel losses up to 89 dB. The technique we report can not only be directly used in ground-based applications, but could also lay the groundwork for future satellite time-frequency dissemination.
ABSTRACT
Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.
Subject(s)
Cytoplasmic Granules/genetics , DNA Helicases/genetics , Poly-ADP-Ribose Binding Proteins/genetics , Protein Biosynthesis , RNA Helicases/genetics , RNA Recognition Motif Proteins/genetics , Stress, Physiological/genetics , 5' Untranslated Regions , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/pathology , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA Helicases/metabolism , Female , HCT116 Cells , HeLa Cells , Humans , Male , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Spermatogonia/cytology , Spermatogonia/pathology , Testis/cytology , Testis/metabolismABSTRACT
Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Calcium Signaling , Cyclic Nucleotide-Gated Cation Channels , Plant Stomata , Protein Kinases , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Calcium/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Cyclic Nucleotide-Gated Cation Channels/genetics , Mutation , Phosphorylation , Plant Stomata/genetics , Plant Stomata/physiology , Plant Stomata/metabolism , Plant Stomata/drug effects , Protein Kinases/metabolism , Protein Kinases/geneticsABSTRACT
T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.
Subject(s)
Immunity, Humoral , Phosphatidylethanolamines/metabolism , Receptors, CXCR5/immunology , T-Lymphocytes, Helper-Inducer/immunology , Animals , B-Lymphocytes/immunology , CRISPR-Cas Systems , Cell Differentiation , Cytidine Diphosphate , Female , Gene Expression Regulation , Humans , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phosphotransferases (Alcohol Group Acceptor) , RNA Nucleotidyltransferases , Signal TransductionABSTRACT
Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Nutrients , Protein Interaction Maps , T-Lymphocytes, Regulatory , Animals , Female , Male , Mice , Carrier Proteins/metabolism , CRISPR-Cas Systems/genetics , Forkhead Transcription Factors/metabolism , Genome/genetics , Homeostasis , Immune Tolerance , Inflammation/pathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Neoplasms/immunology , Nuclear Proteins/metabolism , Nutrients/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , S-Phase Kinase-Associated Proteins/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Trans-Activators/metabolismABSTRACT
Anti-CRISPR proteins (Acrs) targeting CRISPR-Cas9 systems represent natural "off switches" for Cas9-based applications. Recently, AcrIIC1, AcrIIC2, and AcrIIC3 proteins were found to inhibit Neisseria meningitidis Cas9 (NmeCas9) activity in bacterial and human cells. Here we report biochemical and structural data that suggest molecular mechanisms of AcrIIC2- and AcrIIC3-mediated Cas9 inhibition. AcrIIC2 dimer interacts with the bridge helix of Cas9, interferes with RNA binding, and prevents DNA loading into Cas9. AcrIIC3 blocks the DNA loading step through binding to a non-conserved surface of the HNH domain of Cas9. AcrIIC3 also forms additional interactions with the REC lobe of Cas9 and induces the dimerization of the AcrIIC3-Cas9 complex. While AcrIIC2 targets Cas9 orthologs from different subtypes, albeit with different efficiency, AcrIIC3 specifically inhibits NmeCas9. Structure-guided changes in NmeCas9 orthologs convert them into anti-CRISPR-sensitive proteins. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms and guidelines for designing regulatory tools in Cas9-based applications.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , DNA/genetics , Gene Editing , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , CRISPR-Associated Protein 9/antagonists & inhibitors , DNA/chemistry , Humans , Neisseria meningitidis/enzymology , Neisseria meningitidis/geneticsABSTRACT
The endosomal sorting complexes required for transport (ESCRTs) are responsible for membrane remodeling in many cellular processes, such as multivesicular body biogenesis, viral budding, and cytokinetic abscission. ESCRT-III, the most abundant ESCRT subunit, assembles into flat spirals as the primed state, essential to initiate membrane invagination. However, the three-dimensional architecture of ESCRT-III flat spirals remained vague for decades due to highly curved filaments with a small diameter and a single preferred orientation on the membrane. Here, we unveiled that yeast Snf7, a component of ESCRT-III, forms flat spirals on the lipid monolayers using cryogenic electron microscopy. We developed a geometry-constrained Euler angle-assigned reconstruction strategy and obtained moderate-resolution structures of Snf7 flat spirals with varying curvatures. Our analyses showed that Snf7 subunits recline on the membrane with N-terminal motifs α0 as anchors, adopt an open state with fused α2/3 helices, and bend α2/3 gradually from the outer to inner parts of flat spirals. In all, we provide the orientation and conformations of ESCRT-III flat spirals on the membrane and unveil the underlying assembly mechanism, which will serve as the initial step in understanding how ESCRTs drive membrane abscission.
Subject(s)
Cryoelectron Microscopy , Endosomal Sorting Complexes Required for Transport , Saccharomyces cerevisiae Proteins , Cell Membrane/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomal Sorting Complexes Required for Transport/chemistry , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/ultrastructureABSTRACT
Organic electrodes mainly consisting of C, O, H, and N are promising candidates for advanced batteries. However, the sluggish ionic and electronic conductivity limit the full play of their high theoretical capacities. Here, we integrate the idea of metal-support interaction in single-atom catalysts with π-d hybridization into the design of organic electrode materials for the applications of lithium (LIBs) and potassium-ion batteries (PIBs). Several types of transition metal single atoms (e.g., Co, Ni, Fe) with π-d hybridization are incorporated into the semiconducting covalent organic framework (COF) composite. Single atoms favorably modify the energy band structure and improve the electronic conductivity of COF. More importantly, the electronic interaction between single atoms and COF adjusts the binding affinity and modifies ion traffic between Li/K ions and the active organic units of COFs as evidenced by extensive in situ and ex situ characterizations and theoretical calculations. The corresponding LIB achieves a high reversible capacity of 1,023.0 mA h g-1 after 100 cycles at 100 mA g-1 and 501.1 mA h g-1 after 500 cycles at 1,000 mA g-1. The corresponding PIB delivers a high reversible capacity of 449.0 mA h g-1 at 100 mA g-1 after 150 cycles and stably cycled over 500 cycles at 1,000 mA g-1. This work provides a promising route to engineering organic electrodes.