ABSTRACT
Different functional regions of brain are fundamental for basic neurophysiological activities. However, the regional specification remains largely unexplored during human brain development. Here, by combining spatial transcriptomics (scStereo-seq) and scRNA-seq, we built a spatiotemporal developmental atlas of multiple human brain regions from 6-23 gestational weeks (GWs). We discovered that, around GW8, radial glia (RG) cells have displayed regional heterogeneity and specific spatial distribution. Interestingly, we found that the regional heterogeneity of RG subtypes contributed to the subsequent neuronal specification. Specifically, two diencephalon-specific subtypes gave rise to glutamatergic and GABAergic neurons, whereas subtypes in ventral midbrain were associated with the dopaminergic neurons. Similar GABAergic neuronal subtypes were shared between neocortex and diencephalon. Additionally, we revealed that cell-cell interactions between oligodendrocyte precursor cells and GABAergic neurons influenced and promoted neuronal development coupled with regional specification. Altogether, this study provides comprehensive insights into the regional specification in the developing human brain.
Subject(s)
Brain , Transcriptome , Humans , Dopaminergic Neurons , GABAergic Neurons , Mesencephalon , Neocortex , Brain/growth & development , Brain/metabolismABSTRACT
Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (â¼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.
Subject(s)
Evolution, Molecular , Genome , Perissodactyla/genetics , Animals , Demography , Gene Flow , Genetic Variation , Geography , Heterozygote , Homozygote , Host Specificity , Markov Chains , Mutation/genetics , Phylogeny , Species Specificity , Time FactorsABSTRACT
Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32+CD172a+ DC3, distinct from DC2, arising from Ly6C+ monocyte-DC progenitors (MDPs) through Lyz2+Ly6C+CD11c- pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7+Ly6C+CD11c+ pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s.
Subject(s)
Monocytes , Stem Cells , Mice , Humans , Animals , Phenotype , Cells, Cultured , Dendritic Cells , Cell DifferentiationABSTRACT
ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.
Subject(s)
KATP Channels/chemistry , KATP Channels/metabolism , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Cryoelectron Microscopy , Humans , Hydrolases/chemistry , Hydrolases/metabolism , Mammals/metabolism , Mesocricetus , Mice , Models, Molecular , Phosphoinositide Phospholipase C/chemistry , Phosphoinositide Phospholipase C/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/metabolism , Sulfonylurea Receptors/chemistry , Sulfonylurea Receptors/metabolismABSTRACT
Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Infections/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , B-Lymphocytes/immunology , HIV Antibodies/chemistry , HIV Envelope Protein gp120/immunology , HIV Infections/prevention & control , HIV-1/immunology , Humans , Models, Molecular , Molecular Sequence Data , Sequence AlignmentABSTRACT
The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.
Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Amino Acid Sequence , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , CD4 Antigens/metabolism , Complementarity Determining Regions , Epitopes, B-Lymphocyte , HIV Envelope Protein gp120/immunology , Humans , Models, Molecular , Molecular Sequence Data , Sequence AlignmentABSTRACT
Force-controlled release of small molecules offers great promise for the delivery of drugs and the release of healing or reporting agents in a medical or materials context1-3. In polymer mechanochemistry, polymers are used as actuators to stretch mechanosensitive molecules (mechanophores)4. This technique has enabled the release of molecular cargo by rearrangement, as a direct5,6 or indirect7-10 consequence of bond scission in a mechanophore, or by dissociation of cage11, supramolecular12 or metal complexes13,14, and even by 'flex activation'15,16. However, the systems described so far are limited in the diversity and/or quantity of the molecules released per stretching event1,2. This is due to the difficulty in iteratively activating scissile mechanophores, as the actuating polymers will dissociate after the first activation. Physical encapsulation strategies can be used to deliver a larger cargo load, but these are often subject to non-specific (that is, non-mechanical) release3. Here we show that a rotaxane (an interlocked molecule in which a macrocycle is trapped on a stoppered axle) acts as an efficient actuator to trigger the release of cargo molecules appended to its axle. The release of up to five cargo molecules per rotaxane actuator was demonstrated in solution, by ultrasonication, and in bulk, by compression, achieving a release efficiency of up to 71% and 30%, respectively, which places this rotaxane device among the most efficient release systems achieved so far1. We also demonstrate the release of three representative functional molecules (a drug, a fluorescent tag and an organocatalyst), and we anticipate that a large variety of cargo molecules could be released with this device. This rotaxane actuator provides a versatile platform for various force-controlled release applications.
Subject(s)
Delayed-Action Preparations , Rotaxanes , Delayed-Action Preparations/chemical synthesis , Delayed-Action Preparations/chemistry , Polymers/chemistry , Rotaxanes/chemistry , Pharmaceutical Preparations/chemistry , Fluorescent Dyes/chemistryABSTRACT
Phagocyte NADPH oxidase, a protein complex with a core made up of NOX2 and p22 subunits, is responsible for transferring electrons from intracellular NADPH to extracellular oxygen1. This process generates superoxide anions that are vital for killing pathogens1. The activation of phagocyte NADPH oxidase requires membrane translocation and the binding of several cytosolic factors2. However, the exact mechanism by which cytosolic factors bind to and activate NOX2 is not well understood. Here we present the structure of the human NOX2-p22 complex activated by fragments of three cytosolic factors: p47, p67 and Rac1. The structure reveals that the p67-Rac1 complex clamps onto the dehydrogenase domain of NOX2 and induces its contraction, which stabilizes the binding of NADPH and results in a reduction of the distance between the NADPH-binding domain and the flavin adenine dinucleotide (FAD)-binding domain. Furthermore, the dehydrogenase domain docks onto the bottom of the transmembrane domain of NOX2, which reduces the distance between FAD and the inner haem. These structural rearrangements might facilitate the efficient transfer of electrons between the redox centres in NOX2 and lead to the activation of phagocyte NADPH oxidase.
Subject(s)
NADPH Oxidase 2 , Phagocytes , Humans , Electrons , Enzyme Activation , Flavin-Adenine Dinucleotide/metabolism , Heme/chemistry , Heme/metabolism , NADP/metabolism , NADPH Oxidase 2/chemistry , NADPH Oxidase 2/metabolism , Phagocytes/enzymology , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Superoxides/metabolism , Protein BindingABSTRACT
Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.
Subject(s)
Carcinoma, Hepatocellular , Genome, Human , High-Throughput Nucleotide Sequencing , Liver Neoplasms , Mutation , Whole Genome Sequencing , Humans , Aristolochic Acids/metabolism , Carcinogenesis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , China , Chromothripsis , Disease Progression , DNA, Circular/genetics , East Asian People/genetics , Evolution, Molecular , Genome, Human/genetics , Hepatitis B virus/genetics , INDEL Mutation/genetics , Liver/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/virology , Mutation/genetics , Neoplasm Metastasis/genetics , Open Reading Frames/genetics , Reproducibility of ResultsABSTRACT
Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.
Subject(s)
Receptors, AMPA/chemistry , Receptors, AMPA/metabolism , Animals , Cryoelectron Microscopy , Crystallography, X-Ray , Fluorouracil/analogs & derivatives , Fluorouracil/metabolism , Gene Knockout Techniques , Kainic Acid/metabolism , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Tertiary , Rats , Receptors, AMPA/agonists , Receptors, AMPA/geneticsABSTRACT
Uncoupling protein 1 (UCP1) conducts protons through the inner mitochondrial membrane to uncouple mitochondrial respiration from ATP production, thereby converting the electrochemical gradient of protons into heat1,2. The activity of UCP1 is activated by endogenous fatty acids and synthetic small molecules, such as 2,4-dinitrophenol (DNP), and is inhibited by purine nucleotides, such as ATP3-5. However, the mechanism by which UCP1 binds to these ligands remains unknown. Here we present the structures of human UCP1 in the nucleotide-free state, the DNP-bound state and the ATP-bound state. The structures show that the central cavity of UCP1 is open to the cytosolic side. DNP binds inside the cavity, making contact with transmembrane helix 2 (TM2) and TM6. ATP binds in the same cavity and induces conformational changes in TM2, together with the inward bending of TM1, TM4, TM5 and TM6 of UCP1, resulting in a more compact structure of UCP1. The binding site of ATP overlaps with that of DNP, suggesting that ATP competitively blocks the functional engagement of DNP, resulting in the inhibition of the proton-conducting activity of UCP1.
Subject(s)
2,4-Dinitrophenol , Adenosine Triphosphate , Uncoupling Protein 1 , Humans , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Protons , Uncoupling Protein 1/chemistry , Uncoupling Protein 1/metabolism , Fatty Acids/metabolism , 2,4-Dinitrophenol/chemistry , 2,4-Dinitrophenol/metabolism , Protein Conformation , Cell Membrane/metabolism , Cytosol/metabolismABSTRACT
The open-circuit voltage (VOC) deficit in perovskite solar cells is greater in wide-bandgap (over 1.7 eV) cells than in perovskites of roughly 1.5 eV (refs. 1,2). Quasi-Fermi-level-splitting measurements show VOC-limiting recombination at the electron-transport-layer contact3-5. This, we find, stems from inhomogeneous surface potential and poor perovskite-electron transport layer energetic alignment. Common monoammonium surface treatments fail to address this; as an alternative, we introduce diammonium molecules to modify perovskite surface states and achieve a more uniform spatial distribution of surface potential. Using 1,3-propane diammonium, quasi-Fermi-level splitting increases by 90 meV, enabling 1.79 eV perovskite solar cells with a certified 1.33 V VOC and over 19% power conversion efficiency (PCE). Incorporating this layer into a monolithic all-perovskite tandem, we report a record VOC of 2.19 V (89% of the detailed balance VOC limit) and over 27% PCE (26.3% certified quasi-steady state). These tandems retained more than 86% of their initial PCE after 500 h of operation.
ABSTRACT
Human sodium-glucose cotransporter 2 (hSGLT2) mediates the reabsorption of the majority of filtrated glucose in the kidney1. Pharmacological inhibition of hSGLT2 by oral small-molecule inhibitors, such as empagliflozin, leads to enhanced excretion of glucose and is widely used in the clinic to manage blood glucose levels for the treatment of type 2 diabetes1. Here we determined the cryogenic electron microscopy structure of the hSGLT2-MAP17 complex in the empagliflozin-bound state to an overall resolution of 2.95 Å. Our structure shows eukaryotic SGLT-specific structural features. MAP17 interacts with transmembrane helix 13 of hSGLT2. Empagliflozin occupies both the sugar-substrate-binding site and the external vestibule to lock hSGLT2 in an outward-open conformation, thus inhibiting the transport cycle. Our work provides a framework for understanding the mechanism of SLC5A family glucose transporters and also develops a foundation for the future rational design and optimization of new inhibitors targeting these transporters.
Subject(s)
Sodium-Glucose Transporter 2 Inhibitors , Sodium-Glucose Transporter 2 , Cryoelectron Microscopy , Glucose/metabolism , Humans , Sodium-Glucose Transporter 2/chemistry , Sodium-Glucose Transporter 2/metabolism , Sodium-Glucose Transporter 2/ultrastructure , Sodium-Glucose Transporter 2 Inhibitors/chemistry , Sodium-Glucose Transporter 2 Inhibitors/pharmacologyABSTRACT
Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.
Subject(s)
Chitin , Cryoelectron Microscopy , Acetylglucosamine/metabolism , Aminoglycosides/pharmacology , Binding Sites , Cell Membrane/metabolism , Chitin/biosynthesis , Chitin/chemistry , Chitin/metabolism , Chitin/ultrastructure , Chitin Synthase/metabolism , Phytophthora/enzymology , Uridine Diphosphate/metabolism , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
Tumor heterogeneity presents a challenge for inferring clonal evolution and driver gene identification. Here, we describe a method for analyzing the cancer genome at a single-cell nucleotide level. To perform our analyses, we first devised and validated a high-throughput whole-genome single-cell sequencing method using two lymphoblastoid cell line single cells. We then carried out whole-exome single-cell sequencing of 90 cells from a JAK2-negative myeloproliferative neoplasm patient. The sequencing data from 58 cells passed our quality control criteria, and these data indicated that this neoplasm represented a monoclonal evolution. We further identified essential thrombocythemia (ET)-related candidate mutations such as SESN2 and NTRK1, which may be involved in neoplasm progression. This pilot study allowed the initial characterization of the disease-related genetic architecture at the single-cell nucleotide level. Further, we established a single-cell sequencing method that opens the way for detailed analyses of a variety of tumor types, including those with high genetic complex between patients.
Subject(s)
Clonal Evolution , Gene Expression Profiling , High-Throughput Nucleotide Sequencing/methods , Janus Kinase 2/genetics , Myeloproliferative Disorders/genetics , Myeloproliferative Disorders/pathology , Single-Cell Analysis/methods , Thrombocythemia, Essential/genetics , Exome , Genome, Human , Humans , Male , Middle Aged , MutationABSTRACT
Intestinal stromal cells are known to modulate the propagation and differentiation of intestinal stem cells1,2. However, the precise cellular and molecular mechanisms by which this diverse stromal cell population maintains tissue homeostasis and repair are poorly understood. Here we describe a subset of intestinal stromal cells, named MAP3K2-regulated intestinal stromal cells (MRISCs), and show that they are the primary cellular source of the WNT agonist R-spondin 1 following intestinal injury in mice. MRISCs, which are epigenetically and transcriptomically distinct from subsets of intestinal stromal cells that have previously been reported3-6, are strategically localized at the bases of colon crypts, and function to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage through enhanced R-spondin 1 production. Mechanistically, this MAP3K2 specific function is mediated by a previously unknown reactive oxygen species (ROS)-MAP3K2-ERK5-KLF2 axis to enhance production of R-spondin 1. Our results identify MRISCs as a key component of an intestinal stem cell niche that specifically depends on MAP3K2 to augment WNT signalling for the regeneration of damaged intestine.
Subject(s)
Intestinal Mucosa/cytology , MAP Kinase Kinase Kinase 2/metabolism , Stem Cell Niche , Stromal Cells/cytology , Animals , Antigens, CD34 , Colitis/pathology , Colitis/prevention & control , Epigenesis, Genetic , Female , Intestinal Mucosa/pathology , Kruppel-Like Transcription Factors/metabolism , Male , Mice , Reactive Oxygen Species/metabolism , Tetraspanin 28 , Thrombospondins/biosynthesis , Thrombospondins/metabolism , Thy-1 AntigensABSTRACT
We developed an analysis pipeline that can extract microbial sequences from spatial transcriptomic (ST) data and assign taxonomic labels, generating a spatial microbial abundance matrix in addition to the default host expression matrix, enabling simultaneous analysis of host expression and microbial distribution. We called the pipeline spatial metatranscriptome (SMT) and applied it on both human and murine intestinal sections and validated the spatial microbial abundance information with alternative assays. Biological insights were gained from these novel data that showed host-microbe interaction at various spatial scales. Finally, we tested experimental modification that can increase microbial capture while preserving host spatial expression quality and, by use of positive controls, quantitatively showed the capture efficiency and recall of our methods. This proof-of-concept work shows the feasibility of SMT analysis and paves the way for further experimental optimization and application.
Subject(s)
Gene Expression Profiling , Transcriptome , Humans , Animals , MiceABSTRACT
SUR2, similar to SUR1, is a regulatory subunit of the ATP-sensitive potassium channel (KATP), which plays a key role in numerous important physiological processes and is implicated in various diseases. Recent structural studies have revealed that, like SUR1, SUR2 can undergo ligand-dependent dynamic conformational changes, transitioning between an inhibitory inward-facing conformation and an activating occluded conformation. In addition, SUR2 possesses a unique inhibitory Regulatory helix (R helix) that is absent in SUR1. The binding of the activating Mg-ADP to NBD2 of SUR2 competes with the inhibitory Mg-ATP, thereby promoting the release of the R helix and initiating the activation process. Moreover, the signal generated by Mg-ADP binding to NBD2 might be directly transmitted to the TMD of SUR2, prior to NBD dimerization. Furthermore, the C-terminal 42 residues (C42) of SUR2 might allosterically regulate the kinetics of Mg-nucleotide binding on NBD2. These distinctive properties render SUR2 intricate sensors for intracellular Mg-nucleotides.
Subject(s)
Nucleotides , Potassium Channels, Inwardly Rectifying , Nucleotides/metabolism , Sulfonylurea Receptors/chemistry , Sulfonylurea Receptors/metabolism , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/metabolism , Adenosine Triphosphate/metabolismABSTRACT
Pharmacological vitamin C (VC) is a potential natural compound for cancer treatment. However, the mechanism underlying its antitumor effects remains unclear. In this study, we found that pharmacological VC significantly inhibits the mTOR (including mTORC1 and mTORC2) pathway activation and promotes GSK3-FBXW7-mediated Rictor ubiquitination and degradation by increasing the cellular ROS. Moreover, we identified that HMOX1 is a checkpoint for pharmacological-VC-mediated mTOR inactivation, and the deletion of FBXW7 or HMOX1 suppresses the regulation of pharmacological VC on mTOR activation, cell size, cell viability, and autophagy. More importantly, it was observed that the inhibition of mTOR by pharmacological VC supplementation in vivo produces positive therapeutic responses in tumor growth, while HMOX1 deficiency rescues the inhibitory effect of pharmacological VC on tumor growth. These results demonstrate that VC influences cellular activities and tumor growth by inhibiting the mTOR pathway through Rictor and HMOX1, which may have therapeutic potential for cancer treatment.
Subject(s)
Ascorbic Acid , Neoplasms , Humans , F-Box-WD Repeat-Containing Protein 7/metabolism , Ascorbic Acid/pharmacology , Glycogen Synthase Kinase 3/metabolism , Rapamycin-Insensitive Companion of mTOR Protein/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Mechanistic Target of Rapamycin Complex 2/genetics , Mechanistic Target of Rapamycin Complex 2/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Transcription Factors/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolismABSTRACT
After acquiring competence for selected cell fates, embryonic primordia may remain plastic for variable periods before tissue identity is irrevocably determined (commitment). We investigated the chromatin basis for these developmental milestones in mouse endoderm, a tissue with recognizable rostro-caudal patterning and transcription factor (TF)-dependent interim plasticity. Foregut-specific enhancers are as accessible and active in early midgut as in foregut endoderm, and intestinal enhancers and identity are established only after ectopic cis-regulatory elements are decommissioned. Depletion of the intestinal TF CDX2 before this cis element transition stabilizes foregut enhancers, reinforces ectopic transcriptional programs, and hence imposes foregut identities on the midgut. Later in development, as the window of chromatin plasticity elapses, CDX2 depletion weakens intestinal, without strengthening foregut, enhancers. Thus, midgut endoderm is primed for heterologous cell fates, and TFs act on a background of shifting chromatin access to determine intestinal at the expense of foregut identity. Similar principles likely govern other fate commitments.