Biology subjects

Yan, N.

Publications and source records attributed to Yan, N..

33 records · Page 2Linked to original sources

bioRxiv2021

GABAergic neuronal IL-4R mediates T cell effect on memory

Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying effects of T cells on synaptic function and episodic memory. Depletion of CD4 T cells led to memory deficits and impaired long term potentiation. Severe combined immune-deficient (SCID) mice exhibited amnesia, which was reversible by repopulation with T cells from wild-type but not from IL-4-knockout mice. This rescue was mediated via IL-4 receptors (IL-4R) expressed on neurons. Exploration of snRNAseq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways and genes regulated by immune cells and molecules. IL-4R knockout in inhibitory neurons impaired contextual fear memory, suggesting participation of an IL-4-associated switch in regulating synaptic function and promoting contextual fear memory. These findings provide insights into neuroimmune interactions at the transcriptional and functional levels in neurons.

neuroscience↗

bioRxiv2021

Prediction of occult tumor progression via platelet RNAs in a mouse melanoma model: a potential new platform of cancer screening for early detection of cancer

Cancer screening provides the opportunity to detect cancer early, ideally before symptom onset and metastasis, and offers an increased opportunity for a better prognosis. The ideal biomarkers for cancer screening should discriminate individuals who have not developed invasive cancer yet but are destined to do so from healthy subjects1,2. However, most cancers lack effective screening recommendations. Therefore, further studies on novel screening strategies are urgently required. Here, our proof-of-concept study shows blood platelets could be a platform for liquid biopsy-based early cancer detection. By using a simple suboptimal inoculation melanoma mouse model, we identified differentially expressed RNAs in platelet signatures of mice injected with a suboptimal number of cancer cells (eDEGs) compared with mice with macroscopic melanomas and negative controls. These RNAs were strongly enriched in pathways related to immune response and regulation. Moreover, 36 genes selected from the eDEGs via bioinformatics analyses were verified in a mouse validation cohort via quantitative real-time PCR. LASSO regression was employed to generate the prediction models with gene expression signatures as the best predictors for occult tumor progression in mice. The prediction models showed great diagnostic efficacy and predictive value in our murine validation cohort, and could discriminate mice with occult tumors from control group (area under curve (AUC) of 0.935 (training data) and 0.912 (testing data)) (gene signature including Cd19, Cdkn1a, S100a9, Tap1, and Tnfrsf1b) and also from macroscopic tumor group (AUC of 0.920 (training data) and 0.936 (testing data)) (gene signature including Ccr7, Cd4, Kmt2d, and Ly6e). Our study provides evidence for potential clinical relevance of blood platelets as a platform for liquid biopsy-based early detection of cancer. Furthermore, the eDEGs are mostly immune-related, not tumor-specific. Hence it is possible platelets-based liquid biopsy could enable simultaneous early detection of cancers from multiple organs of origin3. It is also feasible to determine the origin of cancer since platelet profiles are influenced by tumor type3.

cancer biology↗

bioRxiv2021

Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the Long QT Syndrome

Nav1.5 is the primary voltage-gated Na+ (Nav) channel in the heart. Mutations of Nav1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Nav1.5-E1784K variant at an overall resolution of 3.3 [A]. Structural mapping of 91 and 178 point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are mainly clustered to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our "door wedge" model for fast inactivation. The structural analysis presented here, with a focus on the impact of disease mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Nav1.5 channelopathies.

biophysics↗

bioRxiv2020

Long-term survival of salmon-attached SARS-CoV-2 at 4 degree as a potential source of transmission in seafood markets

Several outbreaks of COVID-19 were associated with seafood markets, raising concerns that fish-attached SARS-CoV-2 may exhibit prolonged survival in low-temperature environments. Here we showed that salmon-attached SARS-CoV-2 at 4{degrees}C could remain infectious for more than one week, suggesting that fish-attached SARS-CoV-2 may be a source of transmission.

microbiology↗

bioRxiv2020

Orthosteric-allosteric dual inhibitors of PfHT1 as selective anti-malarial agents

Artemisinin-resistant malaria parasites have emerged and been spreading, posing a significant public health challenge. Anti-malarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a "selective starvation" strategy by selectively inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in Plasmodium falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. Comparison of the crystal structures of human GLUT3 and PfHT1 bound to C3361, a PfHT1-specific moderate inhibitor, revealed an inhibitor binding-induced pocket that presented a promising druggable site. We thereby designed small-molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship (SAR) studies, the TH-PF series was identified to selectively inhibit PfHT1 over GLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously targeting the orthosteric and allosteric sites of a transporter. Significance statementBlocking sugar uptake in P. falciparum by selectively inhibiting the hexose transporter PfHT1 kills the blood-stage parasites without affecting the host cells, indicating PfHT1 as a promising therapeutic target. Here, we report the development of novel small-molecule inhibitors that are selectively potent to the malaria parasites over human cell lines by simultaneously targeting the orthosteric and the allosteric binding sites of PfHT1. Our findings established the basis for the rational design of next-generation anti-malarial drugs.

pharmacology and toxicology↗

bioRxiv2020

Structure Basis of Cav1.1 Modulation by Dihydropyridine Compounds

1,4-Dihydropyridines (DHP), the most commonly used antihypertensives, function by inhibiting the L-type voltage-gated Ca2+ (Cav) channels. DHP compounds exhibit chirality-specific antagonistic or agonistic effects. Recent structural elucidation of rabbit Cav1.1 bound to an achiral drug nifedipine reveals the general binding mode for DHP drugs, but the molecular basis for chiral specificity remains elusive. Here, we report five cryo-EM structures of nanodisc-embedded Cav1.1 in the presence of the bestselling drug amlodipine, a DHP antagonist (R)-(+)-Bay K8644, and a titration of its agonistic enantiomer (S)-(-)-Bay K8644 at resolutions of 2.9-3.4 [A]. The amlodipine-bound structure reveals the molecular basis for the high efficacy of the drug. All structures with the addition of the Bay K8644 enantiomers exhibit similar inactivated conformations, suggesting that the agonistic effect of (S)-(-)-Bay K8644 might be transient. The similarity of these structures to that obtained in detergent micelles alleviates the concerns about potential structural perturbation by detergents.

molecular biology↗

bioRxiv2020

Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+ channels in nanodisc

NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the cryo-EM analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Navs, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaces the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 [A] and 3.2 [A] resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 [A], the local resolution of the toxin has been improved from ~ 6 [A] to ~ 4 [A]. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Nav channels in a membrane environment.

biophysics↗

bioRxiv2020

Structure and mechanism of a primate ferroportin

Ferroportin is the only cellular iron exporter in human and essential for iron homoeostasis. Mutations in ferroportin are associated with hemochromatosis or ferroportin diseases characterized by a paradoxical combination of anemia and abnormal accumulation of iron in cells. Ferroportin is also the target of hepcidin, which is a hormone that downregulates ferroportin activity. However, due to a lack of three-dimensional structures, the mechanism of iron transport by ferroportin and its regulation by hepcidin remains unclear. Here we present the structure of a ferroportin from the primate Philippine tarsier (TsFpn) at 3.0 [A] resolution determined by cryo-electron microscopy. TsFpn has a structural fold common to major facilitator superfamily of transporters and the current structure is in an outward-open conformation. The structure identifies two potential ion binding sites with each site coordinated by two residues. Functional studies demonstrate that TsFpn is a H+/Fe2+ antiporter and that transport of one Fe2+ is coupled to the transport of two H+ in the opposite direction such that the transport cycle is electroneutral. Further studies show that the two ion binding sites affect transport of H+ and Fe2+ differently. The structure also provides mechanistic interpretation for mutations that cause ferroportin diseases.

biochemistry↗

bioRxiv2020

Structure and mechanism of human diacylglycerol acyltransferase 1

Human diacylglycerol O-acyltransferase-1 (hDGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage. The lack of 3-dimensional structure has limited our understanding of substrate recognition and mechanism of catalysis, and hampers rational targeting of hDGAT1 for therapeutic purposes. Here we present the structure of hDGAT1 in complex with a substrate oleoyl Coenzyme A at 3.1 [A] resolution. hDGAT1 forms a homodimer and each protomer has nine transmembrane helices that carve out a hollow chamber in the lipid bilayer. The chamber encloses highly conserved catalytic residues and has separate entrances for the two substrates fatty acyl Coenzyme A and diacylglycerol. The N-terminus of hDGAT1 makes extensive interactions with the neighboring protomer, and is required for enzymatic activity.

biochemistry↗

bioRxiv2020

Structural basis for catalysis and substrate specificity of human ACAT1

Acyl-coenzyme A: cholesterol acyltransferases (ACATs) catalyze acyl transfer from acyl-coenzyme A (CoA) to cholesterol to generate cholesteryl ester, which is the primary form for cellular storage and plasma transport of cholesterol. Because of their close relationship with cholesterol metabolism, ACATs represent potential drug target for the treatment of atherosclerosis and other cholesterol-related disorders. Here we present the cryo-EM structure of human ACAT1 at 3.3 [A] resolution for dimer of dimers and 3.0 [A] for a dimer. Each protomer consists of nine transmembrane segments that enclose a cytosolic (C) and a transmembrane (T) tunnel. The tunnels, each accommodating an elongated density, converge at the predicted catalytic site. Structure-guided mutational analyses suggest the cytosolic and lateral entry for acyl-CoA and cholesterol, respectively. Our structural, biochemical, and mass spectrometric characterizations reveal the catalytic mechanism and substrate preference for unsaturated acyl chain by ACAT1.

biochemistry↗

bioRxiv2019

Structural basis for pore blockade of the human cardiac sodium channel Nav1.5 by tetrodotoxin and quinidine

Among the nine subtypes of human voltage-gated Na+ (Nav) channels, tetrodotoxin (TTX)-resistant Nav1.5 is the primary one in heart. More than 400 point mutations have been identified in Nav1.5 that are associated with cardiac disorders exemplified by type 3 long QT syndrome and Brugada syndrome, making Nav1.5 a common target for class I antiarrythmic drugs. Here we present the cryo-EM structures of Nav1.5 bound to quinidine and TTX with resolutions at 3.2 [A] and 3.3 [A], respectively, for the pore domain. The structures allow mapping of 278 disease-related mutations in the resolved region, establishing the framework for mechanistic investigation of these pathogenic mutations. Quinidine is positioned right below the selectivity filter and coordinated by residues from repeats I, III, and IV. Pore blockade is achieved through both direct obstruction of the ion permeation path and induced rotation of a Tyr residue that leads to intracellular gate closure. Structural comparison of Nav1.5 with our previously reported Nav1.2/1.4/1.7 reveals the molecular determinant for the different susceptibility to TTX by TTX-resistant and -sensitive Nav subtypes, and highlights the functional significance of the underexplored and least conserved extracellular loops.

biophysics↗

bioRxiv2019

High Yield Monolayer Graphene Grids for Near-Atomic Resolution Cryo-Electron Microscopy

Cryogenic electron microscopy (cryo-EM) has become one of the most powerful techniques to reveal the atomic structures and working mechanisms of biological macromolecules. New designs of the cryo-EM grids--aimed at preserving thin, uniform vitrified ice and improving protein adsorption--have been considered a promising approach to achieving higher resolution with the minimal amount of materials and data. Here, we describe a method for preparing graphene cryo-EM grids with 99% monolayer graphene coverage that allows for more than 70% grid squares for effective data acquisition with improved image quality and protein density. Using our graphene grids, we have achieved 2.6 [A] resolution for streptavidin, with a molecular weight of 52 kDa, from 11,000 particles. Our graphene grids increase the density of examined soluble, membrane, and lipo-proteins by at least five times, affording the opportunity for structural investigation of challenging proteins which cannot be produced in large quantity. In addition, our method employs only simple tools that most structural biology laboratories can access. Moreover, our approach allows for customized grid designs targeting specific proteins, due to its broad compatibility with a variety of nanomaterials. Significance statementSingle particle cryogenic electron microscopy (cryo-EM) represents the cutting-edge technology to determine three-dimensional atomic structures of bio-macromolecules. However, issues of cryo-sample preparation limit the cryo-EM to achieve higher resolution. Here, we demonstrated a high yield, monolayer graphene supporting film to improve the cryo-sample quality. Using our approach, we have achieved so far, the highest resolution structure of the smallest protein by cryo-EM with the minimal number of datasets. Our technique paves the way for universal cryo-sample preparation for near-atomic resolution cryo-EM.

biophysics↗

bioRxiv2019

Metabolic Control of Viral Infection through PPAR-α Regulation of STING Signaling

Peroxisomal proliferator activated receptors (PPARs) are sensors of dietary lipids and pharmacological targets in the treatment of metabolic disorders. PPAR ligands are also immunosuppressive. However, their function during infection is debated and the mechanisms that underlie their immunoregulatory properties are unclear. We investigated the consequences of PPAR activation during herpesvirus infection. We found that activation of PPAR- increased herpesvirus replication, suppressed type I interferon production and induced reactive oxygen species (ROS). We discovered that ROS induced by PPAR- stimulation suppressed the cytoplasmic DNA sensing pathway after direct activation of stimulator of interferon (STING), the ER adapter downstream of cytoplasmic DNA recognition. Although high ROS induces inflammasome activation and cytokine production, we found that ROS inhibited interferon production after cytoplasmic DNA recognition. Treatment of mice with a clinically relevant agonist of PPAR- increased herpesvirus replication and pathogenesis, comparable to levels observed previously in type I interferon receptor knockout mice. These findings reveal that activation of PPAR- regulates immunity to cytoplasmic DNA and DNA virus infection through inhibition of interferon. Moreover, these results demonstrate that STING signaling and interferon production is regulated by ROS.

immunology↗

bioRxiv2019

Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators

The type-2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report four cryo-EM structures of porcine RyR2 bound to distinct modulators that collectively provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the Central domain that facilitates the dilation of S6 bundle, but is insufficient to open the pore. The small molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the Central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state. Caffeine locks the Central domain in a constitutively contracted state, while further addition of ATP opens the channel by strengthening the coupling between the U-motif and S6. Our study marks an important step towards mechanistic understanding of the complicated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

biochemistry↗

bioRxiv2018

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of oligomeric Hh receptor Patched (Ptch) by the secreted and post-translationally modified ligand Hh relieves suppression of the signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp). The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers and each dimer binds to one ShhNp through two distinct inhibitory interfaces. Ptch1-A binds to ShhNp through the well-characterized Ca2+-mediated interface on the globular domain of ShhNp, and Ptch1-B primarily interacts with the N-terminal peptide and the palmitoyl moiety. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1-B. Our structure elucidates the tetrameric assembly of Ptch1 and suggests asymmetric mode of actions of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

biophysics↗