Publications by authors named "Guisheng Zhong"

44 Publications

Elucidation of Distinct Modular Assemblies of Smoothened Receptor by Bitopic Ligand Measurement.

J Med Chem 2021 Sep 7;64(18):13830-13840. Epub 2021 Sep 7.

iHuman Institute, ShanghaiTech University, Ren Building, 393 Middle Huaxia Road, Shanghai 201210, China.

Class F G protein-coupled receptors are characterized by a large extracellular domain (ECD) in addition to the common transmembrane domain (TMD) with seven α-helixes. For smoothened receptor (SMO), structural studies revealed dissected ECD and TMD, and their integrated assemblies. However, distinct assemblies were reported under different circumstances. Using an unbiased approach based on four series of cross-conjugated bitopic ligands, we explore the relationship between the active status and receptor assembly. Different activity dependency on the linker length for these bitopic ligands corroborates the various occurrences of SMO assembly. These results reveal a rigid "near" assembly for active SMO, which is in contrast to previous results. Conversely, inactive SMO adopts a free ECD, which would be remotely captured at "far" assembly by cholesterol. Altogether, we propose a mechanism of cholesterol flow-caused SMO activation involving an erection of ECD from far to near assembly.
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http://dx.doi.org/10.1021/acs.jmedchem.1c01220DOI Listing
September 2021

Rational Remodeling of Atypical Scaffolds for the Design of Photoswitchable Cannabinoid Receptor Tools.

J Med Chem 2021 Sep 3;64(18):13752-13765. Epub 2021 Sep 3.

iHuman Institute, ShanghaiTech University, Pudong, Shanghai 201210, China.

Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.
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http://dx.doi.org/10.1021/acs.jmedchem.1c01088DOI Listing
September 2021

Multiregional profiling of the brain transmembrane proteome uncovers novel regulators of depression.

Sci Adv 2021 Jul 21;7(30). Epub 2021 Jul 21.

iHuman Institute, ShanghaiTech University, Shanghai 201210, China.

Transmembrane proteins play vital roles in mediating synaptic transmission, plasticity, and homeostasis in the brain. However, these proteins, especially the G protein-coupled receptors (GPCRs), are underrepresented in most large-scale proteomic surveys. Here, we present a new proteomic approach aided by deep learning models for comprehensive profiling of transmembrane protein families in multiple mouse brain regions. Our multiregional proteome profiling highlights the considerable discrepancy between messenger RNA and protein distribution, especially for region-enriched GPCRs, and predicts an endogenous GPCR interaction network in the brain. Furthermore, our new approach reveals the transmembrane proteome remodeling landscape in the brain of a mouse depression model, which led to the identification of two previously unknown GPCR regulators of depressive-like behaviors. Our study provides an enabling technology and rich data resource to expand the understanding of transmembrane proteome organization and dynamics in the brain and accelerate the discovery of potential therapeutic targets for depression treatment.
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http://dx.doi.org/10.1126/sciadv.abf0634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294761PMC
July 2021

The zinc finger protein Miz1 suppresses liver tumorigenesis by restricting hepatocyte-driven macrophage activation and inflammation.

Immunity 2021 06 25;54(6):1168-1185.e8. Epub 2021 May 25.

Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China; Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China. Electronic address:

Chronic inflammation plays a central role in hepatocellular carcinoma (HCC), but the contribution of hepatocytes to tumor-associated inflammation is not clear. Here, we report that the zinc finger transcription factor Miz1 restricted hepatocyte-driven inflammation to suppress HCC, independently of its transcriptional activity. Miz1 was downregulated in HCC mouse models and a substantial fraction of HCC patients. Hepatocyte-specific Miz1 deletion in mice generated a distinct sub-group of hepatocytes that produced pro-inflammatory cytokines and chemokines, which skewed the polarization of the tumor-infiltrating macrophages toward pro-inflammatory phenotypes to promote HCC. Mechanistically, Miz1 sequestrated the oncoprotein metadherin (MTDH), preventing MTDH from promoting transcription factor nuclear factor κB (NF-κB) activation. A distinct sub-group of pro-inflammatory cytokine-producing hepatocytes was also seen in a subset of HCC patients. In addition, Miz1 expression inversely correated with disease recurrence and poor prognosis in HCC patients. Our findings identify Miz1 as a tumor suppressor that prevents hepatocytes from driving inflammation in HCC.
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http://dx.doi.org/10.1016/j.immuni.2021.04.027DOI Listing
June 2021

Structural basis for CD97 recognition of the decay-accelerating factor CD55 suggests mechanosensitive activation of adhesion GPCRs.

J Biol Chem 2021 Jan-Jun;296:100776. Epub 2021 May 14.

Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China. Electronic address:

The adhesion G protein-coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF). Herein, we designed a chimeric expression construct with the EGF domains of CD97 and the SCR domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55-CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55-CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein-coupled receptors.
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http://dx.doi.org/10.1016/j.jbc.2021.100776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191316PMC
August 2021

Organized cannabinoid receptor distribution in neurons revealed by super-resolution fluorescence imaging.

Nat Commun 2020 11 11;11(1):5699. Epub 2020 Nov 11.

iHuman Institute, ShanghaiTech University, 201210, Shanghai, China.

G-protein-coupled receptors (GPCRs) play important roles in cellular functions. However, their intracellular organization is largely unknown. Through investigation of the cannabinoid receptor 1 (CB), we discovered periodically repeating clusters of CB hotspots within the axons of neurons. We observed these CB hotspots interact with the membrane-associated periodic skeleton (MPS) forming a complex crucial in the regulation of CB signaling. Furthermore, we found that CB hotspot periodicity increased upon CB agonist application, and these activated CB displayed less dynamic movement compared to non-activated CB. Our results suggest that CB forms periodic hotspots organized by the MPS as a mechanism to increase signaling efficacy upon activation.
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http://dx.doi.org/10.1038/s41467-020-19510-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7659323PMC
November 2020

Correction: Recent development of AAV-based gene therapies for inner ear disorders.

Gene Ther 2020 Aug;27(7-8):406

iHuman Institute, ShanghaiTech University, Shanghai, 201210, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41434-020-00186-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608405PMC
August 2020

Enhancer Reprogramming within Pre-existing Topologically Associated Domains Promotes TGF-β-Induced EMT and Cancer Metastasis.

Mol Ther 2020 09 1;28(9):2083-2095. Epub 2020 Jun 1.

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; iHuman Institute, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Transcription growth factor β (TGF-β) signaling-triggered epithelial-to-mesenchymal transition (EMT) process is associated with tumor stemness, metastasis, and chemotherapy resistance. However, the epigenomic basis for TGF-β-induced EMT remains largely unknown. Here we reveal that HDAC1-mediated global histone deacetylation and the gain of specific histone H3 lysine 27 acetylation (H3K27ac)-marked enhancers are essential for the TGF-β-induced EMT process. Enhancers gained upon TGF-β treatment are linked to gene activation of EMT markers and cancer metastasis. Notably, dynamic enhancer gain or loss mainly occurs within pre-existing topologically associated domains (TADs) in epithelial cells, with minimal three-dimensional (3D) genome architecture reorganization. Through motif enrichment analysis of enhancers that are lost or gained upon TGF-β stimulation, we identify FOXA2 as a key factor to activate epithelial-specific enhancer activity, and we also find that TEAD4 forms a complex with SMAD2/3 to mediate TGF-β signaling-triggered mesenchymal enhancer reprogramming. Together, our results implicate that key transcription-factor (TF)-mediated enhancer reprogramming modulates the developmental transition in TGF-β signaling-associated cancer metastasis.
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http://dx.doi.org/10.1016/j.ymthe.2020.05.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474343PMC
September 2020

Recent development of AAV-based gene therapies for inner ear disorders.

Gene Ther 2020 08 18;27(7-8):329-337. Epub 2020 May 18.

iHuman Institute, ShanghaiTech University, Shanghai, 201210, China.

Gene therapy for auditory diseases is gradually maturing. Recent progress in gene therapy treatments for genetic and acquired hearing loss has demonstrated the feasibility in animal models. However, a number of hurdles, such as lack of safe viral vector with high efficiency and specificity, robust deafness large animal models, translating animal studies to clinic etc., still remain to be solved. It is necessary to overcome these challenges in order to effectively recover auditory function in human patients. Here, we review the progress made in our group, especially our efforts to make more effective and cell type-specific viral vectors for targeting cochlea cells.
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http://dx.doi.org/10.1038/s41434-020-0155-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445886PMC
August 2020

A Novel G Protein-Biased and Subtype-Selective Agonist for a G Protein-Coupled Receptor Discovered from Screening Herbal Extracts.

ACS Cent Sci 2020 Feb 23;6(2):213-225. Epub 2020 Jan 23.

iHuman Institute, ShanghaiTech University, Shanghai 201210, China.

Subtype selectivity and functional bias are vital in current drug discovery for G protein-coupled receptors (GPCRs) as selective and biased ligands are expected to yield drug leads with optimal on-target benefits and minimal side-effects. However, structure-based design and medicinal chemistry exploration remain challenging in part because of highly conserved binding pockets within subfamilies. Herein, we present an affinity mass spectrometry approach for screening herbal extracts to identify active ligands of a GPCR, the 5-HT receptor. Using this method, we discovered a naturally occurring aporphine 1857 that displayed strong selectivity for activating 5-HT without activating the 5-HT or 5-HT receptors. Remarkably, this novel ligand exhibited exclusive bias toward G protein signaling for which key residues were identified, and it showed comparable efficacy for food intake suppression and weight loss as the antiobesity drug, lorcaserin. Our study establishes an efficient approach to discovering novel GPCR ligands by exploring the largely untapped chemical space of natural products.
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http://dx.doi.org/10.1021/acscentsci.9b01125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047268PMC
February 2020

Calcineurin Signaling Mediates Disruption of the Axon Initial Segment Cytoskeleton after Injury.

iScience 2020 Feb 1;23(2):100880. Epub 2020 Feb 1.

School of Life Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New District, Shanghai 201210, China. Electronic address:

The axon initial segment (AIS) cytoskeleton undergoes rapid and irreversible disruption prior to cell death after injury, and loss of AIS integrity can produce profound neurological effects on the nervous system. Here we described a previously unrecognized mechanism for ischemia-induced alterations in AIS integrity. We show that in hippocampal CA1 pyramidal neurons Na1.6 mostly preserves at the AIS after disruption of the cytoskeleton in a mouse model of middle cerebral artery occlusion. Genetic removal of neurofascin-186 leads to rapid disruption of Na1.6 following injury, indicating that neurofascin is required for Na1.6 maintenance at the AIS after cytoskeleton collapse. Importantly, calcineurin inhibition with FK506 fully protects AIS integrity and sufficiently prevents impairments of spatial learning and memory from injury. This study provides evidence that calcineurin activation is primarily involved in initiating disassembly of the AIS cytoskeleton and that maintaining AIS integrity is crucial for therapeutic strategies to facilitate recovery from injury.
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http://dx.doi.org/10.1016/j.isci.2020.100880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031317PMC
February 2020

Molecular Mechanism for Ligand Recognition and Subtype Selectivity of α Adrenergic Receptor.

Cell Rep 2019 12;29(10):2936-2943.e4

iHuman Institute, ShanghaiTech University, Shanghai 201210, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of β adrenergic receptors (βARs). However, the structure of α adrenergic receptors (αARs) remains to be determined. Here, we report the structure of the human α adrenergic receptor (αAR) with the non-selective antagonist, RS79948, at 2.8 Å. Our structure, mutations, modeling, and functional experiments indicate that a αAR-specific D206-R409-Y405 network plays a role in determining α adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in αAR is involved in receptor activation. Together, our structure of human αAR-RS79948 provides key insight into the mechanism underlying the α adrenergic receptor activation and subtype selectivity.
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http://dx.doi.org/10.1016/j.celrep.2019.10.112DOI Listing
December 2019

Structural Basis of the Diversity of Adrenergic Receptors.

Cell Rep 2019 12;29(10):2929-2935.e4

iHuman Institute, ShanghaiTech University, Shanghai 201210, China; School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the α adrenergic receptor (αAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe and Tyr) to G-protein coupling region (Ile and Lys) are discovered to play a key role in the interplay between partial agonism and biased signaling of αAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors.
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http://dx.doi.org/10.1016/j.celrep.2019.10.088DOI Listing
December 2019

LncRNA- contributes to cardiac fibrosis through --HuR complex in mouse myocardial infarction.

Theranostics 2019 25;9(24):7282-7297. Epub 2019 Sep 25.

Department of Cardiovascular Surgery of the First Affiliated Hospital & Institute for Cardiovascular Science, State Key Laboratory of Radiation Medicine and Protection, Medical College, Soochow University, Suzhou 215000, China.

: As a hallmark of various heart diseases, cardiac fibrosis ultimately leads to end-stage heart failure. Anti-fibrosis is a potential therapeutic strategy for heart failure. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of heart diseases that promise to serve as therapeutic targets. However, few lncRNAs have been directly implicated in cardiac fibrosis. : The lncRNA expression profiles were assessed by microarray in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. The mechanisms and functional significance of lncRNA- in cardiac fibrosis were further investigated with both and models. : We identified 389 differentially expressed lncRNAs in cardiac fibrotic and remote ventricular tissues in mice with myocardial infarction. Among them, a lncRNA () we named was enriched in the nuclei of fibroblasts, and elevated in both myocardial infarction and TGF-β-induced cardiac fibrosis. Knockdown of prevented TGF-β-induced fibroblast-myofibroblast transition, aberrant cell proliferation and secretion of extracellular matrix proteins , and mended the impaired cardiac function in mice suffering myocardial infarction. studies indicated that knockdown of significantly inhibited the expression of its neighboring gene , and vice versa. The overexpression obviously disturbed the regulatory effects of shRNAs in both the cultured cardiac fibroblasts and myocardial infarction-induced fibrosis. Dual-Luciferase assay demonstrated that and mRNA stabilized each other via their complementary binding at the 3'-end. RNA electrophoretic mobility shift assay and RNA immunoprecipitation assay indicated that RNA binding protein HuR could bind to - RNA duplex, whereas the knockdown of dramatically reduced the stabilization of and mRNAs, down-regulated their expression in cardiac fibroblasts, and thus inhibited TGF-β-induced fibrosis. The overexpression partially restrained the phenotype change of cardiac fibroblasts induced by shRNAs, but not that induced by shRNAs. : Our study identifies as a critical regulator of cardiac fibrosis, and demonstrates --HuR complex-mediated mRNA stability is the underlying mechanism of -regulated cardiac fibrosis. Fibroblast-enriched could represent a novel target for anti-fibrotic therapy in heart diseases.
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http://dx.doi.org/10.7150/thno.33920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831303PMC
September 2020

Stress-Induced Metabolic Disorder in Peripheral CD4 T Cells Leads to Anxiety-like Behavior.

Cell 2019 10;179(4):864-879.e19

MOE Laboratory of Biosystem Homeostasis and Protection and Life Sciences Institute, Zhejiang University, Hangzhou 310058, China; Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou 310016, China. Electronic address:

Physical or mental stress leads to neuroplasticity in the brain and increases the risk of depression and anxiety. Stress exposure causes the dysfunction of peripheral T lymphocytes. However, the pathological role and underlying regulatory mechanism of peripheral T lymphocytes in mood disorders have not been well established. Here, we show that the lack of CD4 T cells protects mice from stress-induced anxiety-like behavior. Physical stress-induced leukotriene B4 triggers severe mitochondrial fission in CD4 T cells, which further leads to a variety of behavioral abnormalities including anxiety, depression, and social disorders. Metabolomic profiles and single-cell transcriptome reveal that CD4 T cell-derived xanthine acts on oligodendrocytes in the left amygdala via adenosine receptor A1. Mitochondrial fission promotes the de novo synthesis of purine via interferon regulatory factor 1 accumulation in CD4 T cells. Our study implicates a critical link between a purine metabolic disorder in CD4 T cells and stress-driven anxiety-like behavior.
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http://dx.doi.org/10.1016/j.cell.2019.10.001DOI Listing
October 2019

Second messenger ApA polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells.

Nat Commun 2019 10 11;10(1):4664. Epub 2019 Oct 11.

State Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China.

Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger ApA binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by ApA remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that ApA specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of ApA. These results highlight a direct polymerization signaling mechanism by the second messenger.
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http://dx.doi.org/10.1038/s41467-019-12710-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789022PMC
October 2019

ER-localized Hrd1 ubiquitinates and inactivates Usp15 to promote TLR4-induced inflammation during bacterial infection.

Nat Microbiol 2019 12;4(12):2331-2346

State Key Laboratory of Cell Biology, Key Laboratory of Systems Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Innovation Center for Cell Signaling Network, Shanghai, China.

The special organelle-located MAVS, STING and TLR3 are important for clearing viral infections. Although TLR4 triggers NF-κB activation to produce pro-inflammatory cytokines for bacterial clearance, effectors with special organelle localization have not been identified. Here, we screened more than 280 E3 ubiquitin ligases and discovered that the endoplasmic reticulum-located Hrd1 regulates TLR4-induced inflammation during bacterial infection. Hrd1 interacts directly with the deubiquitinating enzyme Usp15. Unlike the classical function of Hrd1 in endoplasmic reticulum-associated degradation, Usp15 is not degraded but loses its deubiquitinating activity for IκBα deubiquitination, resulting in excessive NF-κB activation. Importantly, Hrd1 deficiency in macrophages protects mice against lipopolysaccharide-induced septic shock, and knockdown of Usp15 in Hrd1-knockout macrophages restores the reduced IL-6 production. This study proposes that there is crosstalk between Hrd1 and TLR4, thereby linking the endoplasmic reticulum-plasma membrane function during bacterial infection.
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http://dx.doi.org/10.1038/s41564-019-0542-2DOI Listing
December 2019

AAV-ie enables safe and efficient gene transfer to inner ear cells.

Nat Commun 2019 08 19;10(1):3733. Epub 2019 Aug 19.

iHuman Institute, ShanghaiTech University, 201210, Shanghai, China.

Hearing loss is the most common sensory disorder. While gene therapy has emerged as a promising treatment of inherited diseases like hearing loss, it is dependent on the identification of gene delivery vectors. Adeno-associated virus (AAV) vector-mediated gene therapy has been approved in the US for treating a rare inherited eye disease but no safe and efficient vectors have been identified that can target the diverse types of inner ear cells. Here, we identify an AAV variant, AAV-inner ear (AAV-ie), for gene delivery in mouse inner ear. Our results show that AAV-ie transduces the cochlear supporting cells (SCs) with high efficiency, representing a vast improvement over conventional AAV serotypes. Furthermore, after AAV-ie-mediated transfer of the Atoh1 gene, we find that many SCs trans-differentiated into new HCs. Our results suggest that AAV-ie is a useful tool for the cochlear gene therapy and for investigating the mechanism of HC regeneration.
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http://dx.doi.org/10.1038/s41467-019-11687-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700137PMC
August 2019

Differentiation of human adipose-derived stem cells into neuron/motoneuron-like cells for cell replacement therapy of spinal cord injury.

Cell Death Dis 2019 08 8;10(8):597. Epub 2019 Aug 8.

East Hospital, School of Medicine, Tongji University, Shanghai, 200120, China.

Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.
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http://dx.doi.org/10.1038/s41419-019-1772-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687731PMC
August 2019

Structural plasticity of actin-spectrin membrane skeleton and functional role of actin and spectrin in axon degeneration.

Elife 2019 05 1;8. Epub 2019 May 1.

Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States.

Axon degeneration sculpts neuronal connectivity patterns during development and is an early hallmark of several adult-onset neurodegenerative disorders. Substantial progress has been made in identifying effector mechanisms driving axon fragmentation, but less is known about the upstream signaling pathways that initiate this process. Here, we investigate the behavior of the actin-spectrin-based Membrane-associated Periodic Skeleton (MPS), and effects of actin and spectrin manipulations in sensory axon degeneration. We show that trophic deprivation (TD) of mouse sensory neurons causes a rapid disassembly of the axonal MPS, which occurs prior to protein loss and independently of caspase activation. Actin destabilization initiates TD-related retrograde signaling needed for degeneration; actin stabilization prevents MPS disassembly and retrograde signaling during TD. Depletion of βII-spectrin, a key component of the MPS, suppresses retrograde signaling and protects axons against degeneration. These data demonstrate structural plasticity of the MPS and suggest its potential role in early steps of axon degeneration.
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http://dx.doi.org/10.7554/eLife.38730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494423PMC
May 2019

Critical role of spectrin in hearing development and deafness.

Sci Adv 2019 04 17;5(4):eaav7803. Epub 2019 Apr 17.

iHuman Institute, ShanghaiTech University, Shanghai, China.

Inner ear hair cells (HCs) detect sound through the deflection of mechanosensory stereocilia. Stereocilia are inserted into the cuticular plate of HCs by parallel actin rootlets, where they convert sound-induced mechanical vibrations into electrical signals. The molecules that support these rootlets and enable them to withstand constant mechanical stresses underpin our ability to hear. However, the structures of these molecules have remained unknown. We hypothesized that αII- and βII-spectrin subunits fulfill this role, and investigated their structural organization in rodent HCs. Using super-resolution fluorescence imaging, we found that spectrin formed ring-like structures around the base of stereocilia rootlets. These spectrin rings were associated with the hearing ability of mice. Further, HC-specific, βII-spectrin knockout mice displayed profound deafness. Overall, our work has identified and characterized structures of spectrin that play a crucial role in mammalian hearing development.
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http://dx.doi.org/10.1126/sciadv.aav7803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469942PMC
April 2019

Identification of natural products as novel ligands for the human 5-HT receptor.

Biophys Rep 2018 9;4(1):50-61. Epub 2018 Mar 9.

1National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.

G protein-coupled receptors (GPCRs) constitute the largest human protein family with over 800 members, which are implicated in many important medical conditions. Serotonin receptors belong to the aminergic GPCR subfamily and play important roles in physiological and psychological activities. Structural biology studies have revealed the structures of many GPCRs in atomic details and provide the basis for the identification and investigation of the potential ligands, which interact with and modulate the receptors. Here, an integrative approach combining a focused target-specific natural compound library, a thermal-shift-based screening method, affinity mass spectrometry, molecular docking, and as well as functional assay, was applied to identify (-)-crebanine and several other aporphine alkaloids as initial hits for a human serotonin receptor subtype, the 5-HT receptor. Further studies illuminated key features of their binding affinity, downstream signaling and tissue reaction, providing a molecular explanation for the interaction between (-)-crebanine and human 5-HT receptor.
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http://dx.doi.org/10.1007/s41048-018-0047-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5860131PMC
March 2018

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains.

ACS Nano 2017 07 19;11(7):6985-6995. Epub 2017 Jun 19.

School of Physical Science and Technology, ShanghaiTech University , Shanghai 201210, China.

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities.
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http://dx.doi.org/10.1021/acsnano.7b02298DOI Listing
July 2017

Elevation in Total Homocysteine Levels in Chinese Patients With Essential Hypertension Treated With Antihypertensive Benazepril.

Clin Appl Thromb Hemost 2016 Mar 30;22(2):191-8. Epub 2014 Dec 30.

Institute of Biomedicine, Anhui Medical University, Hefei, China Division of Epidemiology and Biostatistics, University of Illinois at Chicago School of Public Health, Chicago, IL, USA.

Objective: To investigate the effect of benazepril on plasma homocysteine (Hcy) levels and to analyze the correlation between the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and changes in Hcy levels in response to benazepril.

Methods: A total of 231 patients with mild to moderate essential hypertension were enrolled, and benazepril was orally administered at a dose of 10 mg/d for 2 weeks. Plasma Hcy levels were measured by high-performance liquid chromatography at baseline and after 2 weeks of treatment. Genotyping of the MTHFR C677T polymorphism was performed by TaqMan probe technique.

Results: There was no significant change in Hcy level after benazepril treatment for 2 weeks (P = .97). However, stratified by baseline Hcy levels, the patients with baseline Hcy <10 μmol/L had a significant increase in plasma Hcy levels (P = .003). The results from the multivariable linear regression analysis demonstrated a significant correlation between baseline Hcy levels and the changes in Hcy levels found in both the unadjusted (P = .002) and the adjusted model (P = .004). Strikingly, we found no significant effect modification by the MTHFR C677T polymorphism on the Hcy changes after benazepril treatment. There were also no statistically significant interactions of gene and environment factors (ie, gene smoking and drinking) on the changes in Hcy levels after benazepril treatment.

Conclusion: Benazepril may cause an increase in plasma Hcy levels among patients with hypertension with low baseline Hcy levels, while effect modification by MTHFR C677T genotypes on the changes in Hcy levels in response to benazepril was not significant among patients with essential hypertension.
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http://dx.doi.org/10.1177/1076029614565881DOI Listing
March 2016

Developmental mechanism of the periodic membrane skeleton in axons.

Elife 2014 Dec 23;3. Epub 2014 Dec 23.

Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University, Cambridge, United States.

Actin, spectrin, and associated molecules form a periodic sub-membrane lattice structure in axons. How this membrane skeleton is developed and why it preferentially forms in axons are unknown. Here, we studied the developmental mechanism of this lattice structure. We found that this structure emerged early during axon development and propagated from proximal regions to distal ends of axons. Components of the axon initial segment were recruited to the lattice late during development. Formation of the lattice was regulated by the local concentration of βII spectrin, which is higher in axons than in dendrites. Increasing the dendritic concentration of βII spectrin by overexpression or by knocking out ankyrin B induced the formation of the periodic structure in dendrites, demonstrating that the spectrin concentration is a key determinant in the preferential development of this structure in axons and that ankyrin B is critical for the polarized distribution of βII spectrin in neurites.
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http://dx.doi.org/10.7554/eLife.04581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337613PMC
December 2014

A PIK3C3-ankyrin-B-dynactin pathway promotes axonal growth and multiorganelle transport.

J Cell Biol 2014 Dec;207(6):735-52

Howard Hughes Medical Institute, Chevy Chase, MD 20815 Department of Biochemistry and Department of Radiology, Duke University, Durham, NC 27708

Axon growth requires long-range transport of organelles, but how these cargoes recruit their motors and how their traffic is regulated are not fully resolved. In this paper, we identify a new pathway based on the class III PI3-kinase (PIK3C3), ankyrin-B (AnkB), and dynactin, which promotes fast axonal transport of synaptic vesicles, mitochondria, endosomes, and lysosomes. We show that dynactin associates with cargo through AnkB interactions with both the dynactin subunit p62 and phosphatidylinositol 3-phosphate (PtdIns(3)P) lipids generated by PIK3C3. AnkB knockout resulted in shortened axon tracts and marked reduction in membrane association of dynactin and dynein, whereas it did not affect the organization of spectrin-actin axonal rings imaged by 3D-STORM. Loss of AnkB or of its linkages to either p62 or PtdIns(3)P or loss of PIK3C3 all impaired organelle transport and particularly retrograde transport in hippocampal neurons. Our results establish new functional relationships between PIK3C3, dynactin, and AnkB that together promote axonal transport of organelles and are required for normal axon length.
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http://dx.doi.org/10.1083/jcb.201407063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274267PMC
December 2014

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

Mol Cell Neurosci 2014 Jul 24;61:241-54. Epub 2014 Jul 24.

The Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, United States. Electronic address:

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.
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http://dx.doi.org/10.1016/j.mcn.2014.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134997PMC
July 2014
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