Wonhwa Cho

University of Illinois at Chicago, Chicago, Illinois, United States

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Publications (148)688.59 Total impact

  • Wonhwa Cho · Hyunjin Kim · Yusi Hu
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    ABSTRACT: Membrane–protein interaction plays key roles in a wide variety of biological processes. To facilitate rapid and sensitive measurement of membrane binding of soluble proteins, we developed a fluorescence- based quantitative assay that is universally applicable to all proteins. This fluorescence-quenching assay employs fluorescence protein (FP)-tagged proteins whose fluorescence intensity is greatly decreased when they bind vesicles containing synthetic lipid dark quenchers, such as N-dimethylaminoazobenzenesulfonylphosphatidylethanolamine (dabsyl-PE). This simple assay can be performed with either a spectrofluorometer or a plate reader and optimized for different proteins with various combinations of FPs and quenching lipids. The assay allows rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains and proteins, and also highthroughput screening of small molecules that modulate membrane binding of proteins.
    No preview · Article · Jan 2016
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    Full-text · Dataset · Nov 2015
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    ABSTRACT: Vibrio cholerae, responsible for acute gastroenteritis secretes a large multifunctional-autoprocessing repeat-in-toxin (MARTX) toxin linked to evasion of host immune system, facilitating colonization of small intestine. Unlike other effector domains of the multifunctional toxin that target cytoskeleton, the function of alpha-beta hydrolase (ABH) remained elusive. This study demonstrates that ABH is an esterase/lipase with catalytic Ser-His-Asp triad. ABH binds with high affinity to phosphatidylinositol-3-phosphate (PtdIns3P) and cleaves the fatty acid in PtdIns3P at the sn1 position in vitro making it the first PtdIns3P-specific phospholipase A1 (PLA1). Expression of ABH in vivo reduces intracellular PtdIns3P levels and its PtdIns3P-specific PLA1 activity blocks endosomal and autophagic pathways. In accordance with recent studies acknowledging the potential of extracellular pathogens to evade or exploit autophagy to prevent their clearance and facilitate survival, this is the first report highlighting the role of ABH in inhibiting autophagy and endosomal trafficking induced by extracellular V. cholerae.
    Full-text · Article · Oct 2015 · Nature Communications
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    ABSTRACT: Lipids regulate a wide range of biological activities. Since their local concentrations are tightly controlled in a spatiotemporally specific manner, the simultaneous quantification of multiple lipids is essential for elucidation of the complex mechanisms of biological regulation. Here, we report a new method for the simultaneous in situ quantification of two lipid pools in mammalian cells using orthogonal fluorescent sensors. The sensors were prepared by incorporating two environmentally sensitive fluorophores with minimal spectral overlap separately into engineered lipid-binding proteins. Dual ratiometric analysis of imaging data allowed accurate, spatiotemporally resolved quantification of two different lipids on the same leaflet of the plasma membrane or a single lipid on two opposite leaflets of the plasma membrane of live mammalian cells. This new imaging technology should serve as a powerful tool for systems-level investigation of lipid-mediated cell signaling and regulation.
    Full-text · Article · Dec 2014 · Angewandte Chemie International Edition
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    ABSTRACT: Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examine the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best-characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had direct affinity for PI(4,5)P2 and determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Further, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Dec 2014 · Journal of Biological Chemistry
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    Full-text · Article · Oct 2014 · Angewandte Chemie International Edition in English
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    ABSTRACT: In organellogenesis of the chloroplast from endosymbiotic cyanobacteria, the establishment of protein-targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain and that two lipids, monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling, and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein-interacting ARD into a lipid binding domain.
    Full-text · Article · Sep 2014 · Developmental Cell
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    ABSTRACT: Wnt proteins control diverse biological processes through β-catenin-dependent canonical signalling and β-catenin-independent non-canonical signalling. The mechanisms by which these signalling pathways are differentially triggered and controlled are not fully understood. Dishevelled (Dvl) is a scaffold protein that serves as the branch point of these pathways. Here, we show that cholesterol selectively activates canonical Wnt signalling over non-canonical signalling under physiological conditions by specifically facilitating the membrane recruitment of the PDZ domain of Dvl and its interaction with other proteins. Single-molecule imaging analysis shows that cholesterol is enriched around the Wnt-activated Frizzled and low-density lipoprotein receptor-related protein 5/6 receptors and plays an essential role for Dvl-mediated formation and maintenance of the canonical Wnt signalling complex. Collectively, our results suggest a new regulatory role of cholesterol in Wnt signalling and a potential link between cellular cholesterol levels and the balance between canonical and non-canonical Wnt signalling activities.
    Preview · Article · Jul 2014 · Nature Communications
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    ABSTRACT: Actin polymerization is important for vesicle fission during clathrin-mediated endocytosis (CME), and it has been proposed that actin polymerization may promote vesicle fission during CME by providing direct mechanical forces. However, there is no direct evidence in support of this hypothesis. In the present study, the role of actin polymerization in vesicle fission was tested by analyzing the kinetics of the endocytic tubular membrane neck (the fission-pore) with cell-attached capacitance measurements to detect CME of single vesicles in a millisecond time resolution in mouse chromaffin cells. Inhibition in dynamin GTPase activity increased the fission-pore conductance (Gp), supporting the mechanical role of dynamin GTPase in vesicle fission. However, disruptions in actin polymerization did not alter the fission-pore conductance Gp, thus arguing against the force-generating role of actin polymerization in vesicle fission during CME. Similar to disruptions of actin polymerization, cholesterol depletion results in an increase in the fission-pore duration, indicating a role for cholesterol-dependent membrane reorganization in vesicle fission. Further experiments suggested that actin polymerization and cholesterol might function in vesicle fission during CME in the same pathway. Our results thus support a model in which actin polymerization promotes vesicle fission during CME by inducing cholesterol-dependent membrane reorganization.
    Full-text · Article · Oct 2013 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    ABSTRACT: Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mole% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology (PH) domains, an epsin N-terminal homology (ENTH) domain, and a phox (PX) domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.
    Preview · Article · Sep 2013 · The Journal of Lipid Research
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    ABSTRACT: The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI's cytoplasmic carboxy terminus to the canonical peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or target peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and non-canonical (peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.
    No preview · Article · May 2013 · Journal of Biological Chemistry
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb85
    No preview · Conference Paper · Apr 2013
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb89
    No preview · Conference Paper · Apr 2013
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb88
    No preview · Conference Paper · Apr 2013
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    ABSTRACT: Immunological synapse (IS) is a complex supramolecular structure formed at the interface between T cells and antigen presenting cells (APCs) during T cell antigen recognition. Microfabricated platforms have made great contributions to our understanding of the assembly dynamics and functional roles of the T cell synapses over the last decade. Here, we review three different types of microfabricated platforms developed to modulate and monitor the T cell synapse assembly. Firstly, multi-protein micropatterned surfaces presenting key ligands for T cell activation that can modulate the spatial distribution of receptors/signaling molecules in T cell synapses are described. Secondly, micropatterned supported bilayers that can modulate the dynamics of T cell receptor (TCR) microclusters are introduced. Lastly, T-APC pair arrays that allow for improved fluorescence live cell imaging are discussed. WIREs Nanomed Nanobiotechnol 2013, 5:67–74. doi: 10.1002/wnan.1182 For further resources related to this article, please visit the WIREs website.
    No preview · Article · Jan 2013 · Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology
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    ABSTRACT: Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.
    Full-text · Article · Dec 2012 · Nature Communications
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    ABSTRACT: Protrudin is a FYVE (Fab 1, YOTB, Vac 1 and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia (HSP). Our image-based screening of lipid binding domain (LBD) library revealed novel plasma membrane (PM) localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance (SPR) analysis showed that this FYVE domain preferentially binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol-3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and PM via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.
    No preview · Article · Oct 2012 · Journal of Biological Chemistry
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    Youngdae Yoon · Xiuqi Zhang · Wonhwa Cho
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    ABSTRACT: Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P2 specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H0) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P2-dependent membrane penetration of H0 is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P2-independent manner. Depletion of PtdIns(4,5)P2 from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P2 concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.
    Full-text · Article · Aug 2012 · Journal of Biological Chemistry
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    ABSTRACT: Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.
    Full-text · Article · Jul 2012 · Journal of Biological Chemistry
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    ABSTRACT: Cytokinesis is the process of partitioning the cytoplasm of a dividing cell, thereby completing mitosis. Cytokinesis in the plant cell is achieved by the formation of a new cell wall between daughter nuclei using components carried in Golgi-derived vesicles that accumulate at the midplane of the phragmoplast and fuse to form the cell plate. Proteins that play major roles in the development of the cell plate in plant cells are not well defined. Here, we report that an AP180 amino-terminal homology/epsin amino-terminal homology domain-containing protein from Arabidopsis (Arabidopsis thaliana) is involved in clathrin-coated vesicle formation from the cell plate. Arabidopsis Epsin-like Clathrin Adaptor1 (AtECA1; At2g01600) and its homologous proteins AtECA2 and AtECA4 localize to the growing cell plate in cells undergoing cytokinesis and also to the plasma membrane and endosomes in nondividing cells. AtECA1 (At2g01600) does not localize to nascent cell plates but localizes at higher levels to expanding cell plates even after the cell plate fuses with the parental plasma membrane. The temporal and spatial localization patterns of AtECA1 overlap most closely with those of the clathrin light chain. In vitro protein interaction assays revealed that AtECA1 binds to the clathrin H chain via its carboxyl-terminal domain. These results suggest that these AP180 amino-terminal homology/epsin amino-terminal homology domain-containing proteins, AtECA1, AtECA2, and AtECA4, may function as adaptors of clathrin-coated vesicles budding from the cell plate.
    Full-text · Article · May 2012 · Plant physiology

Publication Stats

6k Citations
688.59 Total Impact Points

Institutions

  • 1994-2015
    • University of Illinois at Chicago
      • Department of Chemistry
      Chicago, Illinois, United States
  • 2011
    • University of Texas Medical Branch at Galveston
      Galveston, Texas, United States
  • 2009
    • Rensselaer Polytechnic Institute
      Троя, New York, United States
  • 2002
    • University of Southampton
      • Centre for Biological Sciences
      Southampton, England, United Kingdom
  • 2001
    • University of Chicago
      Chicago, Illinois, United States