Wonhwa Cho

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

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Publications (156)722.89 Total impact

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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.
    Angewandte Chemie International Edition 10/2014; · 11.34 Impact Factor
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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.
    Developmental Cell 09/2014; 30(5):598-609. · 12.86 Impact Factor
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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.
    Nature Communications 01/2014; 5:4393. · 10.74 Impact Factor
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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.
    Journal of Neuroscience 10/2013; 33(40):15793-15798. · 6.91 Impact Factor
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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.
    The Journal of Lipid Research 09/2013; · 4.39 Impact Factor
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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.
    Journal of Biological Chemistry 05/2013; · 4.65 Impact Factor
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb88
    Experimental Biology Conference. EB 2O13, Boston; 04/2013
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb85
    Experimental Biology Conference - EB 2O13, Boston; 04/2013
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    ABSTRACT: http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/lb89
    Experimental Biology Conference. EB 2O13, Boston; 04/2013
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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.
    Nature Communications 12/2012; 3:1249. · 10.74 Impact Factor
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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 2012 doi: 10.1002/wnan.1182 For further resources related to this article, please visit the WIREs website.
    Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 08/2012; · 5.68 Impact Factor
  • 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)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) 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)P(2)-dependent membrane penetration of H(0) 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)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) 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)P(2) concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.
    Journal of Biological Chemistry 08/2012; 287(41):34078-90. · 4.65 Impact Factor
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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.
    Journal of Biological Chemistry 07/2012; 287(36):30518-28. · 4.65 Impact Factor
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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.
    Plant physiology 05/2012; 159(3):1013-25. · 6.56 Impact Factor
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    ABSTRACT: Emerging evidence indicates that membrane lipids regulate protein networking by directly interacting with protein-interaction domains (PIDs). As a pilot study to identify and functionally annodate lipid-binding PIDs on a genomic scale, we performed experimental and computational studies of PDZ domains. Characterization of 70 PDZ domains showed that ~40% had submicromolar membrane affinity. Using a computational model built from these data, we predicted the membrane-binding properties of 2,000 PDZ domains from 20 species. The accuracy of the prediction was experimentally validated for 26 PDZ domains. We also subdivided lipid-binding PDZ domains into three classes based on the interplay between membrane- and protein-binding sites. For different classes of PDZ domains, lipid binding regulates their protein interactions by different mechanisms. Functional studies of a PDZ domain protein, rhophilin 2, suggest that all classes of lipid-binding PDZ domains serve as genuine dual-specificity modules regulating protein interactions at the membrane under physiological conditions.
    Molecular cell 03/2012; 46(2):226-37. · 14.61 Impact Factor
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    ABSTRACT: Signaling lipids control many of the most important biological pathways, typically by recruiting cognate protein binding targets to cell surfaces, thereby regulating both their function and subcellular localization. A critical family of signaling lipids is that of the phosphatidylinositol polyphosphates (PIP(n)s), which is composed of seven isomers that vary based on phosphorylation pattern. A key protein that is activated upon PIP(n) binding is Akt, which then plays important roles in regulating the cell cycle, and is thus aberrant in disease. Characterization of protein-PIP(n) binding interactions is hindered by the complexity of the membrane environment and of the PIP(n) structures. Herein, we describe two rapid assays of use for characterizing protein-PIP(n) binding interactions. First, a microplate-based binding assay was devised to characterize the binding of effectors to immobilized synthetic PIP(n) headgroup-biotin conjugates corresponding to all seven isomers. The assay was implemented for simultaneous analysis of Akt-PH domain, indicating PI(3,4,5)P(3) and PI(3,4)P(2) as the primary ligands. In addition, density-dependant studies indicated that the amount of ligand immobilized on the surface affected the amplitude of protein binding, but not the affinity, for Akt-PH. Since the PIP(n) ligand motifs used in this analysis lack the membrane environment and glycerolipid backbone, yet still exhibit high-affinity protein binding, these results narrow down the structural requirements for Akt recognition. Additionally, binding detection was also achieved through microarray analysis via the robotic pin printing of ligands onto glass slides in a miniaturized format. Here, fluorescence-based detection provided sensitive detection of binding using minimal amounts of materials. Due to their high-throughput and versatile attributes, these assays provide invaluable tools for probing and perturbing protein-membrane binding interactions.
    Chemistry and Physics of Lipids 12/2011; 165(2):207-15. · 2.59 Impact Factor
  • Nathan Lucas, Wonhwa Cho
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    ABSTRACT: 3-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3'-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1.
    Journal of Biological Chemistry 12/2011; 286(48):41265-72. · 4.65 Impact Factor
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    ABSTRACT: In this presentation we review our recent work using x-ray reflectivity to determine the configuration of membrane-bound proteins. The reflectivity data is analyzed in terms of the known crystallographic structure of proteins and a slab model representing the lipid layer to yield an electron density profile of the lipid/protein system. Our recent modified analysis methodology for the lipid/protein system is concisely described in this report. In addition, some results of the configuration of the membrane-bound proteins cPLA2α-C2, p40phox-PX, and PKCα-C2 are highlighted.
    Journal of Applied Physics 11/2011; 110(10). · 2.21 Impact Factor
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    ABSTRACT: Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.
    Biochemistry 11/2011; 50(51):11143-61. · 3.38 Impact Factor
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    ABSTRACT: Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we report a new chemical method of quantitative lipid imaging using sensors engineered by labelling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2))--a key signalling lipid in diverse cellular processes--was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain, a protein that selectively binds PtdIns(4,5)P(2). The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signalling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P(2) concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.
    Nature Chemistry 11/2011; 3(11):868-74. · 21.76 Impact Factor

Publication Stats

4k Citations
722.89 Total Impact Points

Institutions

  • 1994–2014
    • University of Illinois at Chicago
      • • Department of Chemistry
      • • Department of Bioengineering
      Chicago, Illinois, United States
  • 2011
    • University of Texas Medical Branch at Galveston
      Galveston, Texas, United States
  • 2009–2011
    • University of Tennessee
      • Department of Chemistry
      Knoxville, TN, United States
  • 2004–2007
    • Virginia Commonwealth University
      • Department of Biochemistry and Molecular Biology
      Richmond, VA, United States
  • 2001–2007
    • University of Chicago
      • Department of Medicine
      Chicago, IL, United States
    • Kanazawa Medical University
      • Department of Internal Medicine (III)
      Kanazawa, Ishikawa, Japan