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Phoebe L. Stewart
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA

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Journal article
Published: 27 April 2021 in mBio
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The bacterial RND superfamily of efflux pumps mediate resistance to a variety of biocides, including Cu(I) and Ag(I) ions. Here we report four cryo-EM structures of the trimeric CusA pump in the presence of Cu(I). Combined with MD simulations, our data indicate that each CusA protomer within the trimer recognizes and extrudes Cu(I) independently.

ACS Style

Mitchell A. Moseng; Meinan Lyu; Tanadet Pipatpolkai; Przemyslaw Glaza; Corey C. Emerson; Phoebe L. Stewart; Phillip J. Stansfeld; Edward W. Yu. Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export. mBio 2021, 12, 1 .

AMA Style

Mitchell A. Moseng, Meinan Lyu, Tanadet Pipatpolkai, Przemyslaw Glaza, Corey C. Emerson, Phoebe L. Stewart, Phillip J. Stansfeld, Edward W. Yu. Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export. mBio. 2021; 12 (2):1.

Chicago/Turabian Style

Mitchell A. Moseng; Meinan Lyu; Tanadet Pipatpolkai; Przemyslaw Glaza; Corey C. Emerson; Phoebe L. Stewart; Phillip J. Stansfeld; Edward W. Yu. 2021. "Cryo-EM Structures of CusA Reveal a Mechanism of Metal-Ion Export." mBio 12, no. 2: 1.

Journal article
Published: 15 January 2021 in Viruses
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Adenovirus (AdV) infection elicits a strong immune response with the production of neutralizing antibodies and opsonization by complement and coagulation factors. One anti-hexon neutralizing antibody, called 9C12, is known to activate the complement cascade, resulting in the deposition of complement component C4b on the capsid, and the neutralization of the virus. The mechanism of AdV neutralization by C4b is independent of downstream complement proteins and involves the blockage of the release of protein VI, which is required for viral escape from the endosome. To investigate the structural basis underlying how C4b blocks the uncoating of AdV, we built a model for the complex of human adenovirus type-5 (HAdV5) with 9C12, together with complement components C1 and C4b. This model positions C4b near the Arg-Gly-Asp (RGD) loops of the penton base. There are multiple amino acids in the RGD loop that might serve as covalent binding sites for the reactive thioester of C4b. Molecular dynamics simulations with a multimeric penton base and C4b indicated that stabilizing interactions may form between C4b and multiple RGD loops. We propose that C4b deposition on one RGD loop leads to the entanglement of C4b with additional RGD loops on the same penton base multimer and that this entanglement blocks AdV uncoating.

ACS Style

Corey Emerson; Phoebe Stewart. Structure-Based Modeling of Complement C4 Mediated Neutralization of Adenovirus. Viruses 2021, 13, 111 .

AMA Style

Corey Emerson, Phoebe Stewart. Structure-Based Modeling of Complement C4 Mediated Neutralization of Adenovirus. Viruses. 2021; 13 (1):111.

Chicago/Turabian Style

Corey Emerson; Phoebe Stewart. 2021. "Structure-Based Modeling of Complement C4 Mediated Neutralization of Adenovirus." Viruses 13, no. 1: 111.

Research article
Published: 25 November 2020 in Science Translational Medicine
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Oncolytic virus therapy is a cancer treatment modality that has the potential to improve outcomes for patients with currently incurable malignancies. Although intravascular delivery of therapeutic viruses provides access to disseminated tumors, this delivery route exposes the virus to opsonizing and inactivating factors in the blood, which limit the effective therapeutic virus dose and contribute to activation of systemic toxicities. When human species C adenovirus HAdv-C5 is delivered intravenously, natural immunoglobulin M (IgM) antibodies and coagulation factor X rapidly opsonize HAdv-C5, leading to virus sequestration in tissue macrophages and promoting infection of liver cells, triggering hepatotoxicity. Here, we showed that natural IgM antibody binds to the hypervariable region 1 (HVR1) of the main HAdv-C5 capsid protein hexon. Using compound targeted mutagenesis of hexon HVR1 loop and other functional sites that mediate virus-host interactions, we engineered and obtained a high-resolution cryo–electron microscopy structure of an adenovirus vector, Ad5-3M, which resisted inactivation by blood factors, avoided sequestration in liver macrophages, and failed to trigger hepatotoxicity after intravenous delivery. Systemic delivery of Ad5-3M to mice with localized or disseminated lung cancer led to viral replication in tumor cells, suppression of tumor growth, and prolonged survival. Thus, compound targeted mutagenesis of functional sites in the virus capsid represents a generalizable approach to tailor virus interactions with the humoral and cellular arms of the immune system, enabling generation of “designer” viruses with improved therapeutic properties.

ACS Style

Svetlana Atasheva; Corey C. Emerson; Jia Yao; Cedrick Young; Phoebe L. Stewart; Dmitry M. Shayakhmetov. Systemic cancer therapy with engineered adenovirus that evades innate immunity. Science Translational Medicine 2020, 12, eabc6659 .

AMA Style

Svetlana Atasheva, Corey C. Emerson, Jia Yao, Cedrick Young, Phoebe L. Stewart, Dmitry M. Shayakhmetov. Systemic cancer therapy with engineered adenovirus that evades innate immunity. Science Translational Medicine. 2020; 12 (571):eabc6659.

Chicago/Turabian Style

Svetlana Atasheva; Corey C. Emerson; Jia Yao; Cedrick Young; Phoebe L. Stewart; Dmitry M. Shayakhmetov. 2020. "Systemic cancer therapy with engineered adenovirus that evades innate immunity." Science Translational Medicine 12, no. 571: eabc6659.

Journal article
Published: 16 April 2020 in Journal of Virology
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Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent for Kaposi’s sarcoma (KS), the most common malignancy in HIV/AIDS patients. Oral transmission through saliva is considered the most common route for spreading the virus among HIV/AIDS patients. However, the role of HIV-specific components in the cotransfection of KSHV is unclear. We demonstrate that exosomes purified from the saliva of HIV-positive patients and secreted by HIV-infected T-cell lines promote KSHV infectivity in immortalized and primary oral epithelial cells. HIV-associated exosomes promote KSHV infection, which depends on HIV trans -activation response element (TAR) RNA and EGFR of oral epithelial cells, which can be targeted for reducing KSHV infection. These results reveal that HIV-associated exosomes are a risk factor for KSHV infection in the HIV-infected population.

ACS Style

Lechuang Chen; Zhimin Feng; Guoxiang Yuan; Corey C. Emerson; Phoebe L. Stewart; Fengchun Ye; Ge Jin. Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor. Journal of Virology 2020, 94, 1 .

AMA Style

Lechuang Chen, Zhimin Feng, Guoxiang Yuan, Corey C. Emerson, Phoebe L. Stewart, Fengchun Ye, Ge Jin. Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor. Journal of Virology. 2020; 94 (9):1.

Chicago/Turabian Style

Lechuang Chen; Zhimin Feng; Guoxiang Yuan; Corey C. Emerson; Phoebe L. Stewart; Fengchun Ye; Ge Jin. 2020. "Human Immunodeficiency Virus-Associated Exosomes Promote Kaposi’s Sarcoma-Associated Herpesvirus Infection via the Epidermal Growth Factor Receptor." Journal of Virology 94, no. 9: 1.

Journal article
Published: 05 September 2019 in Pathogens and Immunity
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Background: Human papillomavirus (HPV) is linked to nearly all cases of cervical cancer. Despite available vaccines, a deeper understanding of the immune response to HPV is needed. Human α-defensin 5 (HD5), an innate immune effector peptide, blocks infection of multiple serotypes of HPV, including high-risk HPV16. While a common mechanism of α-defensin anti-viral activity against nonenveloped viruses such as HPV has emerged, there is limited understanding of how α-defensins bind to viral capsids to block infection. Methods: We have used cryo-electron microscopy (cryoEM), mass spectrometry (MS) crosslinking and differential lysine modification studies, and molecular dynamics (MD) simulations to probe the interaction of HPV16 pseudovirions (PsVs) with HD5. Results: CryoEM single particle reconstruction did not reveal HD5 density on the capsid surface. Rather, increased density was observed under the capsid shell in the presence of HD5. MS studies indicate that HD5 binds near the L1 and L2 capsid proteins and specifically near the C-terminal region of L1. MD simulations indicate that favorable electrostatic interactions can be formed between HD5 and the L1 C-terminal tail.Conclusions: A model is presented for how HD5 affects HPV16 structure and cell entry. In this model, HD5 binds to disordered regions of L1 and L2 protruding from the icosahedrally ordered capsid. HD5 acts to cement interactions between L1 and L2 and leads to a closer association of the L2/genome core with the L1 capsid. This model provides a structural rationale for our prior observation that HD5 interferes with the separation of L1 from the L2/genome complex during cell entry.

ACS Style

Neetu M. Gulati; Masaru Miyagi; Mayim E. Wiens; Jason G. Smith; Phoebe L. Stewart. α-Defensin HD5 Stabilizes Human Papillomavirus 16 Capsid/Core Interactions. Pathogens and Immunity 2019, 4, 196 -234.

AMA Style

Neetu M. Gulati, Masaru Miyagi, Mayim E. Wiens, Jason G. Smith, Phoebe L. Stewart. α-Defensin HD5 Stabilizes Human Papillomavirus 16 Capsid/Core Interactions. Pathogens and Immunity. 2019; 4 (2):196-234.

Chicago/Turabian Style

Neetu M. Gulati; Masaru Miyagi; Mayim E. Wiens; Jason G. Smith; Phoebe L. Stewart. 2019. "α-Defensin HD5 Stabilizes Human Papillomavirus 16 Capsid/Core Interactions." Pathogens and Immunity 4, no. 2: 196-234.

Journal article
Published: 27 August 2019 in mBio
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Acinetobacter baumannii is a successful human pathogen which has emerged as one of the most problematic and highly antibiotic-resistant Gram-negative bacteria worldwide. Multidrug efflux is a major mechanism that A. baumannii uses to counteract the action of multiple classes of antibiotics, such as β-lactams, tetracyclines, fluoroquinolones, and aminoglycosides. Here, we report a cryo-electron microscopy (cryo-EM) structure of the prevalent A. baumannii AdeB multidrug efflux pump, which indicates a plausible pathway for multidrug extrusion. Overall, our data suggest a mechanism for energy coupling that powers up this membrane protein to export antibiotics from bacterial cells. Our studies will ultimately inform an era in structure-guided drug design to combat multidrug resistance in these Gram-negative pathogens.

ACS Style

Chih-Chia Su; Christopher E. Morgan; Sekhar Kambakam; Malligarjunan Rajavel; Harry Scott; Wei Huang; Corey C. Emerson; Derek J. Taylor; Phoebe L. Stewart; Robert A. Bonomo; Edward W. Yu. Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump. mBio 2019, 10, e01295-19 .

AMA Style

Chih-Chia Su, Christopher E. Morgan, Sekhar Kambakam, Malligarjunan Rajavel, Harry Scott, Wei Huang, Corey C. Emerson, Derek J. Taylor, Phoebe L. Stewart, Robert A. Bonomo, Edward W. Yu. Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump. mBio. 2019; 10 (4):e01295-19.

Chicago/Turabian Style

Chih-Chia Su; Christopher E. Morgan; Sekhar Kambakam; Malligarjunan Rajavel; Harry Scott; Wei Huang; Corey C. Emerson; Derek J. Taylor; Phoebe L. Stewart; Robert A. Bonomo; Edward W. Yu. 2019. "Cryo-Electron Microscopy Structure of an Acinetobacter baumannii Multidrug Efflux Pump." mBio 10, no. 4: e01295-19.

Paper
Published: 05 August 2019 in Nanoscale
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We report an ultrasound contrast agent for which we engineered the shell structure to impart much better stability under intense stress and deformation.

ACS Style

Al de Leon; Reshani Perera; Christopher Hernandez; Michaela Cooley; Olive Jung; Selva Jeganathan; Eric Abenojar; Grace Fishbein; Amin Jafari Sojahrood; Corey C. Emerson; Phoebe L. Stewart; Michael C. Kolios; Agata A. Exner. Contrast enhanced ultrasound imaging by nature-inspired ultrastable echogenic nanobubbles. Nanoscale 2019, 11, 15647 -15658.

AMA Style

Al de Leon, Reshani Perera, Christopher Hernandez, Michaela Cooley, Olive Jung, Selva Jeganathan, Eric Abenojar, Grace Fishbein, Amin Jafari Sojahrood, Corey C. Emerson, Phoebe L. Stewart, Michael C. Kolios, Agata A. Exner. Contrast enhanced ultrasound imaging by nature-inspired ultrastable echogenic nanobubbles. Nanoscale. 2019; 11 (33):15647-15658.

Chicago/Turabian Style

Al de Leon; Reshani Perera; Christopher Hernandez; Michaela Cooley; Olive Jung; Selva Jeganathan; Eric Abenojar; Grace Fishbein; Amin Jafari Sojahrood; Corey C. Emerson; Phoebe L. Stewart; Michael C. Kolios; Agata A. Exner. 2019. "Contrast enhanced ultrasound imaging by nature-inspired ultrastable echogenic nanobubbles." Nanoscale 11, no. 33: 15647-15658.

Journal article
Published: 18 October 2017 in Scientific Reports
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Gas microbubbles stabilized with lipids, surfactants, proteins and/or polymers are widely used clinically as ultrasound contrast agents. Because of their large 1-10 µm size, applications of microbubbles are confined to the blood vessels. Accordingly, there is much interest in generating nanoscale echogenic bubbles (nanobubbles), which can enable new uses of ultrasound contrast agents in molecular imaging and drug delivery, particularly for cancer applications. While the interactions of microbubbles with ultrasound have been widely investigated, little is known about the activity of nanobubbles under ultrasound exposure. In this work, we demonstrate that cryo-electron microscopy (cryo-EM) can be used to image nanoscale lipid and polymer-stabilized perfluorocarbon gas bubbles before and after their destruction with high intensity ultrasound. In addition, cryo-EM can be used to observe electron-beam induced dissipation of nanobubble encapsulated perfluorocarbon gas.

ACS Style

Christopher Hernandez; Sahil Gulati; Gabriella Fioravanti; Phoebe L. Stewart; Agata A. Exner. Cryo-EM Visualization of Lipid and Polymer-Stabilized Perfluorocarbon Gas Nanobubbles - A Step Towards Nanobubble Mediated Drug Delivery. Scientific Reports 2017, 7, 13517 .

AMA Style

Christopher Hernandez, Sahil Gulati, Gabriella Fioravanti, Phoebe L. Stewart, Agata A. Exner. Cryo-EM Visualization of Lipid and Polymer-Stabilized Perfluorocarbon Gas Nanobubbles - A Step Towards Nanobubble Mediated Drug Delivery. Scientific Reports. 2017; 7 (1):13517.

Chicago/Turabian Style

Christopher Hernandez; Sahil Gulati; Gabriella Fioravanti; Phoebe L. Stewart; Agata A. Exner. 2017. "Cryo-EM Visualization of Lipid and Polymer-Stabilized Perfluorocarbon Gas Nanobubbles - A Step Towards Nanobubble Mediated Drug Delivery." Scientific Reports 7, no. 1: 13517.

Review
Published: 21 December 2016 in Viruses
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Human adenoviruses are large (150 MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause acute respiratory, gastrointestinal and ocular infections. Despite these disease associations, adenovirus has aided basic and clinical research efforts through studies of its association with cells and as a target of host antiviral responses. This review highlights the knowledge of adenovirus disassembly and nuclear transport gleaned from structural, biophysical and functional analyses of adenovirus interactions with soluble and membrane-associated host molecules.

ACS Style

Glen R. Nemerow; Phoebe L. Stewart. Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity. Viruses 2016, 8, 337 .

AMA Style

Glen R. Nemerow, Phoebe L. Stewart. Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity. Viruses. 2016; 8 (12):337.

Chicago/Turabian Style

Glen R. Nemerow; Phoebe L. Stewart. 2016. "Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity." Viruses 8, no. 12: 337.

Journal article
Published: 01 July 2014 in Journal of Structural Biology
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Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs.

ACS Style

Seth A. Villarreal; Phoebe L. Stewart. CryoEM and image sorting for flexible protein/DNA complexes. Journal of Structural Biology 2014, 187, 76 -83.

AMA Style

Seth A. Villarreal, Phoebe L. Stewart. CryoEM and image sorting for flexible protein/DNA complexes. Journal of Structural Biology. 2014; 187 (1):76-83.

Chicago/Turabian Style

Seth A. Villarreal; Phoebe L. Stewart. 2014. "CryoEM and image sorting for flexible protein/DNA complexes." Journal of Structural Biology 187, no. 1: 76-83.

Journal article
Published: 01 February 2013 in Journal of Biological Chemistry
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Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

ACS Style

Jian Shi; Hanane A. Koteiche; Ezelle T. McDonald; Tara L. Fox; Phoebe L. Stewart; Hassane S. Mchaourab. Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding. Journal of Biological Chemistry 2013, 288, 4819 -4830.

AMA Style

Jian Shi, Hanane A. Koteiche, Ezelle T. McDonald, Tara L. Fox, Phoebe L. Stewart, Hassane S. Mchaourab. Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding. Journal of Biological Chemistry. 2013; 288 (7):4819-4830.

Chicago/Turabian Style

Jian Shi; Hanane A. Koteiche; Ezelle T. McDonald; Tara L. Fox; Phoebe L. Stewart; Hassane S. Mchaourab. 2013. "Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding." Journal of Biological Chemistry 288, no. 7: 4819-4830.