42 results on '"Gwendolyn M. Jang"'
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2. Supplementary Table S1 from Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus–Host Protein Network
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Nevan J. Krogan, Trey Ideker, Jennifer R. Grandis, Jason F. Kreisberg, Jacques Archambault, Toni M. Brand, Priya S. Shah, Gwendolyn M. Jang, Tasha L. Johnson, Danielle L. Swaney, Kathleen E. Franks-Skiba, Jeffrey R. Johnson, John Von Dollen, Andrew M. Gross, Wei Zhang, and Manon Eckhardt
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Previously Reported HPV-Human PPIs. Related to Figures 1 and 2.
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- 2023
3. Supplementary Document S1 from Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus–Host Protein Network
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Nevan J. Krogan, Trey Ideker, Jennifer R. Grandis, Jason F. Kreisberg, Jacques Archambault, Toni M. Brand, Priya S. Shah, Gwendolyn M. Jang, Tasha L. Johnson, Danielle L. Swaney, Kathleen E. Franks-Skiba, Jeffrey R. Johnson, John Von Dollen, Andrew M. Gross, Wei Zhang, and Manon Eckhardt
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This combined Supplementary Document contains Supplementary Notes, Methods and References, Legends to Supplementary Tables S1-S6, and Supplementary Figures S1-S5.
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- 2023
4. HBV rewires liver cancer signaling by altering PP2A complexes
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Adriana Pitea, Rigney E Turnham, Manon Eckhardt, Gwendolyn M Jang, Zhong Xu, Huat C Lim, Alex Choi, John Von Dollen, Rebecca S. Levin, James T Webber, Elizabeth McCarthy, Junjie Hu, Xiaolei Li, Li Che, Gary Chan, R. Katie Kelley, Danielle Swaney, Wei Zhang, Sourav Bandyopadhyay, Fabian J Theis, Xin Chen, Kevan Shokat, Trey Ideker, Nevan J Krogan, and John D Gordan
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SummaryInfection by hepatitis B virus (HBV) increases risk for liver cancer by inducing inflammation, cellular stress and cell death. To elucidate the molecular pathways by which HBV promotes cancer development and progression, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human host proteins in hepatocellular carcinoma (HCC). We find that viral proteins target host factors that are preferentially mutated in non-HBV-associated HCC, implicating cancer pathways whose interaction with HBV plays a role in HCC. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we show that HBx remodels the PP2A phosphatase complex, altering its effect on tumor signaling. HBx excludes striatin-family regulatory subunits from PP2A, causing Hippo kinase activation and unmasking a requirement for mTOR complex 2 to maintain expression of the YAP oncoprotein in HCC. Thus, HBV rewires HCC to expose potentially targetable signaling dependencies.SignificancePrecision medicine has revolutionized cancer treatment but remains elusive for HCC. We used proteomics to define HBV/host interactions and integrated them with HCC mutations. The results implicate modifiers of HCC behavior via remodeling of host complexes and illuminate new biological mechanisms in advanced disease for therapeutic investigation.
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- 2023
5. Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories
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Mehdi Bouhaddou, Ann-Kathrin Reuschl, Benjamin J. Polacco, Lucy G. Thorne, Manisha R. Ummadi, Chengjin Ye, Romel Rosales, Adrian Pelin, Jyoti Batra, Gwendolyn M. Jang, Jiewei Xu, Jack M. Moen, Alicia Richards, Yuan Zhou, Bhavya Harjai, Erica Stevenson, Ajda Rojc, Roberta Ragazzini, Matthew V.X. Whelan, Wilhelm Furnon, Giuditta De Lorenzo, Vanessa Cowton, Abdullah M. Syed, Alison Ciling, Noa Deutsch, Daniel Pirak, Giulia Dowgier, Dejan Mesner, Jane L. Turner, Briana L. McGovern, M. Luis Rodriguez, Rocio Leiva-Rebollo, Alistair S. Dunham, Xiaofang Zhong, Manon Eckhardt, Andrea Fossati, Nicholas Liotta, Thomas Kehrer, Anastasija Cupic, Magda Rutkowska, Nacho Mena, Sadaf Aslam, Alyssa Hoffert, Helene Foussard, John Pham, Molly Lyons, Laura Donahue, Aliesha Griffin, Rebecca Nugent, Kevin Holden, Robert Deans, Pablo Aviles, José Antonio López-Martín, Jose M. Jimeno, Kirsten Obernier, Jacqueline M. Fabius, Margaret Soucheray, Ruth Hüttenhain, Irwin Jungreis, Manolis Kellis, Ignacia Echeverria, Kliment Verba, Paola Bonfanti, Pedro Beltrao, Roded Sharan, Jennifer A. Doudna, Luis Martinez-Sobrido, Arvind Patel, Massimo Palmarini, Lisa Miorin, Kris White, Danielle L. Swaney, Adolfo García-Sastre, Clare Jolly, Lorena Zuliani-Alvarez, Greg J. Towers, and Nevan J. Krogan
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A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.
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- 2022
6. Quantitative proteomic analysis reveals apoE4-dependent phosphorylation of the actin-regulating protein VASP
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Zeynep Cakir, Samuel J. Lord, Yuan Zhou, Gwendolyn M. Jang, Benjamin J. Polacco, Manon Eckhardt, David Jimenez-Morales, Billy W. Newton, Adam L. Orr, Jeffrey R. Johnson, Alexandre da Cruz, R. Dyche Mullins, Nevan J. Krogan, Robert W. Mahley, and Danielle L. Swaney
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Proteomics ,Kinase ,Aging ,Biochemistry & Molecular Biology ,Ubiquitylation ,Apolipoprotein E4 ,Apolipoprotein E3 ,Neurodegenerative ,Alzheimer's Disease ,Biochemistry ,Analytical Chemistry ,Mice ,Apolipoproteins E ,Protein-protein interaction ,Alzheimer Disease ,Acquired Cognitive Impairment ,Animals ,2.1 Biological and endogenous factors ,Phosphorylation ,Aetiology ,Molecular Biology ,Neurosciences ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,Actins ,Brain Disorders ,Neurological ,Dementia ,Alzheimer’s disease - Abstract
Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.
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- 2022
7. Global landscape of HIV-human protein complexes.
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Stefanie Jäger, Peter Cimermancic, Natali Gulbahce, Jeffrey R. Johnson, Kathryn E. McGovern, Starlynn C. Clarke, Michael Shales, Gaelle Mercenne, Lars Pache, Kathy Li, Hilda Hernandez, Gwendolyn M. Jang, Shoshannah L. Roth, Eyal Akiva, John Marlett, Melanie Stephens, Iván D'Orso, Jason Fernandes, Marie E. Fahey, Cathal Mahon, Anthony J. O'Donoghue, Aleksandar Todorovic, John H. Morris, David A. Maltby, Tom Alber, Gerard Cagney, Frederic D. Bushman, John A. Young, Sumit K. Chanda, Wesley I. Sundquist, Tanja Kortemme, Ryan D. Hernandez, Charles S. Craik, Alma Burlingame, Andrej Sali, Alan D. Frankel, and Nevan J. Krogan
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- 2012
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8. Abstract PO017: HBV alters YAP regulation in liver cancer by remodeling PP2A complexes
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John Gordan, Adriana Pitea, Rigney E Turnham, Manon Eckhardt, Gwendolyn M Jang, Huat Lim, Alex L Choi, Sourav Bandyopadhyay, Danielle Swaney, Kevan Shokat, Trey Ideker, and Nevan Krogan
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Cancer Research ,Oncology - Abstract
Viral hepatitis promotes hepatocellular carcinoma (HCC) initiation by inducing inflammation, cellular stress and cell death. However, the cell-intrinsic effects of viral infection in advanced HCC remain unclear. We used affinity purification mass spectrometry to develop a complete map of 145 interactions among Hepatitis B Virus (HBV) and host proteins in the HUH7 HCC tumor cell line, identifying known and novel HBV/host protein-protein interactions. We integrated this map with HCC genomes, identifying 61 proteins with preferential mutation in non-HBV HCC, suggesting that their interaction with HBV plays a role in cancer. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we found that HBx rewires the effect of the PP2A phosphatase on HCC signaling. HBx binding excluded striatin-family regulatory subunits from the PP2A complex, causing Hippo kinase activation. This effect creates a requirement for integrin signaling to mTOR complex 2 to maintain expression of the YAP oncoprotein, critical for HCC growth. Thus, HBV rewires HCC signaling and may promote targetable dependencies. Citation Format: John Gordan, Adriana Pitea, Rigney E Turnham, Manon Eckhardt, Gwendolyn M Jang, Huat Lim, Alex L Choi, Sourav Bandyopadhyay, Danielle Swaney, Kevan Shokat, Trey Ideker, Nevan Krogan. HBV alters YAP regulation in liver cancer by remodeling PP2A complexes [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO017.
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- 2022
9. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes
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Jessica K. Peters, Natalia Jura, Aye C. Thwin, Mehdi Bouhaddou, Fei Li, Jocelyne Lopez, Yang Li, Kuei-Ho Chen, Erron W. Titus, Huong T. Kratochvil, Victor L Lam, Christian B. Billesbølle, Tristan W. Owens, Raphael Trenker, Axel F. Brilot, Danielle L. Swaney, Ursula Schulze-Gahmen, Feng Wang, Gregory E. Merz, Jen Chen, Amy Diallo, Yang Zhang, Adam Frost, Soumya G. Remesh, Junrui Li, Edmond Linossi, Fengbo Zhou, Maliheh Safari, Yifan Cheng, Henry C. Nguyen, Andrea Fossati, Devan Diwanji, Evelyn Hernandez, Aashish Manglik, Nevan J. Krogan, Iris D. Young, Jianhua Zhao, Kyle E. Lopez, Alexandrea N. Rizo, Megan Lo, Thomas H. Pospiech, Gwendolyn M. Jang, Y. Li, Sergei Pourmal, Yuan Zhou, Miles Sasha Dickinson, Un Seng Chio, Kaihua Zhang, Caleigh M. Azumaya, Michael D Paul, Oren S. Rosenberg, Yanxin Liu, Tsz Kin Martin Tsui, Carlos Nowotny, Kate Kim, Kliment A. Verba, Loan Doan, Zanlin Yu, Adrian Pelin, Mariano C Tabios, Lorena Zuliani-Alvarez, Daniel R. Southworth, Melody G. Campbell, Ming Sun, Adamo Mancino, Kaitlin Schaefer, Frank R. Moss, Daniel Asarnow, Meghna Gupta, James S. Fraser, Komal Ishwar Pawar, Amber M. Smith, Robert M. Stroud, Tanja Kortemme, Cristina Puchades, Michelle Moritz, Nadia Herrera, Eric Tse, Mingliang Jin, and David A. Agard
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drug design ,1.1 Normal biological development and functioning ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,viruses ,Mutant ,Computational biology ,medicine.disease_cause ,Article ,Vaccine Related ,Eukaryotic translation ,Underpinning research ,Transcription (biology) ,Biodefense ,medicine ,2.1 Biological and endogenous factors ,Functional studies ,Aetiology ,Lung ,Coronavirus ,SARS-CoV-2 ,Host (biology) ,Chemistry ,Prevention ,RNA ,virus diseases ,Pneumonia ,biochemical phenomena, metabolism, and nutrition ,proteins ,Infectious Diseases ,Emerging Infectious Diseases ,Pneumonia & Influenza ,Generic health relevance ,Infection - Abstract
The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.
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- 2021
10. PEAK3/C19orf35 pseudokinase, a new NFK3 kinase family member, inhibits CrkII through dimerization
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Natalia Jura, Jeffrey R. Johnson, Nevan J. Krogan, Małgorzata Dudkiewicz, Krzysztof Pawłowski, John Von Dollen, Mitchell L. Lopez, Megan Lo, Gwendolyn M. Jang, and Jennifer E. Kung
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Membrane ruffling ,Evolution ,1.1 Normal biological development and functioning ,Amino Acid Motifs ,Regulator ,pseudokinase ,Motility ,Evolution, Molecular ,Protein Domains ,Underpinning research ,Protein Interaction Mapping ,Chlorocebus aethiops ,Animals ,Humans ,2.1 Biological and endogenous factors ,Amino Acid Sequence ,Aetiology ,Protein kinase A ,Cell Shape ,Phylogeny ,Conserved Sequence ,Actin ,Cancer ,CrkII ,Multidisciplinary ,Chemistry ,Kinase Family ,Cell Membrane ,Molecular ,Signal transducing adaptor protein ,protein kinase ,Proto-Oncogene Proteins c-crk ,Protein-Tyrosine Kinases ,NKF3 family ,Cell biology ,Cytoskeletal Proteins ,Family member ,HEK293 Cells ,PNAS Plus ,motility ,COS Cells ,Generic health relevance ,Protein Multimerization ,Protein Binding - Abstract
Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain.
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- 2019
11. A missense variant effect prediction and annotation resource for SARS-CoV-2
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Alistair Dunham, Danielle L. Swaney, Monita Muralidharan, Pedro Beltrao, and Gwendolyn M. Jang
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Annotation ,Resource (project management) ,Disease severity ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Expert analysis ,Missense mutation ,Computational biology ,Biology ,Complement (complexity) ,Conserved sequence - Abstract
The COVID19 pandemic is a global crisis severely impacting many people across the world. An important part of the response is monitoring viral variants and determining the impact they have on viral properties, such as infectivity, disease severity and interactions with drugs and vaccines. In this work we generate and make available computational variant effect predictions for all possible single amino-acid substitutions to SARS-CoV-2 in order to complement and facilitate experiments and expert analysis. The resulting dataset contains predictions from evolutionary conservation and protein and complex structural models, combined with viral phosphosites, experimental results and variant frequencies. We demonstrate predictions’ effectiveness by comparing them with expectations from variant frequency and prior experiments. We then identify higher frequency variants with significant predicted effects as well as finding variants measured to impact antibody binding that are least likely to impact other viral functions. A web portal is available atsars.mutfunc.com, where the dataset can be searched and downloaded.
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- 2021
12. A tyrosine kinase protein interaction map reveals targetable EGFR network oncogenesis in lung cancer
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Sourav Bandyopadhyay, Franziska Haderk, Emilie Gross, Khyati N. Shah, Victor Olivas, D. Ciznadija, Trever G. Bivona, Ido Sloma, Vincent B. Masto, Scott L. Weinrich, Xin Zhao, Nevan J. Krogan, Hsien-Ming Hu, John Jascur, Shigeki Nanjo, Swati Kaushik, Jeffery R. Johnson, and Gwendolyn M. Jang
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MAPK/ERK pathway ,Oncogene ,Chemistry ,Cancer research ,medicine ,Kinome ,Tyrosine ,Carcinogenesis ,medicine.disease_cause ,Phenotype ,Tyrosine kinase ,EGFR inhibitors - Abstract
SUMMARYSignaling networks balance the activities of many physically interacting proteins and perturbations to this network influence downstream signaling, potentially leading to oncogenic states. Using affinity purification-mass spectrometry we defined this network for all 90 human tyrosine kinases revealing 1,463 mostly novel interactions between these key cancer proteins and diverse molecular complexes. Modulation of interactor levels altered growth phenotypes associated with corresponding tyrosine kinase partners suggesting that tumors may alter the stoichiometries of interactors to maximize oncogenic signaling. We show that the levels of EGFR interactors delineates this form of network oncogenesis in 19% of EGFR wild-type lung cancer patients which were mostly otherwise oncogene negative, predicting sensitivity to EGFR inhibitors in vitro and in vivo. EGFR network oncogenesis occurs through mechanistically distinct network alleles often in cooperation with weak oncogenes in the MAPK pathway. Network oncogenesis may be a common and targetable convergent mechanism of oncogenic pathway activation in cancer.HIGHLIGHTSA human tyrosine kinome protein interaction map reveals novel physical and functional associations.Dependence on oncogenic tyrosine kinases is modulated through perturbation of their interactors.EGFR network oncogenesis in up to 19% of EGFR wild-type lung cancers is targetable.EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway.
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- 2020
13. The Global Phosphorylation Landscape of SARS-CoV-2 Infection
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Yiming Cai, Maya Modak, Sebastian Weigang, Emmie de Wit, Jean K. Lim, Alistair Dunham, Benjamin J. Polacco, Qiongyu Li, Svenja Ulferts, Gwendolyn M. Jang, Aurelien Dugourd, David E. Gordon, Jeffrey Z. Guo, Kirsten Obernier, Sophia Bouhaddou, Elizabeth R. Fischer, Anna Gaulton, Jason C.J. Chang, Bjoern Meyer, Diego Quintero, Julian Knerr, Trupti Patil, Emma J. Manners, Michael C. O’Neal, Monita Muralidharan, Joseph Hiatt, Ajda Rojc, James E. Melnyk, Tanja Kortemme, Benjamin R. tenOever, Thomas Vallet, Rémy Robinot, Cassandra Koh, Benjamin E. Nilsson-Payant, Ruth Hüttenhain, Saker Klippsten, Alicia L. Richards, Eloy Felix, Brian K. Shoichet, Beril Tutuncuoglu, Danielle L. Swaney, Veronica V. Rezelj, Jeffery R. Johnson, Margaret Soucheray, Marisa Goff, R. Dyche Mullins, Kris M. White, Erica Stevenson, Jyoti Batra, Christopher J.P. Mathy, Yuan Zhou, Minkyu Kim, Marco Vignuzzi, Claudia Hernandez-Armenta, Kevan M. Shokat, Julio Saez-Rodriguez, Jacqueline M. Fabius, Timothy McBride, Adolfo García-Sastre, Quang Dinh Tran, Alexandra Hardy, Elena Moreno, Alberto Valdeolivas, Mehdi Bouhaddou, Andrew R. Leach, Melanie Ott, Georg Kochs, Pedro Beltrao, Jiewei Xu, Robyn M. Kaake, Merve Cakir, Ying Shi, Nevan J. Krogan, Lisa Miorin, Danish Memon, David J. Broadhurst, Miguel Correa Marrero, Robert Grosse, Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Quantitative Biosciences Institute [UC San Francisco, USA] (QBI), University of California [San Francisco] (UC San Francisco), University of California (UC)-University of California (UC), Gladstone Institutes [San Francisco], European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Howard Hughes Medical Institute (HHMI), University of Freiburg [Freiburg], Virus et Immunité - Virus and immunity (CNRS-UMR3569), Universität Heidelberg [Heidelberg] = Heidelberg University, Heidelberg University Hospital [Heidelberg], Zoic Labs [Culver City, CA], Rocky Mountain Laboratories, Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Centre for Integrative Biological Signalling Studies [Freiburg] (CIBSS), This research was funded by grants from the National Institutes of Health ( P50AI150476 , U19AI135990 , U19AI135972 , R01AI143292 , R01AI120694 , P01A1063302 , and R01AI122747 to N.J.K., 1R01CA221969 and 1R01CA244550 to K.M.S., R01GM133981 to D.L.S., 1F32CA236347-01 to J.E.M., U19AI118610 to J.R.J., and F32CA239333 to M.B.), Defense Advance Research Projects Agency HR0011-19-2-0020 (to N.J.K., A.G.S., and K.M.S.), by the Laboratory for Genomics Research (LGR) Excellence in Research Award (ERA) from the Innovative Genomics Institute at UC Berkeley (grant number 133122P ), by CRIP (Center for Research for Influenza Pathogenesis), a NIAID-supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract HHSN272201400008C ) (to A.G.S.), by supplements to NIAID grant U19AI135972 and DoD grant W81XWH-19-PRMRP-FPA (to A.G.S.), and by the generous support of the JPB Foundation , the Open Philanthropy Project (research grant 2020-215611 [5384] ), and other philanthropic donations (to A.G.S.), by the Laboratoire d’Excellence 'Integrative Biology of Emerging Infectious Diseases' grant ANR-10-LABX-62-IBEID (to M.V.), by the DFG under Germany's Excellence Strategy ( EXC-2189 , project ID 390939984 to R.G.), by a Starting Grant Award from the European Research Council ( ERC-2014-STG 638884 PhosFunc to P.B.), by the Federal Ministry of Education and Research (BMBF, Computational Life Sciences grant 031L0181B to J.S.R.), by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases (to E.R.F. and E.D.W.), and by funding from F. Hoffmann-La Roche and Vir Biotechnology and gifts from The Ron Conway Family . K.M.S. is an investigator of the Howard Hughes Medical Institute., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 638884,H2020,ERC-2014-STG,PhosFunc(2015), Universität Heidelberg [Heidelberg], and Vaccine Research Institute (VRI)
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Proteomics ,MAPK/ERK pathway ,MESH: Angiotensin-Converting Enzyme 2 ,MESH: Casein Kinase II ,PIKFYVE ,0302 clinical medicine ,MESH: Chlorocebus aethiops ,MESH: Protein Kinase Inhibitors ,MESH: Animals ,Casein Kinase II ,Lung ,0303 health sciences ,Kinase ,MESH: Proteomics ,Phosphoproteomics ,antiviral ,Spike Glycoprotein ,Cyclin-Dependent Kinases ,3. Good health ,MESH: HEK293 Cells ,Spike Glycoprotein, Coronavirus ,Phosphorylation ,Infection ,MESH: Pandemics ,p38 mitogen-activated protein kinases ,Pneumonia, Viral ,MESH: Vero Cells ,p38 ,Antiviral Agents ,General Biochemistry, Genetics and Molecular Biology ,Article ,Betacoronavirus ,03 medical and health sciences ,Biodefense ,Humans ,MESH: SARS-CoV-2 ,MESH: Humans ,MESH: Phosphorylation ,Prevention ,MESH: Host-Pathogen Interactions ,fungi ,Receptor Protein-Tyrosine Kinases ,AXL ,Pneumonia ,Virology ,MAPK ,Coronavirus ,MESH: Peptidyl-Dipeptidase A ,MESH: Pneumonia, Viral ,MESH: Phosphatidylinositol 3-Kinases ,A549 Cells ,Vero cell ,Drug Evaluation ,030217 neurology & neurosurgery ,Developmental Biology ,MESH: Coronavirus Infections ,[SDV]Life Sciences [q-bio] ,viruses ,CDK ,Drug Evaluation, Preclinical ,MESH: Spike Glycoprotein, Coronavirus ,Medical and Health Sciences ,p38 Mitogen-Activated Protein Kinases ,Phosphatidylinositol 3-Kinases ,Chlorocebus aethiops ,MESH: COVID-19 ,Viral ,Phosphoinositide-3 Kinase Inhibitors ,mass spectrometry ,biology ,phosphoproteomics ,Biological Sciences ,Preclinical ,MESH: Cyclin-Dependent Kinases ,Infectious Diseases ,Host-Pathogen Interactions ,MESH: Betacoronavirus ,MESH: Drug Evaluation, Preclinical ,MESH: Receptor Protein-Tyrosine Kinases ,MESH: Caco-2 Cells ,Angiotensin-Converting Enzyme 2 ,Coronavirus Infections ,MESH: Antiviral Agents ,casein kinase II ,Peptidyl-Dipeptidase A ,Vaccine Related ,Cyclin-dependent kinase ,Proto-Oncogene Proteins ,Animals ,Pandemics ,Protein Kinase Inhibitors ,Vero Cells ,MESH: Phosphoinositide-3 Kinase Inhibitors ,030304 developmental biology ,SARS-CoV-2 ,COVID-19 ,Axl Receptor Tyrosine Kinase ,MESH: p38 Mitogen-Activated Protein Kinases ,MESH: Proto-Oncogene Proteins ,Emerging Infectious Diseases ,Good Health and Well Being ,HEK293 Cells ,biology.protein ,MESH: A549 Cells ,Caco-2 Cells - Abstract
Summary The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies., Graphical Abstract, Highlights • Phosphoproteomics analysis of SARS-CoV-2-infected cells uncovers signaling rewiring • Infection promotes host p38 MAPK cascade activity and shutdown of mitotic kinases • Infection stimulates CK2-containing filopodial protrusions with budding virus • Kinase activity analysis identifies potent antiviral drugs and compounds, Phosphoproteomics analysis of SARS-CoV-2-infected Vero E6 cells reveals host cellular pathways hijacked by viral infection, leading to the identification of small molecules that target dysregulated pathways and elicit potent antiviral efficacy.
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- 2020
14. Dynamic proteomics profiling ofLegionella pneumophilainfection unveils modulation of the host mitochondrial stress response pathway
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Julia Noack, Danielle L. Swaney, Tom Moss, Erica Stevenson, Shaeri Mukherjee, David Jimenez-Morales, Gwendolyn M. Jang, and Nevan J. Krogan
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biology ,Effector ,Mitochondrial unfolded protein response ,Proteome ,Secretion ,Human pathogen ,Mitochondrion ,biology.organism_classification ,Proteomics ,Legionella pneumophila ,Cell biology - Abstract
SUMMARYThe human pathogenLegionella pneumophila (L.p.)secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of mostL.p.effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome duringL.p.infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show thatL.p.substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmtby a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterizedL.p.effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.
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- 2020
15. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing
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Pedro Beltrao, Phillip P. Sharp, Nevan J. Krogan, Sabrina J. Fletcher, Saker Klippsten, Trey Ideker, Melanie Ott, Bryan L. Roth, Xi Liu, Devin A. Cavero, Djoshkun Shengjuler, Christopher J.P. Mathy, Jason C.J. Chang, Theodore L. Roth, Hannes Braberg, Claudia Hernandez-Armenta, Lisa Miorin, Jyoti Batra, Shizhong Dai, Maliheh Safari, Brian K. Shoichet, Danish Memon, Tia A. Tummino, Marco Vignuzzi, Mark von Zastrow, Manon Eckhardt, Alan D. Frankel, Qiongyu Li, Tanja Kortemme, Nicole A. Wenzell, Zun Zar Chi Naing, Ferdinand Roesch, Nastaran Sadat Savar, Mathieu Hubert, Xi Ping Huang, Elena Moreno, Danica Galonić Fujimori, Jeffrey Z. Guo, Natalia Jura, Kirsten Obernier, Kliment A. Verba, Harmit S. Malik, Hao-Yuan Wang, Michael McGregor, Melanie J. Bennett, Julia Noack, Gwendolyn M. Jang, Paige Haas, Alice Mac Kain, Daniel J. Saltzberg, Mehdi Bouhaddou, Ziyang Zhang, Yongfeng Liu, Inigo Barrio-Hernandez, Yiming Cai, Kris M. White, Kelsey M. Haas, Maya Modak, Stephanie A. Wankowicz, Raphael Trenker, Kevan M. Shokat, Fatima S. Ugur, Shiming Peng, Sai J. Ganesan, Shaeri Mukherjee, Yuan Zhou, Minkyu Kim, John D. Gross, Jack Taunton, Alicia L. Richards, John S. Chorba, Margaret Soucheray, Danielle L. Swaney, Benjamin J. Polacco, Alan Ashworth, Wenqi Shen, Adolfo García-Sastre, Merve Cakir, Ujjwal Rathore, Kala Bharath Pilla, Michael C. O’Neal, Ying Shi, Kevin Lou, Cassandra Koh, Stephen N. Floor, Davide Ruggero, Ilsa T Kirby, Srivats Venkataramanan, Ruth Hüttenhain, Olivier Schwartz, Beril Tutuncuoglu, Christophe d'Enfert, Jose Liboy-Lugo, David A. Agard, Charles S. Craik, Veronica V. Rezelj, Tina Perica, Matthew P. Jacobson, Lorenzo Calviello, Eric Verdin, Yizhu Lin, Jiankun Lyu, Jiewei Xu, Joseph Hiatt, Andrej Sali, Oren S. Rosenberg, Markus Bohn, David E. Gordon, James S. Fraser, Sara Brin Rosenthal, Duygu Kuzuoğlu-Öztürk, Robyn M. Kaake, Jacqueline M. Fabius, Matthew J. O’Meara, Quang Dinh Tran, Advait Subramanian, Thomas Vallet, Bjoern Meyer, James E. Melnyk, Robert M. Stroud, Helene Foussard, Rakesh Ramachandran, David J. Broadhurst, Janet M. Young, and Michael Emerman
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0301 basic medicine ,viruses ,Drug Evaluation, Preclinical ,Plasma protein binding ,Proteomics ,medicine.disease_cause ,Mass Spectrometry ,0302 clinical medicine ,Chlorocebus aethiops ,Protein Interaction Mapping ,Molecular Targeted Therapy ,Protein Interaction Maps ,Cloning, Molecular ,Letter to the Editor ,Coronavirus ,Multidisciplinary ,3. Good health ,Drug repositioning ,030220 oncology & carcinogenesis ,Host-Pathogen Interactions ,Coronavirus Infections ,Protein Binding ,Pneumonia, Viral ,Biology ,Antiviral Agents ,Virus ,Betacoronavirus ,Viral Proteins ,03 medical and health sciences ,Immune system ,Protein Domains ,medicine ,Animals ,Humans ,Receptors, sigma ,Pandemics ,Vero Cells ,SKP Cullin F-Box Protein Ligases ,Innate immune system ,SARS-CoV-2 ,fungi ,HEK 293 cells ,Drug Repositioning ,COVID-19 ,Virology ,Immunity, Innate ,COVID-19 Drug Treatment ,HEK293 Cells ,030104 developmental biology ,Protein Biosynthesis - Abstract
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.
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- 2020
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16. A Quantitative Genetic Interaction Map of HIV Infection
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David C. Crosby, Assen Roguev, Joshua Kane, Michael Shales, Jiewei Xu, Erica Stevenson, Ariane Watson, Alexander Marson, David E. Gordon, Ivan Marazzi, Nevan J. Krogan, Gwendolyn M. Jang, Simin Zheng, Jeffrey Z. Guo, Danielle L. Swaney, Alan D. Frankel, Erik Verschueren, Gerard Cagney, and Kathy Franks-Skiba
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CD4-Positive T-Lymphocytes ,CNOT complex ,Interferon Regulatory Factor-7 ,HIV Infections ,combinatorial genetics ,medicine.disease_cause ,Medical and Health Sciences ,0302 clinical medicine ,vE-MAP ,IRF7 ,Innate ,2.1 Biological and endogenous factors ,Aetiology ,innate immunity ,Genetics ,0303 health sciences ,Mutation ,epistasis map ,Biological Sciences ,Phenotype ,CCR4-NOT ,Infectious Diseases ,interferon stimulated gene ,Host-Pathogen Interactions ,HIV/AIDS ,Infection ,Signal Transduction ,Biology ,Article ,viral infection genetic screen ,03 medical and health sciences ,Genetic ,medicine ,Humans ,Molecular Biology ,Gene ,Transcription factor ,030304 developmental biology ,Innate immune system ,Immunity ,host-pathogen network biology ,Epistasis, Genetic ,Cell Biology ,Immunity, Innate ,Emerging Infectious Diseases ,Good Health and Well Being ,HIV-1 ,Epistasis ,Human genome ,Interferons ,030217 neurology & neurosurgery ,Transcription Factors ,Developmental Biology - Abstract
We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary Tcells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of howdifferent pathogens hijack and rewire the host during infection.
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- 2020
17. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing
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David E. Gordon, Gwendolyn M. Jang, Qiongyu Li, Natalia Jura, Sara Brin Rosenthal, Trey Ideker, Paige Haas, Melanie J. Bennett, Ilsa T Kirby, Adolfo García-Sastre, Michael Emerman, Thomas Vallet, Tina Perica, Lorenzo Calviello, Kirsten Obernier, Kliment A. Verba, Tanja Kortemme, Michael McGregor, Alan Ashworth, Ujjwal Rathore, Ziyang Zhang, Kelsey M. Haas, Rakesh Ramachandran, Mark von Zastrow, Jacqueline M. Fabius, Theodore L. Roth, Daniel J. Saltzberg, Matthew P. Jacobson, Kevin Lou, Ferdinand Roesch, Yizhu Lin, John S. Chorba, Beril Tutuncuoglu, Claudia Hernandez-Armenta, Harmit S. Malik, Janet M. Young, Manon Eckhardt, Srivats Venkataramanan, Jose Liboy-Lugo, Phillip P. Sharp, Jeffrey Z. Guo, Maya Modak, Shaeri Mukherjee, Markus Bohn, Brian K. Shoichet, Olivier Schwartz, Jiewei Xu, James S. Fraser, Andrej Sali, Oren S. Rosenberg, Christopher J.P. Mathy, Charles S. Craik, Benjamin J. Polacco, Melanie Ott, Sai J. Ganesan, Pedro Beltrao, Alicia L. Richards, Helene Foussard, Margaret Soucheray, Joseph Hiatt, Robyn M. Kaake, Danielle L. Swaney, Wenqi Shen, Bjoern Meyer, Kala Bharath Pilla, Zun Zar Chi Naing, Marco Vignuzzi, James E. Melnyk, John D. Gross, Shiming Peng, Mehdi Bouhaddou, Nevan J. Krogan, Merve Cakir, Mathieu Hubert, Stephanie A. Wankowicz, Ying Shi, Davide Ruggero, Kevan M. Shokat, Stephen N. Floor, Jack Taunton, Xi Liu, Ruth Hüttenhain, David A. Agard, Lisa Miorin, Danish Memon, Julia Noack, Raphael Trenker, Hannes Braberg, Shizhong Dai, Tia A. Tummino, Kris M. White, Yuan Zhou, Minkyu Kim, Devin A. Cavero, Jyoti Batra, Advait Subramanian, Danica Galonić Fujimori, and Inigo Barrio-Hernandez
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Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,media_common.quotation_subject ,viruses ,Host factors ,Article ,Vaccine Related ,03 medical and health sciences ,0302 clinical medicine ,Rare Diseases ,Biodefense ,2.2 Factors relating to the physical environment ,Aetiology ,Human proteins ,Lung ,030304 developmental biology ,media_common ,0303 health sciences ,Prevention ,Art ,Pneumonia ,3. Good health ,Good Health and Well Being ,Infectious Diseases ,Emerging Infectious Diseases ,5.1 Pharmaceuticals ,030220 oncology & carcinogenesis ,Protein Interaction Networks ,Molecular targets ,Pneumonia & Influenza ,Development of treatments and therapeutic interventions ,Infection ,Humanities - Abstract
Author(s): Gordon, David E; Jang, Gwendolyn M; Bouhaddou, Mehdi; Xu, Jiewei; Obernier, Kirsten; O'Meara, Matthew J; Guo, Jeffrey Z; Swaney, Danielle L; Tummino, Tia A; Huttenhain, Ruth; Kaake, Robyn M; Richards, Alicia L; Tutuncuoglu, Beril; Foussard, Helene; Batra, Jyoti; Haas, Kelsey; Modak, Maya; Kim, Minkyu; Haas, Paige; Polacco, Benjamin J; Braberg, Hannes; Fabius, Jacqueline M; Eckhardt, Manon; Soucheray, Margaret; Bennett, Melanie J; Cakir, Merve; McGregor, Michael J; Li, Qiongyu; Naing, Zun Zar Chi; Zhou, Yuan; Peng, Shiming; Kirby, Ilsa T; Melnyk, James E; Chorba, John S; Lou, Kevin; Dai, Shizhong A; Shen, Wenqi; Shi, Ying; Zhang, Ziyang; Barrio-Hernandez, Inigo; Memon, Danish; Hernandez-Armenta, Claudia; Mathy, Christopher JP; Perica, Tina; Pilla, Kala B; Ganesan, Sai J; Saltzberg, Daniel J; Ramachandran, Rakesh; Liu, Xi; Rosenthal, Sara B; Calviello, Lorenzo; Venkataramanan, Srivats; Lin, Yizhu; Wankowicz, Stephanie A; Bohn, Markus; Trenker, Raphael; Young, Janet M; Cavero, Devin; Hiatt, Joe; Roth, Theo; Rathore, Ujjwal; Subramanian, Advait; Noack, Julia; Hubert, Mathieu; Roesch, Ferdinand; Vallet, Thomas; Meyer, Bjorn; White, Kris M; Miorin, Lisa; Agard, David; Emerman, Michael; Ruggero, Davide; Garcia-Sastre, Adolfo; Jura, Natalia; von Zastrow, Mark; Taunton, Jack; Schwartz, Olivier; Vignuzzi, Marco; d'Enfert, Christophe; Mukherjee, Shaeri; Jacobson, Matt; Malik, Harmit S; Fujimori, Danica G; Ideker, Trey; Craik, Charles S | Abstract: An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity- purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.
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- 2020
18. Biophysical basis of cellular multi-specificity encoded in a model molecular switch
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Gwendolyn M. Jang, Hannes Braberg, Jiewei Xu, Tina Perica, Christopher J.P. Mathy, Robyn M. Kaake, Mark J. S. Kelly, Yang Zhang, Nevan J. Krogan, Tanja Kortemme, David G. Lambright, Noah Ollikainen, and Danielle L. Swaney
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Molecular switch ,0303 health sciences ,Chemistry ,Systems biology ,Point mutation ,030302 biochemistry & molecular biology ,Allosteric regulation ,GTPase ,Computational biology ,03 medical and health sciences ,Ran ,Small GTPase ,Biological regulation ,030304 developmental biology - Abstract
Molecular switches are central to signal transduction in protein interaction networks. One switch protein can independently regulate distinct cellular processes, but the molecular mechanisms enabling this functional multi-specificity remain unclear. Here we integrate system-scale cellular and biophysical measurements to study how a paradigm switch, the small GTPase Ran/Gsp1, achieves its functional multi-specificity. We make 55 targeted point mutations to individual interactions of Ran/Gsp1 and show through quantitative, systematic genetic and physical interaction mapping that Ran/Gsp1 interface perturbations have widespread cellular consequences that cluster by biological processes but, unexpectedly, not by the targeted interactions. Instead, the cellular consequences of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle, and cycle kinetics are allosterically tuned by distal interface mutations. We propose that the functional multi-specificity of Ran/Gsp1 is encoded by a differential sensitivity of biological processes to different kinetic parameters of the Gsp1 switch cycle, and that Gsp1 partners binding to the sites of distal mutations act as allosteric regulators of the switch. Similar mechanisms may underlie biological regulation by other GTPases and biological switches. Finally, our integrative platform to determine the quantitative consequences of cellular perturbations may help explain the effects of disease mutations targeting central switches.
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- 2020
19. Dynamic Proteomics Profiling of Legionella pneumophila Infection Unveils Modulation of the Host Mitochondrial Stress Response Pathway
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Nevan J. Krogan, Danielle L. Swaney, Shaeri Mukherjee, Erica Stevenson, Tom Moss, Julia Noack, David Jimenez-Morales, and Gwendolyn M. Jang
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Proteostasis ,Effector ,Host–pathogen interaction ,Mitochondrial unfolded protein response ,Proteome ,Integrated stress response ,Mitochondrion ,Biology ,Proteomics ,Cell biology - Abstract
The human pathogen Legionella pneumophila (L.p.) secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of most L.p. effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome during L.p. infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show that L.p. substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmt by a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterized L.p. effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.
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- 2020
20. Enterovirus pathogenesis requires the host methyltransferase SETD3
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Or Gozani, Jan E. Carette, Jeffrey R. Johnson, Peter Sarnow, Erik Verschueren, Jonathan Diep, Harry B. Greenberg, Tracy Young, Eileen Foy, Kristi J. Kobluk, James Zengel, Raul Andino, Christine E. Peters, Kuo-Feng Weng, Jiewei Xu, Alex W. Wilkinson, Ruth Hüttenhain, Siyuan Ding, Nevan J. Krogan, Gwendolyn M. Jang, Joshua E. Elias, Yaw Shin Ooi, Orly Laufman, and Claude M. Nagamine
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viruses ,Viral pathogenesis ,medicine.disease_cause ,Virus Replication ,Applied Microbiology and Biotechnology ,Mice ,CRISPR ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Encephalitis, Viral ,Viral ,Aetiology ,Enterovirus ,0303 health sciences ,Neuromuscular Diseases ,Myelitis ,3. Good health ,Infectious Diseases ,Medical Microbiology ,Histone Methyltransferases ,Encephalitis ,Infection ,Microbiology (medical) ,Immunology ,Biology ,Microbiology ,Article ,03 medical and health sciences ,Viral Proteins ,Viral life cycle ,medicine ,Enterovirus Infections ,Genetics ,Animals ,030304 developmental biology ,030306 microbiology ,Animal ,Viral encephalitis ,RNA ,Cell Biology ,Methyltransferases ,medicine.disease ,Virology ,Acute flaccid myelitis ,Disease Models, Animal ,Good Health and Well Being ,Viral replication ,Disease Models ,Proteolysis ,Central Nervous System Viral Diseases ,CRISPR-Cas Systems - Abstract
Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.
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- 2019
21. Identification of antiviral roles for the exon-junction complex and nonsense-mediated decay in flaviviral infection
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Billy Truong, Priya S. Shah, Billy W. Newton, Minghua Li, Sara Cherry, Holly Ramage, Jeffrey R. Johnson, Nevan J. Krogan, Nathan L. Weinbren, John Von Dollen, Gwendolyn M. Jang, Grace Kim, and Mark Dittmar
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viruses ,RNA-binding protein ,Dengue virus ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Zika virus ,RNA interference ,2.2 Factors relating to the physical environment ,2.1 Biological and endogenous factors ,Viral ,Protein Interaction Maps ,Aetiology ,0303 health sciences ,biology ,virus diseases ,RNA-Binding Proteins ,Exons ,3. Good health ,Flavivirus ,Infectious Diseases ,Codon, Nonsense ,Medical Microbiology ,Host-Pathogen Interactions ,RNA, Viral ,RNA Interference ,Infection ,West Nile virus ,Microbiology (medical) ,Immunology ,Antiviral Agents ,Microbiology ,Virus ,Article ,Flavivirus Infections ,Vaccine Related ,03 medical and health sciences ,Viral Proteins ,Rare Diseases ,Biodefense ,Genetics ,medicine ,Humans ,Codon ,030304 developmental biology ,030306 microbiology ,Prevention ,RNA ,Cell Biology ,Zika Virus ,Dengue Virus ,biology.organism_classification ,Virology ,nervous system diseases ,Vector-Borne Diseases ,Emerging Infectious Diseases ,Good Health and Well Being ,Nonsense ,Exon junction complex ,Capsid Proteins ,Carrier Proteins - Abstract
West Nile virus (WNV) is an emerging mosquito-borne flavivirus, related to dengue virus and Zika virus. To gain insight into host pathways involved in WNV infection, we performed a systematic affinity-tag purification mass spectrometry (APMS) study to identify 259 WNV-interacting human proteins. RNA interference screening revealed 26 genes that both interact with WNV proteins and influence WNV infection. We found that WNV, dengue and Zika virus capsids interact with a conserved subset of proteins that impact infection. These include the exon-junction complex (EJC) recycling factor PYM1, which is antiviral against all three viruses. The EJC has roles in nonsense-mediated decay (NMD), and we found that both the EJC and NMD are antiviral and the EJC protein RBM8A directly binds WNV RNA. To counteract this, flavivirus infection inhibits NMD and the capsid-PYM1 interaction interferes with EJC protein function and localization. Depletion of PYM1 attenuates RBM8A binding to viral RNA, suggesting that WNV sequesters PYM1 to protect viral RNA from decay. Together, these data suggest a complex interplay between the virus and host in regulating NMD and the EJC.
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- 2019
22. Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication
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Gwendolyn M. Jang, Paige Haas, Manu Anantpadma, Laura Satkamp, Olena Shtanko, Eusondia Arnett, Carson B. Schmidt, Robert A. Davey, Gaya K. Amarasinghe, Daisy W. Leung, John Von Dollen, Jyoti Batra, Ann N. Reyes, Judd F. Hultquist, Priya Luthra, Douglas J. LaCount, Dandan Liu, Christopher F. Basler, Nevan J. Krogan, Toni M. Schwarz, Gabriel I. Small, Robyn M. Kaake, and Larry S. Schlesinger
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0301 basic medicine ,RNA viruses ,protein-protein interactions ,Peptide ,Endogeny ,medicine.disease_cause ,Crystallography, X-Ray ,Virus Replication ,Medical and Health Sciences ,Ebola virus ,Transcription (biology) ,Protein Interaction Mapping ,host-pathogen interactions ,2.1 Biological and endogenous factors ,VP30 ,Aetiology ,chemistry.chemical_classification ,Gene knockdown ,Crystallography ,Biological Sciences ,Ebolavirus ,Ubiquitin ligase ,Amino acid ,DNA-Binding Proteins ,Infectious Diseases ,Ebola ,Infection ,Biotechnology ,Ubiquitin-Protein Ligases ,030106 microbiology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,Vaccine Related ,03 medical and health sciences ,Viral Proteins ,Biodefense ,medicine ,Genetics ,Humans ,antiviral factor ,RBBP6 ,virus-host interactions ,Prevention ,Hemorrhagic Fever, Ebola ,Virology ,Nucleoprotein ,030104 developmental biology ,HEK293 Cells ,Emerging Infectious Diseases ,Good Health and Well Being ,chemistry ,Hela Cells ,biology.protein ,X-Ray ,Hemorrhagic Fever ,Carrier Proteins ,HeLa Cells ,Transcription Factors ,Developmental Biology - Abstract
Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
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- 2018
23. Abstract 4891: Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies
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Danielle L. Swaney, John D. Gordan, Elizabeth F. Thayer, Wei Zhang, Manon Eckhardt, Trey Ideker, Huat Chye Lim, John Von Dollen, R. Katie Kelley, Nevan J. Krogan, Gwendolyn M. Jang, Alex Choi, Fabian J. Theis, Rigney E. Turnham, and Adriana Pitea
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Hepatitis B virus ,Cancer Research ,Phosphoproteomics ,DNA virus ,Context (language use) ,Protein phosphatase 2 ,Biology ,medicine.disease_cause ,digestive system diseases ,Ubiquitin ligase ,HBx ,Oncology ,Ubiquitin ,medicine ,biology.protein ,Cancer research - Abstract
Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Advanced HCC has proven particularly difficult to treat because of a scarcity of clear genetic drivers of cancer progression; thus, there are currently no predictive markers that guide HCC therapy. HCC arises in the context of co-morbid hepatitis due to hepatitis B virus (HBV), hepatitis C (HCV) or fatty liver disease. We hypothesize that protein-protein interactions (PPIs) between viral proteins and HCC genes may contribute to tumor initiation and maintenance. In order to characterize these PPIs, we performed affinity purification - mass spectrometry (APMS), defining 145 HBV/host PPIs including known and novel interacting partners. We next used a network propagation algorithm to identify host genes and protein complexes that were preferentially mutated in the absence of HBV infection. HBV is a small DNA virus, with 4 genes of which only one has enzymatic activity, raising a question as to how HBV interaction modifies host behavior. Using AP-MS of host proteins, we found that the HBV X protein (HBx) remodels multiple host protein complexes through direct interaction. These physical effects on complex components result in distinct biochemical behavior from the CRL4 E3 ubiquitin ligase complex as well as the phosphatase PP2A, as determined through global phosphoproteomics and ubiquitin analysis. We show that this remodeling driven by HBx substantially changes cellular protein turnover and downstream signaling dynamics. We followed this up with assessments of cellular viability and proliferation in response to pharmacological inhibition or CRISPRi-based knockdown of HBx effectors. Our data support a model where HBV proteins alter the components and behavior of key regulatory protein complexes in the cell, altering tumor behavior and raising the possibility of precision therapeutics for HCC. Citation Format: John D. Gordan, Adriana Pitea, Manon Eckhardt, Gwendolyn Jang, Rigney E. Turnham, Alex L M. Choi, John Von Dollen, Huat C. Lim, Elizabeth F. Thayer, R. Katie Kelley, Danielle L. Swaney, Wei Zhang, Fabian J. Theis, Trey Ideker, Nevan J. Krogan. Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4891.
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- 2020
24. CRL4 AMBRA1 targets Elongin C for ubiquitination and degradation to modulate CRL5 signaling
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Jordan Ye, Gwendolyn M. Jang, Lily Burton, John D. Gross, Yi Liang Liu, Tasha L. Johnson, Alexander Marson, Billy W. Newton, Jayanta Debnath, Joseph Hiatt, David E. Gordon, Robert M. Stroud, Judd F. Hultquist, Kurt M. Reichermeier, Dan Du, Jeffrey R. Johnson, Nevan J. Krogan, Ruth Hüttenhain, and Si-Han Chen
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0301 basic medicine ,General Immunology and Microbiology ,biology ,General Neuroscience ,Signal transducing adaptor protein ,Proteomics ,Substrate degradation ,General Biochemistry, Genetics and Molecular Biology ,3. Good health ,Ubiquitin ligase ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,Adapter (genetics) ,Ubiquitin ,biology.protein ,Ligase activity ,Receptor ,Molecular Biology - Abstract
Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4 AMBRA 1 (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4 AMBRA 1 is required to disrupt the assembly and attenuate the ligase activity of human CRL5 SOCS 3 and HIV‐1 CRL5 VIF complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4 AMBRA 1 modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5 SOCS 3 and CRL5 VIF , respectively. Thus, by discovering a substrate of CRL4 AMBRA 1 , ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway.
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- 2018
25. The HIV-1 Tat protein recruits a ubiquitin ligase to reorganize the 7SK snRNP for transcriptional activation
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Gwendolyn M. Jang, Billy W. Newton, Tyler B. Faust, Amit Weiss, Yang Li, Curtis W. Bacon, Bhargavi Jayaraman, Iván D'Orso, Nevan J. Krogan, and Alan D. Frankel
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0301 basic medicine ,Nuclear Localization Signals ,RNA polymerase II ,0302 clinical medicine ,Ubiquitin ,Models ,7SK RNA ,host-pathogen interactions ,Positive Transcriptional Elongation Factor B ,Biology (General) ,Ribonucleoprotein ,biology ,Chemistry ,General Neuroscience ,RNA-Binding Proteins ,General Medicine ,Ribonucleoproteins, Small Nuclear ,Chromatin ,Active Transport ,3. Good health ,Cell biology ,Ubiquitin ligase ,Protein Transport ,Ribonucleoproteins ,Medicine ,HIV/AIDS ,tat Gene Products, Human Immunodeficiency Virus ,RNA Interference ,tat Gene Products ,Infection ,Human Immunodeficiency Virus ,Research Article ,Human ,Protein Binding ,Transcriptional Activation ,QH301-705.5 ,Science ,1.1 Normal biological development and functioning ,Active Transport, Cell Nucleus ,chemical biology ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Small Nuclear ,Biochemistry and Chemical Biology ,Underpinning research ,Genetics ,Humans ,biochemistry ,snRNP ,Amino Acid Sequence ,human ,transcription elongation ,Cell Nucleus ,7SK snRNP ,General Immunology and Microbiology ,Ubiquitination ,non-degradative ubiquitination ,Biological ,nuclear import ,030104 developmental biology ,HEK293 Cells ,Hela Cells ,Ubiquitin-Conjugating Enzymes ,biology.protein ,HIV-1 ,Cyclin-dependent kinase 9 ,Generic health relevance ,Biochemistry and Cell Biology ,030217 neurology & neurosurgery ,Dichlororibofuranosylbenzimidazole ,HeLa Cells ,Transcription Factors - Abstract
The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.
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- 2018
26. Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection
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Raphael H. Valdivia, Jessica Sherry, Tasha L. Johnson, Michael Shales, Nevan J. Krogan, Michael N. Starnbach, Joe Dan Dunn, Natali Gulbahce, Jeffery S. Cox, Gwendolyn M. Jang, Andrew J. Olive, Oren S. Rosenberg, Kathleen M. Mirrashidi, Isabelle Derré, John Von Dollen, Andrew Frando, Cherilyn A. Elwell, Erik Verschueren, Bennett H. Penn, Stefanie Jäger, Anusha M. Gopalakrishnan, Joanne N. Engel, and Jeffrey R. Johnson
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Cancer Research ,Proteome ,Retromer ,Sorting Nexins ,Immunology ,Chlamydia trachomatis ,Biology ,urologic and male genital diseases ,medicine.disease_cause ,Proteomics ,Microbiology ,Article ,03 medical and health sciences ,Bacterial Proteins ,Immunology and Microbiology(all) ,Virology ,Protein Interaction Mapping ,medicine ,Humans ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Protein Interaction Maps ,Aetiology ,Molecular Biology ,030304 developmental biology ,Inclusion Bodies ,0303 health sciences ,Membrane tubulation ,030306 microbiology ,Intracellular Membranes ,Chlamydia Infections ,Entry into host ,female genital diseases and pregnancy complications ,3. Good health ,Cell biology ,Infectious Diseases ,Membrane protein ,Medical Microbiology ,Host-Pathogen Interactions ,Sexually Transmitted Infections ,Parasitology ,Infection - Abstract
Summary Chlamydia trachomatis is a leading cause of genital and ocular infections for which no vaccine exists. Upon entry into host cells, C. trachomatis resides within a membrane-bound compartment—the inclusion—and secretes inclusion membrane proteins (Incs) that are thought to modulate the host-bacterium interface. To expand our understanding of Inc function(s), we subjected putative C. trachomatis Incs to affinity purification-mass spectroscopy (AP-MS). We identified Inc-human interactions for 38/58 Incs with enrichment in host processes consistent with Chlamydia's intracellular life cycle. There is significant overlap between Inc targets and viral proteins, suggesting common pathogenic mechanisms among obligate intracellular microbes. IncE binds to sorting nexins (SNXs) 5/6, components of the retromer, which relocalizes SNX5/6 to the inclusion membrane and augments inclusion membrane tubulation. Depletion of retromer components enhances progeny production, revealing that retromer restricts Chlamydia infection. This study demonstrates the value of proteomics in unveiling host-pathogen interactions in genetically challenging microbes.
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- 2015
27. Global Mapping of Herpesvirus-Host Protein Complexes Reveals a Transcription Strategy for Late Genes
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Stefanie Jäger, Gwendolyn M. Jang, Nevan J. Krogan, Kevin Kleffman, Erik Verschueren, Julie Horner, Michael Shales, Jimin Park, Tasha L. Johnson, Jeffrey R. Johnson, William Newton, M. Cyrus Maher, Britt A. Glaunsinger, John Von Dollen, Ryan D. Hernandez, and Zoe H. Davis
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Gene Expression Regulation, Viral ,Transcription, Genetic ,viruses ,RNA polymerase II ,Medical and Health Sciences ,Article ,chemistry.chemical_compound ,Viral Proteins ,Genetic ,Transcription (biology) ,Protein Interaction Mapping ,Genetics ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Humans ,Viral ,Protein Interaction Maps ,Herpesvirus 8 ,Aetiology ,Gene ,Molecular Biology ,Cancer ,Regulation of gene expression ,biology ,TATA-Box Binding Protein ,virus diseases ,Promoter ,Cell Biology ,Biological Sciences ,Cell biology ,Infectious Diseases ,Emerging Infectious Diseases ,HEK293 Cells ,chemistry ,Gene Expression Regulation ,Herpesvirus 8, Human ,Host-Pathogen Interactions ,biology.protein ,HIV/AIDS ,RNA Polymerase II ,Transcription factor II D ,Infection ,Transcription ,DNA ,Human ,Biotechnology ,Developmental Biology - Abstract
Mapping host-pathogen interactions has proven instrumental for understanding how viruses manipulate host machinery and how numerous cellular processes are regulated. DNA viruses such as herpesviruses have relatively large coding capacity and thus can target an extensive network of cellular proteins. To identify the host proteins hijacked by this pathogen, we systematically affinity tagged and purified all 89 proteins of Kaposi's sarcoma-associated herpesvirus (KSHV) from human cells. Mass spectrometry of this material identified over 500 virus-host interactions. KSHV causes AIDS-associated cancers, and its interaction network is enriched for proteins linked to cancer and overlaps with proteins that are also targeted by HIV-1. We found that the conserved KSHV protein ORF24 binds to RNA polymerase II and brings it to viral late promoters by mimicking and replacing cellular TATA-box-binding protein (TBP). This is required for herpesviral late gene expression, a complex and poorly understood phase of the viral lifecycle. Kaposi's sarcoma-associated herpesvirus (KSHV) is a major AIDS-associated pathogen. Davis etal. assemble a KSHV-host protein-protein interaction network that suggests herpesvirus-host evolutionary interplay. Using the network, they describe a hybrid KSHV-human transcription complex that activates viral late genes.
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- 2015
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28. An Mtb-Human Protein-Protein Interaction Map Reveals that Bacterial LpqN Antagonizes CBL, a Host Ubiquitin Ligase that Regulates the Balance Between Anti-Viral and Anti-Bacterial Responses
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C Maher, M Naramura, Gwendolyn M. Jang, BH Penn, Samantha L. Bell, S Jaeger, Zoe Netter, Kristina M. Geiger, Alex Choi, Curtis Chen, Ryan D. Hernandez, Yamini M. Ohol, John Von Dollen, Tasha L. Johnson, Jeffery S. Cox, Trevor J Parry, Daniel A. Portnoy, Jeffrey R. Johnson, Michael Shales, Nevan J. Krogan, X Du, and Laurent Coscoy
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0303 health sciences ,Innate immune system ,biology ,Host (biology) ,Mutant ,chemical and pharmacologic phenomena ,respiratory system ,biology.organism_classification ,bacterial infections and mycoses ,Molecular biology ,Ubiquitin ligase ,Cell biology ,Pathogenesis ,Mycobacterium tuberculosis ,03 medical and health sciences ,0302 clinical medicine ,Immunity ,biology.protein ,Anti bacterial ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SUMMARYAlthough macrophages are armed with potent anti-bacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage-intrinsic bacterial control, and the Mtb strategies to overcome them are poorly understood. To further study these processes, we used a systematic affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two new factors involved in Mtb pathogenesis - the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl-/- macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between anti-bacterial and anti-viral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map as a resource for developing a deeper understanding of the intricate interactions between Mtb and its host.
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- 2017
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29. Multiple Routes to Oncogenesis Are Promoted by the Human Papillomavirus-Host Protein Network
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Trey Ideker, Jacques Archambault, Wei Zhang, Toni M. Brand, Danielle L. Swaney, Gwendolyn M. Jang, Tasha L. Johnson, Jason F. Kreisberg, Kathleen Franks-Skiba, John Von Dollen, Jennifer R. Grandis, Jeffrey R. Johnson, Manon Eckhardt, Priya S. Shah, Andrew M. Gross, and Nevan J. Krogan
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0301 basic medicine ,Viral protein ,Carcinogenesis ,Computational biology ,Biology ,medicine.disease_cause ,Article ,Protein–protein interaction ,03 medical and health sciences ,Interaction network ,medicine ,Biomarkers, Tumor ,Humans ,Protein Interaction Maps ,Papillomaviridae ,Mutation ,Histone ubiquitination ,Papillomavirus Infections ,HPV infection ,medicine.disease ,KEAP1 ,030104 developmental biology ,Oncology ,Head and Neck Neoplasms ,Host-Pathogen Interactions ,Carcinoma, Squamous Cell - Abstract
We have mapped a global network of virus–host protein interactions by purification of the complete set of human papillomavirus (HPV) proteins in multiple cell lines followed by mass spectrometry analysis. Integration of this map with tumor genome atlases shows that the virus targets human proteins frequently mutated in HPV− but not HPV+ cancers, providing a unique opportunity to identify novel oncogenic events phenocopied by HPV infection. For example, we find that the NRF2 transcriptional pathway, which protects against oxidative stress, is activated by interaction of the NRF2 regulator KEAP1 with the viral protein E1. We also demonstrate that the L2 HPV protein physically interacts with the RNF20/40 histone ubiquitination complex and promotes tumor cell invasion in an RNF20/40-dependent manner. This combined proteomic and genetic approach provides a systematic means to study the cellular mechanisms hijacked by virally induced cancers. Significance: In this study, we created a protein–protein interaction network between HPV and human proteins. An integrative analysis of this network and 800 tumor mutation profiles identifies multiple oncogenesis pathways promoted by HPV interactions that phenocopy recurrent mutations in cancer, yielding an expanded definition of HPV oncogenic roles. Cancer Discov; 8(11); 1474–89. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1333
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- 2017
30. An Mtb-Human Protein-Protein Interaction Map Identifies a Switch between Host Antiviral and Antibacterial Responses
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Zoe Netter, Jeffrey R. Johnson, Dan A. Portnoy, Laurent Coscoy, Samantha L. Bell, Kristina M. Geiger, Chen Chen, Tasha L. Johnson, Bhopal Mohapatra, Xiaotang Du, Gwendolyn M. Jang, Matthew D. Storck, Jeffery S. Cox, Bennett H. Penn, Nevan J. Krogan, Guillaume Golovkine, Alex Choi, Yamini M. Ohol, Trevor J Parry, Hamid Band, Michael Shales, Ryan D. Hernandez, Cyrus Maher, Stefanie Jäger, and John Von Dollen
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0301 basic medicine ,Mutant ,Chlamydia trachomatis ,host-pathogen interaction ,Medical and Health Sciences ,mycobacterium ,protein-protein interaction ,Mice ,0302 clinical medicine ,Ubiquitin ,Protein Interaction Mapping ,2.1 Biological and endogenous factors ,2.2 Factors relating to the physical environment ,Protein Interaction Maps ,Lymphocytes ,Proto-Oncogene Proteins c-cbl ,Aetiology ,LpqN ,Tumor ,biology ,Biological Sciences ,respiratory system ,Ubiquitin ligase ,Anti-Bacterial Agents ,Infectious Diseases ,Herpesvirus 8, Human ,Host-Pathogen Interactions ,HIV/AIDS ,Infection ,Human ,Protein Binding ,Signal Transduction ,Cbl ,Virulence Factors ,Host–pathogen interaction ,Primary Cell Culture ,chemical and pharmacologic phenomena ,macrophage ,Antiviral Agents ,Article ,Cell Line ,Microbiology ,Protein–protein interaction ,Vaccine Related ,Mycobacterium tuberculosis ,03 medical and health sciences ,Rare Diseases ,Bacterial Proteins ,Biodefense ,Cell Line, Tumor ,ubiquitin ,Tuberculosis ,Animals ,Humans ,Herpesvirus 8 ,Molecular Biology ,Innate immune system ,Prevention ,Macrophages ,HIV ,Cell Biology ,biology.organism_classification ,bacterial infections and mycoses ,Good Health and Well Being ,030104 developmental biology ,RAW 264.7 Cells ,Gene Expression Regulation ,biology.protein ,030217 neurology & neurosurgery ,Developmental Biology ,Mycobacterium - Abstract
Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis - the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl(−/−) macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.
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- 2017
31. CRL4
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Si-Han, Chen, Gwendolyn M, Jang, Ruth, Hüttenhain, David E, Gordon, Dan, Du, Billy W, Newton, Jeffrey R, Johnson, Joseph, Hiatt, Judd F, Hultquist, Tasha L, Johnson, Yi-Liang, Liu, Lily A, Burton, Jordan, Ye, Kurt M, Reichermeier, Robert M, Stroud, Alexander, Marson, Jayanta, Debnath, John D, Gross, and Nevan J, Krogan
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Interleukin-6 ,Ubiquitin-Protein Ligases ,Elongin ,Ubiquitination ,HIV Infections ,Articles ,HEK293 Cells ,Suppressor of Cytokine Signaling 3 Protein ,Proteolysis ,HIV-1 ,vif Gene Products, Human Immunodeficiency Virus ,Humans ,Adaptor Proteins, Signal Transducing ,Signal Transduction - Abstract
Multi‐subunit cullin‐RING ligases (CRLs) are the largest family of ubiquitin E3 ligases in humans. CRL activity is tightly regulated to prevent unintended substrate degradation or autocatalytic degradation of CRL subunits. Using a proteomics strategy, we discovered that CRL4(AMBRA) (1) (CRL substrate receptor denoted in superscript) targets Elongin C (ELOC), the essential adapter protein of CRL5 complexes, for polyubiquitination and degradation. We showed that the ubiquitin ligase function of CRL4(AMBRA) (1) is required to disrupt the assembly and attenuate the ligase activity of human CRL5(SOCS) (3) and HIV‐1 CRL5(VIF) complexes as AMBRA1 depletion leads to hyperactivation of both CRL5 complexes. Moreover, CRL4(AMBRA) (1) modulates interleukin‐6/STAT3 signaling and HIV‐1 infectivity that are regulated by CRL5(SOCS) (3) and CRL5(VIF), respectively. Thus, by discovering a substrate of CRL4(AMBRA) (1), ELOC, the shared adapter of CRL5 ubiquitin ligases, we uncovered a novel CRL cross‐regulation pathway.
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- 2017
32. PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription
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Yang Li, Shumin Yang, Alan D. Frankel, Amit Weiss, Nevan J. Krogan, Jeffrey R. Johnson, Gwendolyn M. Jang, and Tyler B. Faust
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Gene Expression Regulation, Viral ,Transcriptional Activation ,0301 basic medicine ,Transcription, Genetic ,Ubiquitin-Protein Ligases ,RNA polymerase II ,Virus Replication ,Article ,Cell Line ,Jurkat Cells ,03 medical and health sciences ,Ubiquitin ,Transcription (biology) ,RNA interference ,Cell Line, Tumor ,Humans ,Positive Transcriptional Elongation Factor B ,Promoter Regions, Genetic ,P-TEFb ,Polymerase ,Multidisciplinary ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Ubiquitination ,RNA ,Molecular biology ,3. Good health ,Ubiquitin ligase ,Cell biology ,HEK293 Cells ,030104 developmental biology ,HIV-1 ,biology.protein ,RNA, Viral ,tat Gene Products, Human Immunodeficiency Virus ,RNA Polymerase II ,Protein Processing, Post-Translational ,HeLa Cells - Abstract
Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire.
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- 2017
33. Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis
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Gwendolyn M. Jang, Billy W. Newton, Laurence G. Webb, Vinod Balasubramaniam, Laura Satkamp, Gabriel N. Iglesias, Holly Ramage, Tongtong Zhu, Ana Fernandez-Sesma, Nichole Link, Erica Stevenson, Marine J. Petit, Michel Tassetto, Leila Shokat, Raul Andino, Katherine S. Pollard, Jack Taunton, Phillip P. Sharp, Amanda Everitt, Nevan J. Krogan, Hugo J. Bellen, Danielle L. Swaney, Andrea V. Gamarnik, John Von Dollen, Sara Cherry, Ivan Marazzi, Sebastian Aguirre, Priya S. Shah, A. Jeremy Willsey, Michael Shales, Orly Laufman, Ruth Hüttenhain, Tierney Baum, Jeffrey R. Johnson, Shashank Tripathi, and Adolfo García-Sastre
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0301 basic medicine ,Microcephaly ,viruses ,Viral Nonstructural Proteins ,Dengue virus ,medicine.disease_cause ,Medical and Health Sciences ,Zika virus ,Dengue fever ,Dengue ,0302 clinical medicine ,flavivirus ,Interferon ,Protein Interaction Mapping ,2.2 Factors relating to the physical environment ,2.1 Biological and endogenous factors ,microcephaly ,Aetiology ,Tumor ,biology ,Zika Virus Infection ,interferon stimulated genes ,Nuclear Proteins ,virus diseases ,Biological Sciences ,endoplasmic reticulum ,Flavivirus ,Infectious Diseases ,Drosophila ,Infection ,medicine.drug ,PAF1C ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Vaccine Related ,03 medical and health sciences ,proteomics ,Rare Diseases ,ANKLE2 ,Biodefense ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Gene ,dengue virus ,Prevention ,Membrane Proteins ,Zika Virus ,Dengue Virus ,biochemical phenomena, metabolism, and nutrition ,Sec61 ,biology.organism_classification ,medicine.disease ,Virology ,Vector-Borne Diseases ,Emerging Infectious Diseases ,Good Health and Well Being ,Culicidae ,HEK293 Cells ,030104 developmental biology ,Transcription preinitiation complex ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with invivo models, can be used to unravel pathogenic mechanisms.
- Published
- 2018
34. Abstract 3297: A tyrosine kinase interactome reveals network states that guide the use of targeted therapies in cancer
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Xin Zhao, Hsien-Ming Hu, Erik Verschueren, John Jascur, Khyati N. Shah, Nevan J. Krogan, Jeffrey R. Johnson, Sourav Bandyopadhyay, John Von Dollen, Swati Kaushik, and Gwendolyn M. Jang
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Cancer Research ,medicine.medical_treatment ,Cancer ,Biology ,medicine.disease ,medicine.disease_cause ,Oncogene Addiction ,Interactome ,Targeted therapy ,Oncology ,Cancer cell ,medicine ,Cancer research ,KRAS ,Erlotinib ,Tyrosine kinase ,medicine.drug - Abstract
Characterization of the genomic landscapes of cancer patients has provided valuable insights into the key oncogenic drivers and revolutionized the concept of precision treatment of patients. However, a key limitation is that targetable alterations are only found in a small fraction of patients. This is due to the fact that the majority of cancer drugs are developed against specific oncogenes. However, oncogenes do not act in isolation but rather function as a part of complex protein interactions that can alter oncogene activity and dependence. We hypothesize that a systems approach to read the cellular activity of oncogenic proteins by mapping interaction network states of cancer cells can aid in patient stratification for targeted therapy. To identify interaction networks centered on the major class of cancer drug targets, we experimentally mapped protein-protein interaction (PPI) networks of all 90 human tyrosine kinases (TK) using proteomics approach of affinity purification and mass spectrometry. This analysis identified 1,458 high confidence interactors of TK in HEK293 cells. Detailed analyses of this interactome revealed the diverse cellular localizations and novel associations of TK with multiple protein complexes, suggesting a broader functional role in cellular signaling than previously appreciated. To map the cellular activity of TK in cancer patients, we developed a novel computational approach to integrate PPI networks with genomic data from cancer patients profiled in TCGA. Application to lung adenocarcinoma samples identified that activity of EGFR interactors could be used to define an EGFR network state that was highly predictive of the presence of EGFR mutation. Intriguingly, our analysis identified that 23% of EGFR wild-type samples were positive for the EGFR network state, suggesting a role for EGFR in lung cancer beyond EGFR mutant cases. Furthermore, this state was highly predictive of erlotinib sensitivity in EGFR wild-type lung PDX and cell lines. We identified that many KRAS and NF1 mutant NSCLC samples were EGFR network state positive and displayed evidence of EGFR activation identified by RPPA. These results indicate that a network state approach can precisely expand the pool of patients that may benefit from EGFR TK inhibitors (TKi). Finally, we tested whether components of TK networks were critical for their function by performing synthetic lethal RNAi screens in cell lines with mutation in key TK and identified many kinase interactors as drug sensitizers. Our results indicate that integration of high-throughput genomic datasets with the PPI networks provides an effective tool to understand complex oncogenic network states of cancer cells and provides a high-resolution readout of tumor cell dependence. This work provides the most complete interaction map for TK to date and is a valuable resource to probe mechanisms of oncogene addiction to improve the utility of TKi in cancer. Citation Format: Swati Kaushik, Gwendolyn Jang, Hsien-Ming Hu, Khyati Shah, Xin Zhao, John Jascur, John Von Dollen, Erik Verschueren, Jeffrey Johnson, Nevan Krogan, Sourav Bandyopadhyay. A tyrosine kinase interactome reveals network states that guide the use of targeted therapies in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3297.
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- 2018
35. Alternative polyadenylation signals in the 3′ non-coding region of a voltage-gated potassium channel gene are major determinants of mRNA isoform expression
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Bert L. Semler, George A. Gutman, Gwendolyn M. Jang, Brian S. Tanaka, and Alan L. Goldin
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Gene isoform ,Polyadenylation ,Xenopus ,Molecular Sequence Data ,Cleavage and polyadenylation specificity factor ,Biology ,Article ,Mice ,Genetics ,Animals ,Humans ,Protein Isoforms ,Coding region ,RNA, Messenger ,3' Untranslated Regions ,Cells, Cultured ,Regulation of gene expression ,Messenger RNA ,Base Sequence ,Three prime untranslated region ,Alternative splicing ,General Medicine ,Molecular biology ,Rats ,Kv1.4 Potassium Channel ,HeLa Cells - Abstract
We investigated the role of the 3' non-coding region of a mouse voltage-gated potassium channel mRNA (mKv1.4 mRNA) in post-transcriptional regulation of gene expression. In contrast to an earlier report from studies carried out in Xenopus oocytes, we found that 3' non-coding region sequences of mKv1.4 mRNAs did not significantly affect expression of a heterologous reporter RNA in vitro or in mammalian cells/cell lines. Instead, our data revealed a possible role for alternative polyadenylation mediated by distinct determinants approximately 0.2 kb and approximately 1.2 kb downstream of the Kv1.4 coding region. The use of the downstream polyadenylation signal correlated with the synthesis of a larger Kv1.4 mRNA isoform that was more abundantly expressed than the smaller mRNA species, whose expression was regulated by the upstream polyadenylation signal. Our results suggest that the relative strengths of the polyadenylation signals are major determinants of overall Kv1.4 mRNA abundance in cells.
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- 2008
36. An internal ribosome entry site mediates translation of lymphoid enhancer factor-1
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Judith Jimenez, Gwendolyn M. Jang, Marian L. Waterman, and Bert L. Semler
- Subjects
RNA Caps ,Gene isoform ,Untranslated region ,Cytoplasm ,Lymphoid Enhancer-Binding Factor 1 ,Genetic Vectors ,Molecular Sequence Data ,Biology ,Ribosome ,Article ,Dogs ,Cell Line, Tumor ,Chlorocebus aethiops ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,Enhancer ,Molecular Biology ,Conserved Sequence ,Messenger RNA ,Base Sequence ,fungi ,Wnt signaling pathway ,Molecular biology ,DNA-Binding Proteins ,Internal ribosome entry site ,COS Cells ,embryonic structures ,Cattle ,5' Untranslated Regions ,Ribosomes ,HeLa Cells ,Transcription Factors ,Lymphoid enhancer-binding factor 1 - Abstract
The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5′-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5′-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator β-catenin. Truncated LEF-1 lacks the β-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5′-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5′-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5′-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5′-UTR shows that maximal IRES activity requires the majority of the 5′-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.
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- 2005
37. Structurally Distinct Elements Mediate Internal Ribosome Entry within the 5′-Noncoding Region of a Voltage-gated Potassium Channel mRNA
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Lily T. Hoang, George A. Gutman, Gwendolyn M. Jang, Louis Leong, Ping H. Wang, and Bert L. Semler
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5' Flanking Region ,RNase P ,5' flanking region ,Biology ,Biochemistry ,Ribosome ,Cell Line ,Mice ,Ribonucleases ,Protein biosynthesis ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,Molecular Biology ,Messenger RNA ,RNA ,Cell Biology ,Voltage-gated potassium channel ,Molecular biology ,Cell biology ,Gene Expression Regulation ,Polypyrimidine tract ,Potassium Channels, Voltage-Gated ,Protein Biosynthesis ,Mutation ,Kv1.4 Potassium Channel ,Nucleic Acid Conformation ,Ribosomes - Abstract
The approximately 1.2-kb 5'-noncoding region (5'-NCR) of mRNA species encoding mouse Kv1.4, a member of the Shaker-related subfamily of voltage-gated potassium channels, was shown to mediate internal ribosome entry in cells derived from brain, heart, and skeletal muscle, tissues known to express Kv1.4 mRNA species. We also show that the upstream approximately 1.0 kb and the downstream approximately 0.2 kb of the Kv1.4 5'-NCR independently mediated internal ribosome entry; however, separately, these sequences were less efficient in mediating internal ribosome entry than when together in the complete (and contiguous) 5'-NCR. Using enzymatic structure probing, the 3'-most approximately 0.2 kb was predicted to form three distinct stem-loop structures (stem-loops X, Y, and Z) and two defined single-stranded regions (loops Psi and Omega) in the presence and absence of the upstream approximately 1.0 kb. Although the systematic deletion of sequences within the 3'-most approximately 0.2 kb resulted in distinct changes in expression, enzymatic structure probing indicated that local RNA folding was not completely altered. Structure probing analysis strongly suggested an interaction between stem-loop X and a downstream polypyrimidine tract; however, opposing changes in activity were observed when sequences within these two regions were independently deleted. Moreover, deletions correlating with positive as well as negative changes in expression altered RNase cleavage within stem-loop X, indicating that this structure may be an integral element. Therefore, these findings indicate that Kv1.4 expression is mediated through a complex interplay between many distinct RNA regions.
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- 2004
38. Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA
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Elizabeth Quezada, David S. Booth, Alan D. Frankel, Gwendolyn M. Jang, Tyler B. Faust, Iván D'Orso, and Alexander W. Pastuszak
- Subjects
Gene Expression Regulation, Viral ,Models, Molecular ,Transcriptional Activation ,1.1 Normal biological development and functioning ,Molecular Sequence Data ,RNA polymerase II ,HIV Infections ,Biology ,Medical and Health Sciences ,Cell Line ,Small Nuclear ,Transcription (biology) ,Models ,7SK RNA ,Genetics ,Humans ,snRNP ,Positive Transcriptional Elongation Factor B ,Viral ,Amino Acid Sequence ,P-TEFb ,Molecular Biology ,Transcription factor ,Conserved Sequence ,Ribonucleoprotein ,HIV Long Terminal Repeat ,Molecular ,HIV ,RNA-Binding Proteins ,Cell Biology ,Articles ,Biological Sciences ,Ribonucleoproteins, Small Nuclear ,Molecular biology ,Cell biology ,Ribonucleoproteins ,Gene Expression Regulation ,Generic Health Relevance ,Mutation ,biology.protein ,RNA ,RNA, Viral ,tat Gene Products, Human Immunodeficiency Virus ,tat Gene Products ,Infection ,Human Immunodeficiency Virus ,Developmental Biology ,Transcription Factors - Abstract
Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase. Here, we show that P-TEFb artificially recruited to the nascent transcript is not competent for transcription but rather remains inactive due to its assembly with the 7SK snRNP. Tat supplied in trans is able to displace the kinase inhibitor Hexim1 from the snRNP and activate P-TEFb, thereby uncoupling Tat requirements for kinase activation and TAR binding. By combining comprehensive mutagenesis of Tat with multiple cell-based reporter assays that probe the activity of Tat in different arrangements, we genetically defined a transition step in which preassembled Tat:P-TEFb complexes switch to TAR. We propose that a conserved network of residues in Tat has evolved to control this transition and thereby switch the host elongation machinery to viral transcription.
- Published
- 2012
39. Purification and characterization of HIV–human protein complexes
- Author
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Gwendolyn M. Jang, Stefanie Jäger, Jason D Fernandes, Tom Alber, Qiang Zhou, Alan D. Frankel, Seemay Chou, Peter Cimermancic, Nevan J. Krogan, Joshua Kane, Nanhai He, Natali Gulbahce, and Iván D'Orso
- Subjects
Recombinant Fusion Proteins ,Human Immunodeficiency Virus Proteins ,Human immunodeficiency virus (HIV) ,HIV Infections ,Computational biology ,Genome, Viral ,Host-Derived Cellular Factors ,Biology ,Virus-host interaction ,medicine.disease_cause ,Proteomics ,Transfection ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Article ,Chromatography, Affinity ,Jurkat Cells ,Viral entry ,Protein Interaction Mapping ,medicine ,Humans ,Cloning, Molecular ,Molecular Biology ,Extramural ,Virology ,Viral genomes ,Host-Pathogen Interactions ,HIV-1 ,Systematic mapping ,Subcellular Fractions - Abstract
To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.
- Published
- 2010
40. Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis
- Author
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David M. Brown, Bert L. Semler, Steven E. Kauder, Christopher T. Cornell, Vincent R. Racaniello, and Gwendolyn M. Jang
- Subjects
viruses ,Immunology ,RNA-dependent RNA polymerase ,Mice, Transgenic ,Biology ,medicine.disease_cause ,Virus Replication ,Microbiology ,Virus ,Mice ,Neuroblastoma ,Virology ,medicine ,Protein biosynthesis ,Tumor Cells, Cultured ,Animals ,Humans ,3' Untranslated Regions ,Virulence ,Poliovirus ,Viral translation ,Viral protein processing ,RNA ,Membrane Proteins ,Molecular biology ,Virus-Cell Interactions ,Viral replication ,Insect Science ,Protein Biosynthesis ,RNA, Viral ,Receptors, Virus ,Gene Deletion ,HeLa Cells ,Poliomyelitis - Abstract
We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.
- Published
- 2004
41. Solid-Phase Fluorescence Immunoassay for the Detection of Antibiotic Residues in Milk
- Author
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Marcos Piani, Richard Michele Rocco, Krista K. Hara-Mikami, Danton Kai-Yu Leung, Amit Kumar, Shanta Kharadia, Gwendolyn M. Jang, Larry Sheldon Jang, and Caroline Yu
- Subjects
Chromatography ,medicine.drug_class ,Chemistry ,Phase (matter) ,Antibiotics ,medicine ,Fluorescence immunoassay - Published
- 1996
42. The HIV-1 Tat protein recruits a ubiquitin ligase to reorganize the 7SK snRNP for transcriptional activation
- Author
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Tyler B Faust, Yang Li, Curtis W Bacon, Gwendolyn M Jang, Amit Weiss, Bhargavi Jayaraman, Billy W Newton, Nevan J Krogan, Iván D'Orso, and Alan D Frankel
- Subjects
non-degradative ubiquitination ,transcription elongation ,nuclear import ,7SK snRNP ,host-pathogen interactions ,HIV-1 ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
The HIV-1 Tat protein hijacks P-TEFb kinase to activate paused RNA polymerase II (RNAP II) at the viral promoter. Tat binds additional host factors, but it is unclear how they regulate RNAP II elongation. Here, we identify the cytoplasmic ubiquitin ligase UBE2O as critical for Tat transcriptional activity. Tat hijacks UBE2O to ubiquitinate the P-TEFb kinase inhibitor HEXIM1 of the 7SK snRNP, a fraction of which also resides in the cytoplasm bound to P-TEFb. HEXIM1 ubiquitination sequesters it in the cytoplasm and releases P-TEFb from the inhibitory 7SK complex. Free P-TEFb then becomes enriched in chromatin, a process that is also stimulated by treating cells with a CDK9 inhibitor. Finally, we demonstrate that UBE2O is critical for P-TEFb recruitment to the HIV-1 promoter. Together, the data support a unique model of elongation control where non-degradative ubiquitination of nuclear and cytoplasmic 7SK snRNP pools increases P-TEFb levels for transcriptional activation.
- Published
- 2018
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