DOCSLIB.ORG

Regulation of Hsp90 Client Proteins by a Cullin5-RING E3 Ubiquitin Ligase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of Hsp90 Client Proteins by a Cullin5-RING E3 Ubiquitin Ligase Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase Elana S. Ehrlicha, Tao Wanga, Kun Luoa, Zuoxiang Xiaoa, Anna Maria Niewiadomskaa, Tara Martineza, Wanping Xub, Len Neckersb, and Xiao-Fang Yua,1 aDepartment of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; and bUrologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892 Edited by William J. Muller, McGill University, Montreal, Canada, and accepted by the Editorial Board September 23, 2009 (received for review October 22, 2008) We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the Here we demonstrate that Cul5 regulates Hsp90 clients. Our heat shock protein 90 (Hsp90) chaperone complex. Hsp90 partici- results indicate that Cul5 interacts with the Hsp90 chaperone pates in the folding of its client proteins into their functional complex and the Hsp90 client ErbB2 and demonstrate that Cul5 conformation. Many Hsp90 clients have been reported to be is recruited to the site of ErbB2 on the plasma membrane, aberrantly expressed in a number of cancers. We demonstrate Cul5 thereby inducing its polyubiquitination and proteasome- interaction with members of the Hsp90 chaperone complex as well mediated degradation. Other Hsp90 client proteins, such as as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the HIF1-␣, were also regulated by Cul5. We observed Cul5- site of ErbB2 at the plasma membrane and subsequent induction of mediated degradation of ErbB2 to occur in the absence of the polyubiquitination and proteasomal degradation. We also dem- traditional Cul5 adaptors ElonginB and ElonginC, suggesting onstrate Cul5 involvement in regulation of another Hsp90 client, that a component of the Hsp90 chaperone complex may serve Hif-1␣. We observed Cul5 degradation of ErbB2 to occur indepen- this function. This is an example of a link between Hsp90 and the dently of ElonginB-ElonginC function. The involvement of Cul5 in Cullin family of E3 ubiquitin ligases. This is also a report of an Hsp90 client regulation has implications in the effectiveness of ElonginB-ElonginC-independent Cul5 E3 ubiquitin ligase. Hsp90 targeted chemotherapy, which is currently undergoing Hsp90 is a well-established target for chemotherapy (16). Our CELL BIOLOGY clinical trials. The link between Cul5 and Hsp90 client regulation data now provide an explanation for the decrease or loss of Cul5 may represent an avenue for cancer drug development. expression that is observed in a number of cancers (17). Our data also suggest that Cul5 levels could potentially influence suscep- chaperone ͉ erbb2 tibility to certain cancers and the effectiveness of anti-cancer treatment with geldanamycin or its derivatives, which are cur- rently in human clinical trials. he regulation of client proteins by heat shock protein 90 T(Hsp90) plays an important role in critical cellular processes Results such as cell cycle control and apoptosis. Dysregulation is linked to Cul5 Interacts with the Hsp90 Chaperone Complex. While the cellular cancer and neurological diseases (1, 2). Hsp90 is a molecular function of Cul5 is poorly understood, it is known to be hijacked chaperone responsible for the correct folding of proteins, allowing by the HIV type 1 (HIV-1) Vif protein to suppress the antiviral them to attain their proper functional conformations (3). Many protein Apobec3G (A3G) (18–20) and by adenovirus E4orf6 client proteins of Hsp90 are overexpressed oncogenes that are and E1B55K to degrade p53, Mre11, and DNA ligase IV (18, critical for the transformed phenotype observed in tumors (4–6). 21–25). To identify potential cellular partners of the Cul5 E3 Clients of Hsp90 are also regulated by the ubiquitin/proteasome ligase, we immunoprecipitated HA-tagged Cul5 and identified system (7). However, the identity of the cellular E3 ubiquitin proteins that specifically co-immunoprecipitated with Cul5-HA ligase(s) that regulate(s) Hsp90 client proteins is still elusive. but not with a cmyc-tagged Cul5 control protein. In repeated The Cullin family of RING E3 ubiquitin ligases are modular experiments, Cul5-HA, but not Cul5-myc, co-precipitated enzymes that act as a scaffolding to bring a specific substrate Hsp70, as indicated by mass spectrometry analysis (Fig. 1A). within close proximity to the E2 ubiquitin conjugating enzyme, Interaction of Hsp70 with Cul5-HA but not Cul5-myc was thereby facilitating ubiquitination and subsequent proteasomal confirmed by Western blotting with an anti-Hsp70 antibody (Fig. degradation (8, 9). There are seven known human Cullin pro- 1B). We also observed an interaction between Hsp90 and teins, Cul1, 2, 3, 4a, 4b, 5, and 7, with diverse functions from cell Cul5-HA (Fig. 1B), suggesting that Cul5 may be involved in the cycle regulation, to DNA repair, to regulation of developmental regulation of Hsp90 client proteins. Hsp90 is a chaperone that is processes. Substrate specificity is determined by the combina- required for the maturation and function of a number of classes torial nature of E3 ligase assembly. In the case of Cul1, diversity of proteins, namely receptors, kinases, and transcription factors is achieved by the assembly of different F-box substrate receptor (7, 26). Hsp90 assembles with a number of co-chaperones, proteins with Cul1 through a single Skp1 adaptor protein (10). including Hsp70, and coordinates the maturation of its client Each F-box protein determines the specificity of Cul1 substrates. proteins. Client protein maturation occurs via a dynamic ATP- Cul3 is unique in having one protein with two domains, one dependent process (7). ATP hydrolysis or inhibition of Hsp90 functioning as an adaptor forming an interface with Cullin, and the other acting as a substrate receptor (9). Cul2 and 5 are interesting in that they both use ElonginB-C Author contributions: E.S.E. designed research; E.S.E., T.W., K.L., Z.X., and A.M.N. per- formed research; T.M., W.X., and L.N. contributed new reagents/analytic tools; E.S.E., L.N., adaptor proteins, through which they bind a SOCS box contain- and X.-F.Y. analyzed data; and E.S.E. wrote the paper. ing substrate receptor (8). Whether a substrate receptor recruits The authors declare no conflict of interest. Cul2 or Cul5 depends on an additional Cullin binding interface. This article is a PNAS Direct Submission. W.J.M. is a guest editor invited by the Editorial In the case of cellular substrate receptors, Cullin selection is Board. determined by the presence of a VHL or SOCS box motif (11), 1To whom correspondence should be addressed. E-mail: [email protected]. in the case of HIV and SIV, a zinc-stabilized helix mediates Cul5 This article contains supporting information online at www.pnas.org/cgi/content/full/ selection (12–15). 0810571106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810571106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 IP HA 293T cells U87 cells also confirmed in U87cells (Fig. 1C). Immunoprecipitation of A kD B C Cl5Cu - Cul5Cul5- endogenous Hsp70 also co-precipitated endogenous Cul5 (Fig. Cl5Cu Hsp90- Hsp90- Hsp70 1D). Conversely, immunoprecipitation of endogenous Cul5 also 66 Hsp70- Hsp70- co-precipitated endogenous Hsp90 and Hsp70 (Fig. 1E). 121 2 121 2 Hsp90 has been shown to be expressed at high levels, along + - Cul5-HA + - -45 Cul5-HA with a number of its client proteins, in a variety of cancers and 1 2 Cul5-cmyc -+ Cul5-cmyc -+ has therefore become a promising target for intervention (7). Cul5-HA + - IP Cul5:Anti-HA IP Cul5:Anti-HA Cul5-cmyc -++ Treatment of cells with the benzoquinone ansamycin antibiotic Coomassie stain Cell lysate geldanamycin (GA) or its analogs results in the proteasomal EFCuCl5 - degradation of Hsp90 client proteins (16). GA is an ATPase D Hsp90- Ab α : IgG ErbB2 inhibitor that binds the nucleotide-binding pocket with an af- IP: Hsp70 IgG Hsp70- Erbb2- finity greater than that of ATP or ADP, shifting the chaperone Abα : IgG Cul5 Cul5- Cul5Cul5- complex into a conformation that favors client-protein degra- Hsp70- Cul5- 1 2 3 1 2 Hsp90- dation (27). ErbB2 is an Hsp90 client that has been extensively GA -+ - IP Endogenous Hsp70 Hsp70- characterized. This receptor tyrosine kinase is overexpressed in 1231 2 3 IP ErbB2 approximately 30% of breast cancers and is required for tumor GA -+ - cell proliferation. Treatment of ErbB2- overexpressing cells with IP Endogenous Cul5 GA or its analogs results in rapid proteasomal degradation of Fig. 1. Cul5 interacts with the Hsp90-Hsp70 chaperone complex. (A) Cul5-HA ErbB2 (28, 29). While the U-box-containing E3 ligase, CHIP, and Cul5-cmyc were immunoprecipitated with anti-HA-conjugated agarose has been implicated in the degradation of ErbB2 via Hsp/Hsc70, beads. The eluates were analyzed by SDS-PAGE and Coomassie staining, and GA-mediated ErbB2 degradation still occurs in CHIPϪ/Ϫ cells, bands were identified by mass spectrometry. (B and C) Cul5-HA and Cul5-cmyc suggesting that an additional E3 ligase is also involved in were immunoprecipitated with anti-HA-conjugated agarose beads in (B) 293T GA-mediated degradation of ErbB2 (29). and (C) U87 cells. Eluates were analyzed by SDS-PAGE and Western blotting with antibodies against HA, Hsp90, and Hsp70. (D) 293T cell lysates were To examine the possibility that Cul5, interacting with the incubated with antibody against Hsp70 or IgG and protein A/G agarose. Hsp90 chaperone complex, might serve as an E3 ligase to Protein complexes were immunoprecipitated and analyzed by SDS-PAGE and degrade Hsp90 client proteins, we characterized the interaction Western blotting with antibodies against Hsp70 and Cul5. (E) 293T cells were of Cul5 with ErbB2. We treated 293T cells transfected with treated with GA or DMSO as indicated. Protein complexes were immunopre- ErbB2 or empty vector with 2 ␮M GA or control DMSO and 5 cipitated with antibodies against Cul5 and protein A/G agarose.
Load more

Recommended publications
  • Transcriptome Analyses of Rhesus Monkey Pre-Implantation Embryos Reveal A
    Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptome analyses of rhesus monkey pre-implantation embryos reveal a reduced capacity for DNA double strand break (DSB) repair in primate oocytes and early embryos Xinyi Wang 1,3,4,5*, Denghui Liu 2,4*, Dajian He 1,3,4,5, Shengbao Suo 2,4, Xian Xia 2,4, Xiechao He1,3,6, Jing-Dong J. Han2#, Ping Zheng1,3,6# Running title: reduced DNA DSB repair in monkey early embryos Affiliations: 1 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 2 Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 3 Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China 4 University of Chinese Academy of Sciences, Beijing, China 5 Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China 6 Primate Research Center, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, China * Xinyi Wang and Denghui Liu contributed equally to this work 1 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press # Correspondence: Jing-Dong J. Han, Email: [email protected]; Ping Zheng, Email: [email protected] Key words: rhesus monkey, pre-implantation embryo, DNA damage 2 Downloaded from genome.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press ABSTRACT Pre-implantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA) and cell fate commitment.
    [Show full text]
  • 538.Full.Pdf
    The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation This information is current as of September 24, 2021. Amrita Kumar, Huixia Wu, Lauren S. Collier-Hyams, Young-Man Kwon, Jason M. Hanson and Andrew S. Neish J Immunol 2009; 182:538-546; ; doi: 10.4049/jimmunol.182.1.538 http://www.jimmunol.org/content/182/1/538 Downloaded from References This article cites 75 articles, 30 of which you can access for free at: http://www.jimmunol.org/content/182/1/538.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1 Amrita Kumar,* Huixia Wu,* Lauren S. Collier-Hyams,* Young-Man Kwon,* Jason M.
    [Show full text]
  • Filamins but Not Janus Kinases Are Substrates of the Asb2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells
    Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells Isabelle Lamsoul1,2, Monique Erard1,2, Peter F. M. van der Ven3, Pierre G. Lutz1,2* 1 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne BP64182, F-31077 Toulouse, France, 2 Universite´ de Toulouse, UPS, IPBS, F- 31077 Toulouse, France, 3 Department of Molecular Cell Biology, Institute of Cell Biology, University of Bonn, Bonn, Germany Abstract The ASB2a protein is the specificity subunit of an E3 ubiquitin ligase complex involved in hematopoietic differentiation and is proposed to exert its effects by regulating the turnover of specific proteins. Three ASB2a substrates have been described so far: the actin-binding protein filamins, the Mixed Lineage Leukemia protein, and the Janus kinases 2 and 3. To determine the degradation of which substrate drives ASB2a biological effects is crucial for the understanding of ASB2a functions in hematopoiesis. Here, we show that neither endogenous nor exogenously expressed ASB2a induces degradation of JAK proteins in hematopoietic cells. Furthermore, we performed molecular modeling to generate the first structural model of an E3 ubiquitin ligase complex of an ASB protein bound to one of its substrates. Citation: Lamsoul I, Erard M, van der Ven PFM, Lutz PG (2012) Filamins but Not Janus Kinases Are Substrates of the ASB2a Cullin-Ring E3 Ubiquitin Ligase in Hematopoietic Cells. PLoS ONE 7(8): e43798. doi:10.1371/journal.pone.0043798 Editor: Zhengqi Wang, Emory University, United States of America Received March 21, 2012; Accepted July 26, 2012; Published August 20, 2012 Copyright: ß 2012 Lamsoul et al.
    [Show full text]
  • Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration
    5668 • The Journal of Neuroscience, April 21, 2010 • 30(16):5668–5676 Cellular/Molecular Cullin 5 Regulates Cortical Layering by Modulating the Speed and Duration of Dab1-Dependent Neuronal Migration Sergi Simó, Yves Jossin, and Jonathan A. Cooper Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Themultilayeredmammalianneocortexdevelopsbythecoordinatedimmigrationanddifferentiationofcellsthatareproducedatdistant sites. Correct layering requires an extracellular protein, Reelin (Reln), an intracellular signaling molecule, Disabled-1 (Dab1), and an E3 ubiquitinligase,Cullin-5(Cul5).RelnactivatesDab1,whichisthendegradedbyCul5.HerewetestwhetherCul5regulatesneuronlayering by affecting Dab1 stability or other mechanisms. We find that a stabilized mutant Dab1, which resists Cul5-dependent degradation, causes a similar phenotype to Cul5 deficiency. Moreover, Cul5 has no effect when Dab1 is absent. The effects of Cul5 and Dab1 are cell autonomous, and Cul5 regulates movement of early as well as late cortical neurons. Removing Cul5 increases the speed at which neurons migrate through the cortical plate by reducing the time spent stationary and increasing the speed of individual steps. These results show thatCul5regulatesneuronlayeringbystimulatingDab1degradationandthatCul5controlsmigrationspeedandstoppingpoint,andthey demonstrate the importance of negative feedback in signaling during cortical development. Introduction (Pinto-Lord et al., 1982; Dulabon et al., 2000; Sanada et al., 2004). The mammalian
    [Show full text]
  • Temporal Proteomic Analysis of HIV Infection Reveals Remodelling of The
    1 1 Temporal proteomic analysis of HIV infection reveals 2 remodelling of the host phosphoproteome 3 by lentiviral Vif variants 4 5 Edward JD Greenwood 1,2,*, Nicholas J Matheson1,2,*, Kim Wals1, Dick JH van den Boomen1, 6 Robin Antrobus1, James C Williamson1, Paul J Lehner1,* 7 1. Cambridge Institute for Medical Research, Department of Medicine, University of 8 Cambridge, Cambridge, CB2 0XY, UK. 9 2. These authors contributed equally to this work. 10 *Correspondence: [email protected]; [email protected]; [email protected] 11 12 Abstract 13 Viruses manipulate host factors to enhance their replication and evade cellular restriction. 14 We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a 15 comprehensive time course analysis of >6,500 viral and cellular proteins during HIV 16 infection. To enable specific functional predictions, we categorized cellular proteins regulated 17 by HIV according to their patterns of temporal expression. We focussed on proteins depleted 18 with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be 19 necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the 20 B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). 21 Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent 22 hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. 23 The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral 2 24 lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and 25 conserved Vif function.
    [Show full text]
  • Chemotherapy Induces NEDP1-Mediated Destabilization of MDM2
    Oncogene (2010) 29, 297–304 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc SHORT COMMUNICATION Chemotherapy induces NEDP1-mediated destabilization of MDM2 IR Watson1,2,BKLi1,2, O Roche1, A Blanch2, M Ohh1 and MS Irwin1,2,3 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 2Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada and 3Department of Paediatrics and Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada MDM2 is an E3 ligase that promotes ubiquitin-mediated In response to DNA damage, p53 becomes phos- destruction of p53. Cellular stresses such as DNA damage phorylated by several kinases within the MDM2- can lead to p53 activation due in part to MDM2 binding domain, which prevents MDM2–p53 interac- destabilization. Here, we show that the stability of tion (Bode and Dong, 2004). The stabilization of p53 MDM2 is regulated by an ubiquitin-like NEDD8 pathway then leads to DNA repair, cell cycle arrest, senescence or and identify NEDP1 as a chemotherapy-induced isopepti- apoptosis. Recent studies have shown that MDM2 is dase that deneddylates MDM2, resulting in MDM2 destabilized in response to DNA damage, which promotes destabilization concomitant with p53 activation. Concor- p53 activation (Stommel and Wahl, 2004; Meulmeester dantly, RNAi-mediated knockdown of endogenous et al., 2005). NEDD8 is a ubiquitin-like protein that NEDP1 blocked diminution of MDM2 levels and regulates protein function through covalent modification increased chemoresistance of tumor cells. These findings of substrates such as Cullins, BCA3, EGFR, ribosomal unveil the regulation of MDM2 stability through NEDP1 L11 protein, VHL, p73 and p53 (Xirodimas, 2008).
    [Show full text]
  • E3 Ubiquitin Ligase Cullin-5 Modulates Multiple Molecular and Cellular Responses to Heat Shock Protein 90 Inhibition in Human Cancer Cells
    E3 ubiquitin ligase Cullin-5 modulates multiple molecular and cellular responses to heat shock protein 90 inhibition in human cancer cells Rahul S. Samant, Paul A. Clarke, and Paul Workman1 Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 3, 2014 (received for review December 24, 2013) The molecular chaperone heat shock protein 90 (HSP90) is required Given the link between CUL5 and the HSP90 inhibitor- for the activity and stability of its client proteins. Pharmacologic induced degradation of ERBB2 (12), we have investigated the inhibition of HSP90 leads to the ubiquitin-mediated degradation of role of Cullin-RING ligases with respect to HSP90’s protein kinase clients, particularly activated or mutant oncogenic protein kinases. clients in human cancer cell lines. Our initial focused siRNA Client ubiquitination occurs via the action of one or more E3 screen of 28 Cullin-RING ligase family members identified five ubiquitin ligases. We sought to identify the role of Cullin-RING fam- genes, including CUL5, that were required for ERBB2 degra- ily E3 ubiquitin ligases in the cellular response to HSP90 inhibition. dation following treatment with 17-AAG—which we use here as Through a focused siRNA screen of 28 Cullin-RING ligase family a representative HSP90 inhibitor and chemical tool to promote members, we found that CUL5 and RBX2 were required for degra- client protein turnover. We go on to show for the first time to our dation of several HSP90 clients upon treatment of human cancer knowledge that RNAi silencing of CUL5 reduces the 17-AAG– cells with the clinical HSP90 inhibitor 17-AAG.
    [Show full text]
  • "The Genecards Suite: from Gene Data Mining to Disease Genome Sequence Analyses". In: Current Protocols in Bioinformat
    The GeneCards Suite: From Gene Data UNIT 1.30 Mining to Disease Genome Sequence Analyses Gil Stelzer,1,5 Naomi Rosen,1,5 Inbar Plaschkes,1,2 Shahar Zimmerman,1 Michal Twik,1 Simon Fishilevich,1 Tsippi Iny Stein,1 Ron Nudel,1 Iris Lieder,2 Yaron Mazor,2 Sergey Kaplan,2 Dvir Dahary,2,4 David Warshawsky,3 Yaron Guan-Golan,3 Asher Kohn,3 Noa Rappaport,1 Marilyn Safran,1 and Doron Lancet1,6 1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 2LifeMap Sciences Ltd., Tel Aviv, Israel 3LifeMap Sciences Inc., Marshfield, Massachusetts 4Toldot Genetics Ltd., Hod Hasharon, Israel 5These authors contributed equally to the paper 6Corresponding author GeneCards, the human gene compendium, enables researchers to effectively navigate and inter-relate the wide universe of human genes, diseases, variants, proteins, cells, and biological pathways. Our recently launched Version 4 has a revamped infrastructure facilitating faster data updates, better-targeted data queries, and friendlier user experience. It also provides a stronger foundation for the GeneCards suite of companion databases and analysis tools. Improved data unification includes gene-disease links via MalaCards and merged biological pathways via PathCards, as well as drug information and proteome expression. VarElect, another suite member, is a phenotype prioritizer for next-generation sequencing, leveraging the GeneCards and MalaCards knowledgebase. It au- tomatically infers direct and indirect scored associations between hundreds or even thousands of variant-containing genes and disease phenotype terms. Var- Elect’s capabilities, either independently or within TGex, our comprehensive variant analysis pipeline, help prepare for the challenge of clinical projects that involve thousands of exome/genome NGS analyses.
    [Show full text]
  • A Cullin-RING Ubiquitin Ligase Promotes Thermotolerance As Part of the Intracellular Pathogen Response in Caenorhabditis Elegans
    A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans Johan Paneka, Spencer S. Ganga, Kirthi C. Reddya, Robert J. Luallena, Amitkumar Fulzelea, Eric J. Bennetta, and Emily R. Troemela,1 aDivision of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093 Edited by Gary Ruvkun, Massachusetts General Hospital, Boston, MA, and approved February 24, 2020 (received for review October 22, 2019) Intracellular pathogen infection leads to proteotoxic stress in host most common cause of infection of C. elegans in the wild, with organisms. Previously we described a physiological program in the Nematocida parisii being the most commonly found micro- nematode Caenorhabditis elegans called the intracellular patho- sporidian species in C. elegans (14). N. parisii replicates inside gen response (IPR), which promotes resistance to proteotoxic the C. elegans intestine, and the infection is associated with stress and appears to be distinct from canonical proteostasis path- hallmarks of perturbed proteostasis in the host, such as the ways. The IPR is controlled by PALS-22 and PALS-25, proteins of formation of large ubiquitin aggregates in the intestine (12). unknown biochemical function, which regulate expression of Interestingly, the host transcriptional response to this infec- genes induced by natural intracellular pathogens. We previ- tion is very similar to the host transcriptional response to ously showed that PALS-22 and PALS-25 regulate the mRNA ex- another natural intracellular pathogen of the C. elegans in- pression of the predicted ubiquitin ligase component cullin cul-6, testine, the Orsay virus (12, 15, 16).
    [Show full text]
  • CRL5-Dependent Regulation of the Small Gtpases ARL4C and ARF6 Controls Hippocampal Morphogenesis
    CRL5-dependent regulation of the small GTPases ARL4C and ARF6 controls hippocampal morphogenesis Jisoo S. Hana,1, Keiko Hinoa,1, Wenzhe Lia, Raenier V. Reyesa, Cesar P. Canalesa,2, Adam M. Miltnera, Yasmin Haddadia, Junqing Sunb, Chao-Yin Chenb, Anna La Torrea, and Sergi Simóa,3 aDepartment of Cell Biology and Human Anatomy, University of California, Davis, CA 95616; and bDepartment of Pharmacology, University of California, Davis, CA 95616 Edited by Mary E. Hatten, Rockefeller University, New York, NY, and approved August 3, 2020 (received for review February 14, 2020) The small GTPase ARL4C participates in the regulation of cell migra- Importantly, CRL5 substrate adaptors are dynamically regulated tion, cytoskeletal rearrangements, and vesicular trafficking in epithe- during development, directing CRL5 activity to specific substrates lial cells. The ARL4C signaling cascade starts by the recruitment of the at different times and areas in the CNS (19). In the neocortex, ARF–GEF cytohesins to the plasma membrane, which, in turn, bind CRL5 opposes projection neuron migration by down-regulating and activate the small GTPase ARF6. However, the role of ARL4C– the Reelin/DAB1 signaling pathway as migrating projection neu- cytohesin–ARF6 signaling during hippocampal development remains rons reach the top of the cortical plate (18, 20, 21). In the CNS, elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/RBX2 Reelin/DAB1 signaling participates in several developmental (CRL5) controls the stability of ARL4C and its signaling effectors to processes, including neuron migration, dendritogenesis, axo- regulate hippocampal morphogenesis. Both RBX2 knockout and genesis, and synaptogenesis (22–24). Moreover, CRL5 also con- Cullin 5 knockdown cause hippocampal pyramidal neuron mislocali- trols levels of the tyrosine kinase FYN, which exerts important zation and development of multiple apical dendrites.
    [Show full text]
  • Transcriptional Elongation Factor Elongin a Regulates Retinoic Acid-Induced Gene Expression During Neuronal Differentiation
    Cell Reports Report Transcriptional Elongation Factor Elongin A Regulates Retinoic Acid-Induced Gene Expression during Neuronal Differentiation Takashi Yasukawa,1 Shachi Bhatt,3,4 Tamotsu Takeuchi,6 Junya Kawauchi,7 Hidehisa Takahashi,3,8 Aya Tsutsui,1 Takuya Muraoka,1 Makoto Inoue,7 Masayuki Tsuda,2 Shigetaka Kitajima,7 Ronald C. Conaway,3,5 Joan W. Conaway,3,5 Paul A. Trainor,3,4 and Teijiro Aso1,* 1Department of Functional Genomics 2Institute for Laboratory Animal Research Kochi Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan 3Stowers Institute for Medical Research, Kansas City, MO 64110, USA 4Department of Anatomy and Cell Biology 5Department of Biochemistry and Molecular Biology Kansas University Medical Center, Kansas City, KS 66160, USA 6Department of Immunopathology, Gifu University School of Medicine, 1-1 Yanagido, Gifu, Gifu 501-1194, Japan 7Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113-8510, Japan 8Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638, Japan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2012.09.031 SUMMARY target for gene regulation by a diverse collection of elongation factors that promote efficient elongation of transcripts by pol II Elongin A increases the rate of RNA polymerase II in vitro (Saunders et al., 2006). Elongin is a member of a family (pol II) transcript elongation by suppressing transient of elongation factors that can increase the overall rate of RNA pausing by the enzyme. Elongin A also acts as chain elongation by decreasing the frequency and/or duration a component of a cullin-RING ligase that can target of transient pausing by pol II as it traverses the DNA template stalled pol II for ubiquitylation and proteasome- (Conaway et al., 2000; Shilatifard et al., 2003).
    [Show full text]
  • Cancer Cell Responses to Hsp70 Inhibitor JG-98: Comparison With
    www.nature.com/scientificreports Correction: Author Correction OPEN Cancer cell responses to Hsp70 inhibitor JG-98: Comparison with Hsp90 inhibitors and fnding Received: 17 March 2017 Accepted: 18 October 2017 synergistic drug combinations Published: xx xx xxxx Julia A. Yaglom1, Yongmei Wang2, Amy Li1, Zhenghu Li3, Stephano Monti 1, Ilya Alexandrov4, Xiongbin Lu3 & Michael Y. Sherman5 Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer activities afecting both cancer cells and tumor-associated macrophages. They disrupt Hsp70 interaction with a co-chaperone Bag3 and afect signaling pathways important for cancer development. Due to a prior report that depletion of Hsp70 causes similar responses as depletion of Hsp90, interest to Hsp70 inhibitors as drug prototypes is hampered by potential similarity of their efects to efects of Hsp90 inhibitors. Here, using the Connectivity Map platform we demonstrate that physiological efects of JG-98 are dissimilar from efects of Hsp90 inhibitors, thus justifying development of these compounds. Using gene expression and ActivSignal IPAD platform, we identifed pathways modulated by JG-98. Some of these pathways were afected by JG-98 in Bag3-dependent (e.g. ERK) and some in Bag3- independent manner (e.g. Akt or c-myc), indicating multiple efects of Hsp70 inhibition. Further, we identifed genes that modulate cellular responses to JG-98, developed approaches to predict potent combinations of JG-98 with known drugs, and demonstrated that inhibitors of proteasome, RNApol, Akt and RTK synergize with JG-98. Overall, here we established unique efects of novel Hsp70 inhibitors on cancer cell physiology, and predicted potential drug combinations for pre-clinical development.
    [Show full text]