Adenovirus 12 E4orf6 Inhibits ATR Activation by Promoting TOPBP1 Degradation

Total Page:16

File Type:pdf, Size:1020Kb

Adenovirus 12 E4orf6 Inhibits ATR Activation by Promoting TOPBP1 Degradation Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation Andrew N. Blackforda,1,2, Rakesh N. Patela, Natalie A. Forrestera, Kathrin Theilb, Peter Groitlb, Grant S. Stewarta, A. Malcolm R. Taylora, Iain M. Morganc, Thomas Dobnerb, Roger J. A. Granda,2, and Andrew S. Turnella,2 aSchool of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom; bHeinrich Pette Institute for Experimental Virology and Immunology, 20251 Hamburg, Germany; and cInstitute of Comparative Medicine, University of Glasgow Faculty of Veterinary Medicine, Glasgow G61 1QH, United Kingdom Edited* by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved May 14, 2010 (received for review December 22, 2009) Activation of the cellular DNA damage response is detrimental to Adenovirus type 5 (Ad5) is a linear dsDNA tumor virus that adenovirus (Ad) infection. Ad has therefore evolved a number of provides a useful model system for studying the DNA damage strategies to inhibit ATM- and ATR-dependent signaling pathways response, as ATM and ATR signaling pathways are inhibited during infection. Recent work suggests that the Ad5 E4orf3 protein during infection (11). Ad5 E1B-55K and E4orf6 proteins prevent prevents ATR activation through its ability to mislocalize the MRN ATM activation by promoting the ubiquitin-dependent protea- complex. Here we provide evidence to indicate that Ad12 has somal degradation of MRN (11, 12). Cullin-RING ubiquitin evolved a different strategy from Ad5 to inhibit ATR. We show that ligases (CRLs) are multiprotein complexes required for the pol- Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to yubiquitylation and proteasomal degradation of a large number promote the proteasomal degradation of the ATR activator protein of cellular proteins, to control many diverse processes such as cell topoisomerase-IIβ–binding protein 1 (TOPBP1). Ad12 also uses this cycle progression, limb patterning, and glucose sensing (13). complex to degrade p53 during infection, in contrast to Ad5, which During Ad5 infection, E1B-55K and E4orf6 associate with a CRL requires a CUL5-based ubiquitin ligase. Although Ad12-mediated complex containing CUL5, RBX1, and elongins B and C, and degradation of p53 is dependent upon both E1B-55K and E4orf6, serve to target this CRL to new substrates (14, 15). It has been Ad12-mediated degradation of TOPBP1 is solely dependent on suggested that E1B-55K is the substrate receptor for the complex, E4orf6. We propose that Ad12 E4orf6 has two principal activities: and E4orf6 is the adaptor that recruits the CRL as it has several to recruit the CUL2-based ubiquitin ligase and to act as substrate motifs, termed BC boxes, that bind to the elongin B and C sub- receptor for TOPBP1. In support of the idea that Ad12 E4orf6 spe- units (16, 17). As virtually all detectable Ad5 E1B-55K/E4orf6 fi ci cally prevents ATR activation during infection by targeting associates with the CUL5 complex (15), it is likely that targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can in- cellular proteins for degradation is the major function of E1B- hibit the ATR-dependent phosphorylation of CHK1 in response to 55K and E4orf6 during Ad5 infection (18). replication stress. Taken together, these data provide insights into E1B-55K/E4orf6–dependent degradation of MRN is sufficient how Ad modulates ATR signaling pathways during infection. to prevent ATM activation (11), and this appears to be conserved between some Ad species (19). Interestingly, however, two viral DNA damage | cullin-RING ubiquitin ligases | DNA damage | proteasome serotypes, Ad5 and Ad12, differentially regulate ATR during in- fection (19). The Ad5 E4orf3 protein inhibits ATR activation by aintenance of genome integrity is essential for cell survival. relocalizing and immobilizing MRN subunits before their tar- MCells have therefore evolved a complex set of biochemical geted degradation by E1B-55K/E4orf6 (20), but Ad12 E4orf3 pathways to recognize and repair damaged DNA (1). In addition lacks this activity (21). Yet both viruses inhibit CHK1 phosphor- to recognizing cellular DNA lesions, there is increasing evidence ylation, indicating that full ATR activation is prevented by both to show that this response is also activated by viral DNA during Ad5 and Ad12, but by different strategies (19). infection, and that some DNA repair proteins can inhibit viral Here, we provide evidence to show that a virus can inhibit ATR replication (2). signaling by specifically targeting its activator protein, TOPBP1. The DNA damage response is primarily controlled by two re- Ad12 E4orf6 assembles a CRL complex that targets TOPBP1 for lated protein kinases, ataxia-telangiectasia mutated (ATM) and proteasomal degradation. In contrast to previous work with Ad5, ATM/Rad3-related (ATR). Both kinases have a large and partially we show that this CRL contains CUL2 rather than CUL5, and overlapping set of substrates that includes tumor suppressor and that Ad12 E1B-55K is not required for TOPBP1 degradation. checkpoint proteins such as p53 and BRCA1 (3). However, some Rather, Ad12 E4orf6 interacts directly with TOPBP1 and is proteins, such as the checkpoint effector kinases CHK1 and CHK2, necessary and sufficient to promote its degradation. Finally, we MICROBIOLOGY are specific substrates for ATR and ATM, respectively (4). ATM show that, in uninfected cells exposed to replication stress, ex- predominantly responds to DNA double-strand breaks (DSBs), ogenous expression of Ad12 E4orf6 prevents CHK1 phosphory- which are sensed by the MRE11-RAD50-NBS1 (MRN) complex lation, indicating that by targeting TOPBP1 for degradation, (5). MRN is required for ATM activation, as it stimulates both ATM recruitment to DSBs and phosphorylation of its substrates (5, 6). In contrast, ATR becomes active in response to a much Author contributions: A.N.B., R.J.A.G., and A.S.T. designed research; A.N.B., R.N.P., N.A.F., wider range of genotoxic stresses (4). This is probably accomplished R.J.A.G., and A.S.T. performed research; K.T., P.G., G.S.S., A.M.R.T., I.M.M., and T.D. con- tributed new reagents/analytic tools; A.N.B., R.J.A.G., and A.S.T. analyzed data; and A.N.B., by the recognition of a common signal, ssDNA, which can be R.J.A.G., and A.S.T. wrote the paper. generated at stalled replication forks or by enzymatic processing of The authors declare no conflict of interest. DNA lesions (4). Replication protein A (RPA) binds to tracts of *This Direct Submission article had a prearranged editor. ssDNA and recruits ATR by interacting with its essential binding 1 partner, ATR-interacting protein (ATRIP) (7, 8). ATR activation Present address: Weatherall Institute of Molecular Medicine, University of Oxford, John β– Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom. also requires other proteins, including topoisomerase-II binding 2 To whom correspondence may be addressed. E-mail: [email protected], protein 1 (TOPBP1) (9, 10). TOPBP1 contains a domain that [email protected], or [email protected]. stimulates ATR kinase activity, and so is essential for the phos- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. phorylation of many ATR targets, including CHK1 (9, 10). 1073/pnas.0914605107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0914605107 PNAS | July 6, 2010 | vol. 107 | no. 27 | 12251–12256 Downloaded by guest on September 24, 2021 E4orf6 can directly inhibit ATR in the absence of other viral pathway are recruited to Ad5 and Ad12 viral replication centers in proteins. Taken together, this study establishes Ad12 E4orf6 as infected cells (11, 19–21). Given the apparent differences in ATR a modulator of CRL-dependent ubiquitylation and a regulator of and TOPBP1 regulation in Ad5- and Ad12-infected cells, we ex- ATR/TOPBP1-dependent signaling. amined TOPBP1 localization patterns in these cells. In mock- treated cells, both RPA32 and TOPBP1 were found to be diffusely Results localized in the nucleoplasm, but excluded from nucleoli (Fig. TOPBP1 Is Degraded in Ad12- but Not Ad5-Infected Cells. Ad5 E1B- 2A). However, in Ad5-infected cells, TOPBP1 was relocalized to 55K and E4orf6 proteins promote proteasomal degradation of sites of viral replication, where it colocalized with Ad5 DNA- MRN to inhibit ATM signaling (11). We reasoned that Ad12 might binding protein (DBP), E1B-55K, and RPA32 at early and late also degrade one or more other cellular proteins to inhibit ATR. times after infection (Fig. 2B and Fig. S1). Next, we determined Therefore, we infected HeLa cells with WT Ad5 or Ad12 and TOPBP1 localization in Ad12-infected cells, using RPA32 as examined the steady-state levels of proteins involved in ATR ac- a marker of viral replication centers (21). At early times after tivation at appropriate times postinfection, using MRE11 degra- infection, TOPBP1 was recruited to viral replication sites (Fig. dation by both Ad5 and Ad12 as a control (Fig. 1A). DNA ligase IV S1); significantly, however, this staining pattern was lost at later (LIG4), previously shown to be targeted for degradation in Ad5- times (Fig. 2C), consistent with Western blotting data shown in infected cells (22), was also degraded in Ad12-infected cells, in- Fig. 1. Weak residual TOPBP1 nuclear staining was present at late dicating that, like MRN, it may be a common target for human times after infection, but this was not observed at viral replication Ads. Further analysis revealed that ATR, ATRIP, RPA70, RPA32, centers (Fig. 2C). These results indicate that, although TOPBP1 is and RAD9 levels remained constant in Ad5- and Ad12-infected initially recruited to viral replication sites, it is subsequently de- cells. Slower migrating forms of RAD9 and RPA32 were detected graded in Ad12-infected cells.
Recommended publications
  • Genome-Wide Analysis and Characterization of F-Box Gene Family in Gossypium Hirsutum L Shulin Zhang1,2, Zailong Tian1, Haipeng Li1, Yutao Guo1, Yanqi Zhang1, Jeremy A
    Zhang et al. BMC Genomics (2019) 20:993 https://doi.org/10.1186/s12864-019-6280-2 RESEARCH ARTICLE Open Access Genome-wide analysis and characterization of F-box gene family in Gossypium hirsutum L Shulin Zhang1,2, Zailong Tian1, Haipeng Li1, Yutao Guo1, Yanqi Zhang1, Jeremy A. Roberts3, Xuebin Zhang1* and Yuchen Miao1* Abstract Background: F-box proteins are substrate-recognition components of the Skp1-Rbx1-Cul1-F-box protein (SCF) ubiquitin ligases. By selectively targeting the key regulatory proteins or enzymes for ubiquitination and 26S proteasome mediated degradation, F-box proteins play diverse roles in plant growth/development and in the responses of plants to both environmental and endogenous signals. Studies of F-box proteins from the model plant Arabidopsis and from many additional plant species have demonstrated that they belong to a super gene family, and function across almost all aspects of the plant life cycle. However, systematic exploration of F-box family genes in the important fiber crop cotton (Gossypium hirsutum) has not been previously performed. The genome- wide analysis of the cotton F-box gene family is now possible thanks to the completion of several cotton genome sequencing projects. Results: In current study, we first conducted a genome-wide investigation of cotton F-box family genes by reference to the published F-box protein sequences from other plant species. 592 F-box protein encoding genes were identified in the Gossypium hirsutume acc.TM-1 genome and, subsequently, we were able to present their gene structures, chromosomal locations, syntenic relationships with their parent species. In addition, duplication modes analysis showed that cotton F-box genes were distributed to 26 chromosomes, with the maximum number of genes being detected on chromosome 5.
    [Show full text]
  • 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]
  • Neddylation: a Novel Modulator of the Tumor Microenvironment Lisha Zhou1,2*†, Yanyu Jiang3†, Qin Luo1, Lihui Li1 and Lijun Jia1*
    Zhou et al. Molecular Cancer (2019) 18:77 https://doi.org/10.1186/s12943-019-0979-1 REVIEW Open Access Neddylation: a novel modulator of the tumor microenvironment Lisha Zhou1,2*†, Yanyu Jiang3†, Qin Luo1, Lihui Li1 and Lijun Jia1* Abstract Neddylation, a post-translational modification that adds an ubiquitin-like protein NEDD8 to substrate proteins, modulates many important biological processes, including tumorigenesis. The process of protein neddylation is overactivated in multiple human cancers, providing a sound rationale for its targeting as an attractive anticancer therapeutic strategy, as evidence by the development of NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Neddylation inhibition by MLN4924 exerts significantly anticancer effects mainly by triggering cell apoptosis, senescence and autophagy. Recently, intensive evidences reveal that inhibition of neddylation pathway, in addition to acting on tumor cells, also influences the functions of multiple important components of the tumor microenvironment (TME), including immune cells, cancer-associated fibroblasts (CAFs), cancer-associated endothelial cells (CAEs) and some factors, all of which are crucial for tumorigenesis. Here, we briefly summarize the latest progresses in this field to clarify the roles of neddylation in the TME, thus highlighting the overall anticancer efficacy of neddylaton inhibition. Keywords: Neddylation, Tumor microenvironment, Tumor-derived factors, Cancer-associated fibroblasts, Cancer- associated endothelial cells, Immune cells Introduction Overall, binding of NEDD8 molecules to target proteins Neddylation is a reversible covalent conjugation of an can affect their stability, subcellular localization, conform- ubiquitin-like molecule NEDD8 (neuronal precursor ation and function [4]. The best-characterized substrates cell-expressed developmentally down-regulated protein of neddylation are the cullin subunits of Cullin-RING li- 8) to a lysine residue of the substrate protein [1, 2].
    [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]
  • 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]
  • Expression and Purification of Functional Recombinant CUL2
    www.nature.com/scientificreports OPEN Expression and purifcation of functional recombinant CUL2•RBX1 from E. coli Stephanie Diaz1, Lihong Li1,2, Kankan Wang1 & Xing Liu1,2* Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. CUL2 is the scafold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efciently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-efective systems for the expression and purifcation of recombinant human CUL2 from E. coli cells. The purifed CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation. Protein turnover is a cellular regulatory system defned by the continuous synthesis and decomposition of specifc proteins to maintain the integrity of optimally functioning proteins 1,2. Abnormalities during protein turnover, specifcally during protein degradation, ofen result in human diseases such as cystic fbrosis and liposarcoma.
    [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]
  • Disruption of the Coordination Between Host Circadian Rhythms and Malaria
    bioRxiv preprint doi: https://doi.org/10.1101/791046; this version posted October 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Disruption of the coordination between host circadian rhythms and malaria parasite development alters the duration of the intraerythrocytic cycle Authors Amit K. Subudhi1, Aidan J. O’Donnell2†, Abhinay Ramaprasad1†, Hussein M. Abkallo3, Abhinav Kaushik1, Hifzur R. Ansari1, Alyaa M Abdel-Haleem1, Fathia Ben Rached1, Osamu Kaneko4, Richard Culleton3,*, Sarah E. Reece2,*, Arnab Pain1,5,6* 1Pathogen Genomics Group, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia 2Institute of Evolutionary Biology, and Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3FL, UK 3Malaria Unit, Department of Pathology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan 4Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan 5Center for Zoonosis Control, Global Institution for Collaborative Research and Education (GI-CoRE); Hokkaido University, N20 W10 Kita-ku, Sapporo, 001-0020 Japan 6Lead Contact Current address: Computational Bioscience Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia. †Contributed equally 1 bioRxiv preprint doi: https://doi.org/10.1101/791046; this version posted October 2, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]