Types of Nuclear Localization Signals and Mechanisms of Protein Import
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XPO1E571K Mutation Modifies Exportin 1 Localisation And
cancers Article XPO1E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-Cell Lymphoma Hadjer Miloudi 1, Élodie Bohers 1,2, François Guillonneau 3 , Antoine Taly 4,5 , Vincent Cabaud Gibouin 6,7 , Pierre-Julien Viailly 1,2 , Gaëtan Jego 6,7 , Luca Grumolato 8 , Fabrice Jardin 1,2 and Brigitte Sola 1,* 1 INSERM U1245, Unicaen, Normandie University, F-14000 Caen, France; [email protected] (H.M.); [email protected] (E.B.); [email protected] (P.-J.V.); [email protected] (F.J.) 2 Centre de lutte contre le Cancer Henri Becquerel, F-76000 Rouen, France 3 Plateforme Protéomique 3P5, Université de Paris, Institut Cochin, INSERM, CNRS, F-75014 Paris, France; [email protected] 4 Laboratoire de Biochimie Théorique, CNRS UPR 9030, Université de Paris, F-75005 Paris, France; [email protected] 5 Institut de Biologie Physico-Chimique, Fondation Edmond de Rothschild, PSL Research University, F-75005 Paris, France 6 INSERM, LNC UMR1231, F-21000 Dijon, France; [email protected] (V.C.G.); [email protected] (G.J.) 7 Team HSP-Pathies, University of Burgundy and Franche-Comtée, F-21000 Dijon, France 8 INSERM U1239, Unirouen, Normandie University, F-76130 Mont-Saint-Aignan, France; [email protected] * Correspondence: [email protected]; Tel.: +33-2-3156-8210 Received: 11 September 2020; Accepted: 28 September 2020; Published: 30 September 2020 Simple Summary: Almost 25% of patients with either primary mediastinal B-cell lymphoma (PMBL) or classical Hodgkin lymphoma (cHL) possess a recurrent mutation of the XPO1 gene encoding the major nuclear export protein. -
Targeting the Nuclear Import Receptor Kpnb1 As an Anticancer Therapeutic
Published OnlineFirst February 1, 2016; DOI: 10.1158/1535-7163.MCT-15-0052 Small Molecule Therapeutics Molecular Cancer Therapeutics Targeting the Nuclear Import Receptor Kpnb1as an Anticancer Therapeutic Pauline J. van der Watt1, Alicia Chi1, Tamara Stelma1, Catherine Stowell1, Erin Strydom1, Sarah Carden1, Liselotte Angus1, Kate Hadley1, Dirk Lang2, Wei Wei3, Michael J. Birrer3, John O. Trent4, and Virna D. Leaner1 Abstract Karyopherin beta 1 (Kpnb1) is a nuclear transport receptor tissue origins. Minimum effect on the proliferation of non- that imports cargoes into the nucleus. Recently, elevated Kpnb1 cancer cells was observed at the concentration of INI-43 that expression was found in certain cancers and Kpnb1silencing showed a significant cytotoxic effect on various cervical and with siRNA was shown to induce cancer cell death. This study esophageal cancer cell lines. A rescue experiment confirmed aimed to identify novel small molecule inhibitors of Kpnb1, that INI-43 exerted its cell killing effects, in part, by targeting and determine their anticancer activity. An in silico screen Kpnb1. INI-43 treatment elicited a G2–M cell-cycle arrest in identified molecules that potentially bind Kpnb1 and Inhibitor cancer cells and induced the intrinsic apoptotic pathway. Intra- of Nuclear Import-43, INI-43 (3-(1H-benzimidazol-2-yl)-1-(3- peritoneal administration of INI-43 significantly inhibited the dimethylaminopropyl)pyrrolo[5,4-b]quinoxalin-2-amine) was growth of subcutaneously xenografted esophageal and cervical investigated further as it interfered with the nuclear localization tumor cells. We propose that Kpnb1 inhibitors could have of Kpnb1andknownKpnb1cargoesNFAT,NFkB, AP-1, and therapeutic potential for the treatment of cancer. -
Cselp Functions As the Nuclear Export Receptor for Importin a in Yeast
FEBS 20654 FEBS Letters 433 (1998) 185 190 Cselp functions as the nuclear export receptor for importin a in yeast Markus Kfinzler, Eduard C. Hurt* Biochemie-Zentrum Heidelberg ( BZH), Ruprecht-Karls-Universitgit, lm Neuenheimer Feld 328, 4. OG, 69120 Heidelberg, Germany Received 14 May 1998; revised version' received 14 July 1998 review see [4,5]). The similarity is mainly found in a Ran-GTP Abstract CSEI is essential for yeast cell viability and has been implicated in chromosome segregation. Based on its sequence bindirfg domain at the N-terminus of these proteins [8,9]. similarity, Cselp has been grouped into the family of importin Binding of Ran-GTP has opposite effects on the binding of like nucleocytoplasmic transport receptors with highest homol- cargo by import and export receptors. Complexes between ogy to the recently identified human nuclear export receptor for import receptors and cargo are dissociated by Ran-GTP, importin ~, CAS. We demonstrate here that Cselp physically whereas binding of Ran-GTP and cargo to export receptors interacts with yeast Ran and yeast importin tx (Srplp) in the is cooperative. These properties of import and export recep- yeast two-hybrid system and that recombinant Cselp, Srplp and tors together with a high concentration of Ran-GTP in the Ran-GTP form a trimeric complex in vitro. Re-export of Srplp nucleus trigger release of cargo from import receptors and from the nucleus into the cytoplasm and nuclear uptake of a association of cargo with export receptors in the nucleus. Re- reporter protein containing a classical NLS are inhibited in a lease of export cargo and binding of import cargo in the csel mutant strain. -
T-Cell Receptor (TCR) Signaling Promotes the Assembly of Ranbp2
RESEARCH ARTICLE T-cell receptor (TCR) signaling promotes the assembly of RanBP2/RanGAP1- SUMO1/Ubc9 nuclear pore subcomplex via PKC--mediated phosphorylation of RanGAP1 Yujiao He1, Zhiguo Yang1†, Chen-si Zhao1†, Zhihui Xiao1†, Yu Gong1, Yun-Yi Li1, Yiqi Chen1, Yunting Du1, Dianying Feng1, Amnon Altman2, Yingqiu Li1* 1MOE Key Laboratory of Gene Function and Regulation, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China; 2Center for Cancer Immunotherapy, La Jolla Institute for Immunology, La Jolla, United States Abstract The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport, and its dysfunction has been associated with many diseases. The metazoan NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1, and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in regulation of NPC remain poorly understood. Here, we show that in human and murine T cells, following T-cell receptor (TCR) stimulation, protein kinase C-q (PKC-q) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription *For correspondence: factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-q to the NPC, 504 506 [email protected] where it interacts with and phosphorylates RanGAP1 on Ser and Ser . RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 †These authors contributed and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-q equally to this work as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of Competing interests: The RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC authors declare that no subcomplex. -
In Vivo Loss-Of-Function Screens Identify KPNB1 As a New Druggable
In vivo loss-of-function screens identify KPNB1 as a PNAS PLUS new druggable oncogene in epithelial ovarian cancer Michiko Kodamaa,b,1, Takahiro Kodamaa,c,1,2, Justin Y. Newberga,d, Hiroyuki Katayamae, Makoto Kobayashie, Samir M. Hanashe, Kosuke Yoshiharaf, Zhubo Weia, Jean C. Tiena,g, Roberto Rangela,h, Kae Hashimotob, Seiji Mabuchib, Kenjiro Sawadab, Tadashi Kimurab, Neal G. Copelanda,i, and Nancy A. Jenkinsa,i,2 aCancer Research Program, Houston Methodist Research Institute, Houston, TX 77030; bDepartment of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka 5650871, Japan; cDepartment of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka 5650871, Japan; dDepartment of Molecular Oncology, Moffitt Cancer Center, Tampa, FL 33612; eDepartment of Clinical Cancer Prevention, University of Texas MD Anderson Cancer Center, Houston, TX 77030; fDepartment of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 9518510, Japan; gDepartment of Pathology, Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, MI 48109; hDepartment of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX 77030; and iDepartment of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030 Contributed by Nancy A. Jenkins, July 23, 2017 (sent for review April 3, 2017; reviewed by Roland Rad and Kosuke Yusa) Epithelial ovarian cancer (EOC) is a deadly cancer, and its prognosis has To overcome this problem, several alternative methods have re- not been changed significantly during several decades. To seek new cently been used for cancer gene discovery, including insertional therapeutic targets for EOC, we performed an in vivo dropout screen mutagenesis and RNAi/shRNA/CRISPR/Cas9-based screens. -
Identification of a Potential Mitotic Function for the Mammalian Nup50
Identification of a Potential Mitotic Function for the Mammalian Nup50 A Senior Thesis Presented in Partial Fulfillment of the Requirements for graduation with research distinction in Biology in the undergraduate colleges of The Ohio State University by Jessica El-Hallal The Ohio State University June 2011 Project Advisor: Dr. Stephen Osmani, Department of Molecular Genetics ABSTRACT Mitosis is a conserved process in which the genetic material, DNA, is equally segregated between two daughter cells. DNA is contained in the nucleus of the eukaryotic cell and surrounded by the nuclear envelope. Multi protein complexes known as the Nuclear Pore Complexes (NPCs) embed within the nuclear envelope and regulate the transport of molecules in and out of the nucleus. Surprisingly, in Aspergillus nidulans, the model system used in my study, a nuclear pore complex protein Nup2 undergoes a unique translocation to chromatin during mitosis and is essential for proper mitotic progression. Interestingly, the Nup2 homolog in higher eukaryotes, Nup50, undergoes the same translocation. Therefore, the purpose of this study is to test whether Nup50 can translocate onto chromatin in Aspergillus nidulans and complement the mitotic function of Nup2. In order to test this hypothesis, the Nup50 gene was integrated into A. nidulans using homologous recombination. Four way fusion PCR was used to generate a DNA cassette that contains the Nup50 gene fused to EGFP2 marker and its expression under control of the inducible promoter alcA. Once Nup50 was introduced into A. nidulans, Nup2 was deleted in the background. So far, we have discovered that Nup50 is present in the nucleus at interphase and disperses throughout the cell during mitosis in the absence or presence of the Aspergillus nidulans Nup2. -
Small Gtpase Ran and Ran-Binding Proteins
BioMol Concepts, Vol. 3 (2012), pp. 307–318 • Copyright © by Walter de Gruyter • Berlin • Boston. DOI 10.1515/bmc-2011-0068 Review Small GTPase Ran and Ran-binding proteins Masahiro Nagai 1 and Yoshihiro Yoneda 1 – 3, * highly abundant and strongly conserved GTPase encoding ∼ 1 Biomolecular Dynamics Laboratory , Department a 25 kDa protein primarily located in the nucleus (2) . On of Frontier Biosciences, Graduate School of Frontier the one hand, as revealed by a substantial body of work, Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Ran has been found to have widespread functions since Osaka 565-0871 , Japan its initial discovery. Like other small GTPases, Ran func- 2 Department of Biochemistry , Graduate School of Medicine, tions as a molecular switch by binding to either GTP or Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 , GDP. However, Ran possesses only weak GTPase activ- Japan ity, and several well-known ‘ Ran-binding proteins ’ aid in 3 Japan Science and Technology Agency , Core Research for the regulation of the GTPase cycle. Among such partner Evolutional Science and Technology, Osaka University, 1-3 molecules, RCC1 was originally identifi ed as a regulator of Yamada-oka, Suita, Osaka 565-0871 , Japan mitosis in tsBN2, a temperature-sensitive hamster cell line (3) ; RCC1 mediates the conversion of RanGDP to RanGTP * Corresponding author in the nucleus and is mainly associated with chromatin (4) e-mail: [email protected] through its interactions with histones H2A and H2B (5) . On the other hand, the GTP hydrolysis of Ran is stimulated by the Ran GTPase-activating protein (RanGAP) (6) , in con- Abstract junction with Ran-binding protein 1 (RanBP1) and/or the large nucleoporin Ran-binding protein 2 (RanBP2, also Like many other small GTPases, Ran functions in eukaryotic known as Nup358). -
Destabilization of Ran C-Terminus Promotes GTP Loading and Occurs
bioRxiv preprint doi: https://doi.org/10.1101/305177; this version posted April 20, 2018. 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-ND 4.0 International license. Destabilization of Ran C-terminus promotes GTP loading and occurs in multiple Ran cancer mutations Yuqing Zhang1#, Jinhan Zhou1#, Yuping Tan1, Qiao Zhou1, Aiping Tong1, Xiaofei Shen1,2, Da Jia1,2, Xiaodong Sun3, Qingxiang Sun1* 1Department of Pathology and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu 610041, China 2Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, Division of Neurology, West China Second University Hospital, Sichuan University, Chengdu 610041, China 3 Department of Pharmacology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China #Equal contribution *Corresponding author: [email protected] Abstract Ran (Ras-related nuclear protein) plays several important roles in nucleo-cytoplasmic transport, mitotic spindle formation, nuclear envelope/nuclear pore complex assembly, and other diverse functions in the cytoplasm, as well as in cellular transformation when activated. Unlike other Ras superfamily proteins, Ran contains an auto-inhibitory C-terminal tail, which packs against its G domain and bias Ran towards binding GDP over GTP. The biological importance of this C- terminal tail is not well understood. By disrupting the interaction between the C-terminus and the G domain, we were able to generate Ran mutants that are innately active and potently bind to RanBP1 (Ran Binding Protein 1), nuclear export factor CRM1 and nuclear import factor KPNB1. -
Persistent Transcription-Blocking DNA Lesions Trigger Somatic Growth Attenuation Associated with Longevity
ARTICLES Persistent transcription-blocking DNA lesions trigger somatic growth attenuation associated with longevity George A. Garinis1,2, Lieneke M. Uittenboogaard1, Heike Stachelscheid3,4, Maria Fousteri5, Wilfred van Ijcken6, Timo M. Breit7, Harry van Steeg8, Leon H. F. Mullenders5, Gijsbertus T. J. van der Horst1, Jens C. Brüning4,9, Carien M. Niessen3,9,10, Jan H. J. Hoeijmakers1 and Björn Schumacher1,9,11 The accumulation of stochastic DNA damage throughout an organism’s lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative stress. Our findings suggest that the accumulation of DNA damage in transcribed genes in most if not all tissues contributes to the ageing-associated shift from growth to somatic maintenance that triggers stress resistance and is thought to promote longevity. Ageing represents the progressive functional decline that is exempted levels as a result of pituitary dysfunction (Snell and Ames mice) — have an from evolutionary selection because it largely occurs after reproduc- extended lifespan17–20. -
Distinct Ranbp1 Nuclear Export and Cargo Dissociation Mechanisms
RESEARCH ARTICLE Distinct RanBP1 nuclear export and cargo dissociation mechanisms between fungi and animals Yuling Li1†, Jinhan Zhou1†, Sui Min1†, Yang Zhang2, Yuqing Zhang1, Qiao Zhou1, Xiaofei Shen3, Da Jia3, Junhong Han2, Qingxiang Sun1* 1Department of Pathology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Collaborative Innovation Centre of Biotherapy, Chengdu, China; 2Division of Abdominal Cancer, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Collaborative Innovation Centre for Biotherapy, Chengdu, China; 3Key Laboratory of Birth Defects and Related Diseases of Women and Children, Department of Paediatrics, Division of Neurology, West China Second University Hospital, Sichuan University, Chengdu, China Abstract Ran binding protein 1 (RanBP1) is a cytoplasmic-enriched and nuclear-cytoplasmic shuttling protein, playing important roles in nuclear transport. Much of what we know about RanBP1 is learned from fungi. Intrigued by the long-standing paradox of harboring an extra NES in animal RanBP1, we discovered utterly unexpected cargo dissociation and nuclear export mechanisms for animal RanBP1. In contrast to CRM1-RanGTP sequestration mechanism of cargo dissociation in fungi, animal RanBP1 solely sequestered RanGTP from nuclear export complexes. In fungi, RanBP1, CRM1 and RanGTP formed a 1:1:1 nuclear export complex; in contrast, animal RanBP1, CRM1 and RanGTP formed a 1:1:2 nuclear export complex. The key feature for the two mechanistic changes from fungi to animals was the loss of affinity between RanBP1-RanGTP and *For correspondence: CRM1, since residues mediating their interaction in fungi were not conserved in animals. The [email protected] biological significances of these different mechanisms in fungi and animals were also studied. -
Caspases Mediate Nucleoporin Cleavage, but Not Early Redistribution of Nuclear Transport Factors and Modulation of Nuclear Permeability in Apoptosis
Cell Death and Differentiation (2001) 8, 495 ± 505 ã 2001 Nature Publishing Group All rights reserved 1350-9047/01 $15.00 www.nature.com/cdd Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis E Ferrando-May1, V Cordes2,3, I Biller-Ckovric1, J Mirkovic1, Val-Ala-aspartyl-¯uoromethylketone; DEVD-CHO, N-acetyl-Asp- DGoÈ rlich4 and P Nicotera*,5 Glu-Val-Asp-aldehyde 1 Chair of Molecular Toxicology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany Introduction 2 Karolinska Institutet, Medical Nobel Institute, Department of Cellular and Molecular Biology, S-17177 Stockholm, Sweden The most evident morphological feature of apoptosis is the 3 Division of Cell Biology, Germany Cancer Research Center, D-69120, disassembly of the nucleus, which involves the condensation Heidelberg, Germany 4 of chromatin and its segregation into membrane-enclosed Zentrum fuÈr Molekulare Biologie der UniversitaÈt Heidelberg, D-69120, 1 Heidelberg, Germany particles. Biochemical hallmarks of apoptotic nuclear 5 MRC Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster execution are DNA cleavage in large and small (oligonu- Road, Leicester LE1 9HN, UK cleosomal-sized) fragments, as well as the specific proteo- * Corresponding author: P Nicotera, MRC Toxicology Unit, Hodgkin Building, lysis of several nuclear substrates. Major effectors of University of Leicester, Lancaster Road, Leicester LE1 9HN, UK. apoptotic nuclear changes are members of the cysteine Tel +44-116-2525611; Fax: +44-116-2525616; E-mail: [email protected] protease family of caspases. Nuclear substrates for caspases 2,3 Received 23.11.00; revised 22.12.00; accepted 29.12.00 include nucleoskeletal elements like lamins, and proteins Edited by M Piacentini involved in the organisation and replication of DNA, like SAF- A, MCM3 and RCF140.4±6 Cleavage of nuclear proteins may have important Abstract implications for the apoptotic process. -
Identification of Proteins Interacting with Dysferlin Using the Tandem Affinity Purification Method
The Open Cell Development & Biology Journal, 2008, 1, 17-23 17 Open Access Identification of Proteins Interacting with Dysferlin Using the Tandem Affinity Purification Method Maziar Assadi*,1, Thomas Schindler1, Bernd Muller1, John D. Porter2, Markus A. Ruegg3 and Hanno Langen1 1F. Hoffmann-La Roche, Roche Center for Medical Genomics, Basel, Switzerland 2Case Western Reserve University, Department of Neurology, Cleveland, USA 3University of Basel, Biozentrum, Basel, Switzerland Abstract: Mutations of DYSF, the gene encoding dysferlin, cause two types of muscular dystrophies: limb-girdle muscu- lar dystrophy type 2B and Miyoshi myopathy. Recent work suggests a role of dysferlin in membrane repair and demon- strates that defective membrane repair is a novel mechanism of muscle degeneration. We used the tandem affinity purifi- cation method for the purification of proteins interacting with dysferlin. Three interacting partners were identified by this method (striatin, adaptin alpha, utrophin) and were confirmed by co-immunoprecipitations. All three proteins play a role in vesicle trafficking. Knowing the interacting partners of dysferlin will help to understand how muscle cells repair tears in the sarcolemma and will give a deeper insight into this very important cell function. At the same time the identified proteins could serve as potential candidates for other muscular dystrophies and muscle-related diseases with unknown ae- tiology. INTRODUCTION this machinery. These proteins could serve as potential can- didate for other muscular dystrophies and muscle-diseases Dysferlin is a member of a mammalian gene family shar- with unknown aetiology. Therefore, we attempted to identify ing homology with the Caenorhabditis elegans spermato- the interacting partners of dysferlin and used the tandem genesis factor fer-1 gene, which mediates vesicle fusion to affinity purification (TAP) method for this purpose.