DOCSLIB.ORG

Histone H2B Ubiquitin Ligase RNF20 Is Required for MLL-Rearranged Leukemia

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Histone H2B Ubiquitin Ligase RNF20 Is Required for MLL-Rearranged Leukemia Histone H2B ubiquitin ligase RNF20 is required for MLL-rearranged leukemia Eric Wanga,1, Shinpei Kawaokaa,1, Ming Yub, Junwei Shia,c, Ting Nid, Wenjing Yangd, Jun Zhud, Robert G. Roederb,2, and Christopher R. Vakoca,2 aCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; bLaboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065; cMolecular and Cellular Biology Program, Stony Brook University, Stony Brook, NY 11794; and dGenetics and Development Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Robert G. Roeder, January 18, 2013 (sent for review December 19, 2012) Mixed-lineage leukemia (MLL) fusions are potent oncogenes that (4–6). The translocation partners of MLL can be highly diverse; initiate aggressive forms of acute leukemia. As aberrant tran- however, fusions with AF9 are the most common in AML (1). scriptional regulators, MLL-fusion proteins alter gene expression The aberrant recruitment of AF9-associated proteins to MLL- in hematopoietic cells through interactions with the histone H3 occupied genes (e.g., HOXA9) leads to transcriptional deregu- lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference lation and, consequently, leukemia initiation. Several binding with MLL-fusion cofactors like DOT1L is an emerging therapeutic partners of AF9 are involved in MLL-AF9–mediated leukemo- strategy in this disease. Here, we identify the histone H2B E3 ubiq- genesis, including the super elongation complex (SEC), the uitin ligase ring finger protein 20 (RNF20) as an additional chromatin histone H3K79 methyltransferase DOT1L, and the chromodo- regulatorthatisnecessaryforMLL-fusion–mediated leukemo- main-containing protein CBX8 (7–10). genesis. Suppressing the expression of Rnf20 in diverse models Targeting of MLL-fusion–associated factors has emerged as a of MLL-rearranged leukemia leads to inhibition of cell proliferation, promising therapeutic strategy in this subtype of leukemia. Ge- under tissue culture conditions as well as in vivo. Rnf20 knockdown netic studies have validated that conditional inactivation Dot1l leads to reduced expression of MLL-fusion target genes, effects re- specifically inhibits progression of MLL-rearranged leukemias in sembling Dot1l inhibition. Using ChIP-seq, we found that H2B vivo, in association with reduced expression of MLL-AF9 target ubiquitination is enriched in the body of MLL-fusion target genes, genes (11, 12). Furthermore, a small-molecule inhibitor of DOT1L CELL BIOLOGY correlating with sites of H3K79 methylation and transcription elon- demonstrates antileukemia activity in MLL-rearranged disease gation. Furthermore, Rnf20 is required to maintain local levels of models (13). Genetic or pharmacological disruption of the MLL: H3K79 methylation by Dot1l at Hoxa9 and Meis1.Thesefindings Menin interaction has also been validated as means of sup- support a model whereby cotranscriptional recruitment of Rnf20 at pressing proliferation of MLL-fusion leukemias (5, 14). Target- MLL-fusion target genes leads to amplification of Dot1l-mediated ing of DOT1L or Menin in cells that lack MLL rearrangements H3K79 methylation, thereby rendering leukemia cells dependent leads to remarkably little toxicity, suggesting a potential thera- on Rnf20 to maintain their oncogenic transcriptional program. peutic window for this general approach (13, 14). Ring finger protein 20 (Rnf20) (also called Bre1a) is the major epigenetics | cancer H2B-specific ubiquitin ligase in mammalian cells that targets lysine 120 for monoubiquitination [H2B ubiquitination (H2Bub)] he mixed-lineage leukemia (MLL) protooncogene (also called (15–18). Rnf20 can be recruited to chromatin via the PAF com- TMLL1)wasfirst cloned based on its involvement in chromo- plex, resulting in the accumulation of H2Bub at genes in a tran- somal translocations found in leukemia, occurring at a frequency scription-dependent manner (19–22). Although found broadly of 5–10% in acute myeloid leukemia (AML) and acute lympho- at active genes, H2Bub is not strictly required for transcription blastic leukemia (1). MLL translocations are especially common in elongation, but instead performs specialized roles in regulating infant acute leukemias and in secondary AMLs that arise follow- nucleosome dynamics (22), the DNA damage response (23, 24), ing cancer treatment with topoisomerase II inhibitors (1). In both and the activity of other histone-modifying enzymes (19, 21, of these clinical settings, MLL rearrangements are associated with 22). Regarding the latter, it is known that the presence of H2Bub a poor outcome (1). MLL-rearranged leukemias are notable for on nucleosomes can stimulate the activity of DOT1L in cata- (i) a paucity of additional gene mutations found in these cases lyzing H3K79 methylation in vitro and in vivo through apparent (although NRAS mutations are among the most common coop- allosteric regulation (19, 25). H2Bub also promotes H3K4 erating lesion) (2), (ii) a unique transcriptional signature char- methylation by the SET1 family of lysine methyltransferases (26). acterized by overexpression of homeobox A (HOXA) genes (1), The role of H2Bub in supporting histone methylation in mam- fi and (iii) an absence of available targeted therapies. Regarding malian cells appears to be dependent on the speci ccelltype fi the latter, the current clinical management of MLL-rearranged and/or on the speci c genomic region examined (17, 27, 28). leukemia with cytotoxic chemotherapy is similar to that of other Although substantial evidence indicates cross talk between genetic subtypes of leukemia that fall into the same risk category (1). Hence, a strong rationale exists to define unique therapeutic targets tailored to the molecular pathogenesis of this disease. Author contributions: E.W., S.K., R.G.R., and C.R.V. designed research; E.W., S.K., M.Y., MLL and J.S. performed research; E.W., S.K., M.Y., T.N., W.Y., J.Z., and C.R.V. analyzed data; encodes a chromatin regulator belonging to the SET1 and R.G.R. and C.R.V. wrote the paper. family of histone H3K4 methyltransferases, which form multi- The authors declare no conflict of interest. subunit protein complexes that maintain active transcription (1, Freely available online through the PNAS open access option. 3). MLL translocations found in leukemia generate fusions that Data deposition: The data reported in this paper have been deposited in the Gene Ex- encode an N-terminal fragment of MLL (which lacks the meth- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE43725). yltransferase domain) linked to a C-terminal fragment of various 1E.W. and S.K. contributed equally to this work. partner proteins (1). This N-terminal fragment of MLL physically 2 To whom correspondence may be addressed. E-mail: [email protected] or roeder@ interacts with Menin, CpG-rich DNA, and the polymerase rockefeller.edu. fi associated factor (PAF) complex, which collectively are suf - This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cient for chromatin occupancy at specific genes such as HOXA9 1073/pnas.1301045110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1301045110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 s H2Bub and H3K79 methylation in various contexts, it has yet to 2Een A Ube2a be addressed whether mammalian Rnf20 supports the bi- Ube2b otsi Ube2e1 ological functions performed by Dot1l in vivo. h Dzip3 Here, we identify a role for Rnf20 in the pathogenesis of s 3Een Ring1 MLL-fusion leukemia. Suppression of Rnf20 leads to impaired Rnf2 o leukemia progression in vivo associated with reduced expres- tsih Rnf20 sion of MLL-AF9 target genes, a finding we link to a defect in Rnf40 maintenance of local H3K79 methylation. Hence, our findings sBUD Mysm1 Usp3 implicate Rnf20 as a key requirement for MLL-fusion leukemia e Usp16 n through regulatory cross talk with Dot1l. otsih Usp21 Usp22 Results slor Rpa3 t noc Ren luc Rnf20 Is Required for Proliferation of MLL-Fusion Leukemia Cells. 0102030 Based on the known role of H2Bub in stimulating H3K79 meth- Fold-decrease in GFP% ylation in various systems (19, 25, 29), we hypothesized that Rnf20 might support the leukemogenic function of Dot1l in MLL-rear- B MLL-AF9; NrasG12D AML d2 ranged disease. We approached this question in a systematic 1.0 d4 d6 fashion in which we suppressed expression of several histone d8 d10 monoubiquitination regulators and evaluated the impact on pro- d12 liferation of MLL-fusion+ leukemia cells. We constructed a set of 0.5 45 shRNA vectors targeting the known E2 conjugating enzymes, E3 ligases, and deubiquitinating enzymes with specificity for his- Relative GFP% tone H2A or H2B for monoubiquitination (13 genes in total) (30). 0.0 Each shRNA (linked to a GFP reporter) was retrovirally in- Rnf20 shRpa3 457 troduced into cells derived from a genetically engineered mouse β-actin model of MLL-AF9/NrasG12D AML, shown previously to reca- shRen 2853 2949 3718 1295 3277 1944 3595 713 pitulate an aggressive, chemotherapy-resistant disease subtype shRnf20 (31). The GFP positivity of partially infected cells was tracked C Immortalized fibroblast D shRNF20.2710 fl d2 d6 d10 d4 d12 d20 d28 by ow cytometry over 8 d to monitor the relative rate of accu- d4 d8 d12 d8 d16 d24 mulation of shRNA+/GFP+ cells relative to shRNA−/GFP− 1.0 1.0 cells. Four shRNAs targeting Rnf20 were found to inhibit leu- kemia proliferation/viability compared with a negative control 0.5 0.5 shRNA targeting Renilla luciferase and a positive control shRNA Relative GFP% A Relative GFP% targeting the replication protein A3 (Rpa3) (Fig. 1 ) (32). No- 0.0 0.0 tably, all of the other shRNAs targeting ubiquitination regulators shRen 3277 1944 3595 shRpa3 MOLM13 MV4-11 THP-1 CMK 713 shRnf20 457 human MLL-fusion+ AML led to negligible effects on leukemia proliferation (Fig. 1A). This includes shRNAs targeting Ube2a and Ube2b, which encode Fig. 1. Rnf20 is required for proliferation of MLL-AF9/NrasG12D leukemia Rad6a and Rad6b, respectively, the E2-conjugating enzymes that cells in vitro.
Load more

Recommended publications
  • Ubiquitination Is Not Omnipresent in Myeloid Leukemia Ramesh C
    Editorials Ubiquitination is not omnipresent in myeloid leukemia Ramesh C. Nayak1 and Jose A. Cancelas1,2 1Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center and 2Hoxworth Blood Center, University of Cincinnati Academic Health Center, Cincinnati, OH, USA E-mail: JOSE A. CANCELAS - [email protected] / [email protected] doi:10.3324/haematol.2019.224162 hronic myelogenous leukemia (CML) is a clonal tination of target proteins through their cognate E3 ubiq- biphasic hematopoietic disorder most frequently uitin ligases belonging to three different families (RING, Ccaused by the expression of the BCR-ABL fusion HERCT, RING-between-RING or RBR type E3).7 protein. The expression of BCR-ABL fusion protein with The ubiquitin conjugating enzymes including UBE2N constitutive and elevated tyrosine kinase activity is suffi- (UBC13) and UBE2C are over-expressed in a myriad of cient to induce transformation of hematopoietic stem tumors such as breast, pancreas, colon, prostate, lym- cells (HSC) and the development of CML.1 Despite the phoma, and ovarian carcinomas.8 Higher expression of introduction of tyrosine kinase inhibitors (TKI), the dis- UBE2A is associated with poor prognosis of hepatocellu- ease may progress from a manageable chronic phase to a lar cancer.9 In leukemia, bone marrow (BM) cells from clinically challenging blast crisis phase with a poor prog- pediatric acute lymphoblastic patients show higher levels nosis,2 in which myeloid or lymphoid blasts fail to differ- of UBE2Q2
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • UBE2E1 (Ubch6) [Untagged] E2 – Ubiquitin Conjugating Enzyme
    UBE2E1 (UbcH6) [untagged] E2 – Ubiquitin Conjugating Enzyme Alternate Names: UbcH6, UbcH6, Ubiquitin conjugating enzyme UbcH6 Cat. No. 62-0019-100 Quantity: 100 µg Lot. No. 1462 Storage: -70˚C FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Page 1 of 2 Background Physical Characteristics The enzymes of the ubiquitylation Species: human Protein Sequence: pathway play a pivotal role in a num- GPLGSPGIPGSTRAAAM SDDDSRAST ber of cellular processes including Source: E. coli expression SSSSSSSSNQQTEKETNTPKKKESKVSMSKN regulated and targeted proteasomal SKLLSTSAKRIQKELADITLDPPPNCSAGP degradation of substrate proteins. Quantity: 100 μg KGDNIYEWRSTILGPPGSVYEGGVFFLDIT FTPEYPFKPPKVTFRTRIYHCNINSQGVI Three classes of enzymes are in- Concentration: 1 mg/ml CLDILKDNWSPALTISKVLLSICSLLTDCNPAD volved in the process of ubiquitylation; PLVGSIATQYMTNRAEHDRMARQWTKRYAT activating enzymes (E1s), conjugating Formulation: 50 mM HEPES pH 7.5, enzymes (E2s) and protein ligases 150 mM sodium chloride, 2 mM The residues underlined remain after cleavage and removal (E3s). UBE2E1 is a member of the E2 dithiothreitol, 10% glycerol of the purification tag. ubiquitin-conjugating enzyme family UBE2E1 (regular text): Start bold italics (amino acid and cloning of the human gene was Molecular Weight: ~23 kDa residues 1-193) Accession number: AAH09139 first described by Nuber et al. (1996). UBE2E1 shares 74% sequence ho- Purity: >98% by InstantBlue™ SDS-PAGE mology with UBE2D1 and contains an Stability/Storage: 12 months at -70˚C; N-terminal extension of approximately aliquot as required 40 amino acids. A tumour suppressor candidate, tumour-suppressing sub- chromosomal transferable fragment Quality Assurance cDNA (TSSC5) is located in the re- gion of human chromosome 11p15.5 Purity: Protein Identification: linked with Beckwith-Wiedemann syn- 4-12% gradient SDS-PAGE Confirmed by mass spectrometry.
    [Show full text]
  • Regulation of Canonical Wnt Signalling by the Ciliopathy Protein MKS1 and the E2
    bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.897959; this version posted March 28, 2020. 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. Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1. Katarzyna Szymanska1, Karsten Boldt2, Clare V. Logan1, Matthew Adams1, Philip A. Robinson1+, Marius Ueffing2, Elton Zeqiraj3, Gabrielle Wheway1,4#, Colin A. Johnson1#* *corresponding author: [email protected] ORCID: 0000-0002-2979-8234 # joint last authors + deceased 1 Leeds Institute of Medical Research, School of Medicine, University of Leeds, Leeds, UK 2 Institute of Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany 3 Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK 4 Faculty of Medicine, University of Southampton, Human Development and Health, UK; University Hospital Southampton NHS Foundation Trust, UK 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.08.897959; this version posted March 28, 2020. 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. Abstract A functional primary cilium is essential for normal and regulated signalling. Primary ciliary defects cause a group of developmental conditions known as ciliopathies, but the precise mechanisms of signal regulation by the cilium remain unclear.
    [Show full text]
  • A Systematic Analysis of Nuclear Heat Shock Protein 90 (Hsp90) Reveals A
    Max Planck Institute of Immunobiology und Epigenetics Freiburg im Breisgau A systematic analysis of nuclear Heat Shock Protein 90 (Hsp90) reveals a novel transcriptional regulatory role mediated by its interaction with Host Cell Factor-1 (HCF-1) Inaugural-Dissertation to obtain the Doctoral Degree Faculty of Biology, Albert-Ludwigs-Universität Freiburg im Breisgau presented by Aneliya Antonova born in Bulgaria Freiburg im Breisgau, Germany March 2019 Dekanin: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Referent: Dr. Ritwick Sawarkar Koreferent: Prof. Dr. Rudolf Grosschedl Drittprüfer: Prof. Dr. Andreas Hecht Datum der mündlichen Prüfung: 27.05.2019 ii AFFIDAVIT I herewith declare that I have prepared the present work without any unallowed help from third parties and without the use of any aids beyond those given. All data and concepts taken either directly or indirectly from other sources are so indicated along with a notation of the source. In particular I have not made use of any paid assistance from exchange or consulting services (doctoral degree advisors or other persons). No one has received remuneration from me either directly or indirectly for work which is related to the content of the present dissertation. The work has not been submitted in this country or abroad to any other examination board in this or similar form. The provisions of the doctoral degree examination procedure of the faculty of Biology of the University of Freiburg are known to me. In particular I am aware that before the awarding of the final doctoral degree I am not entitled to use the title of Dr.
    [Show full text]
  • Deubiquitylases in Developmental Ubiquitin Signaling and Congenital Diseases
    Cell Death & Differentiation (2021) 28:538–556 https://doi.org/10.1038/s41418-020-00697-5 REVIEW ARTICLE Deubiquitylases in developmental ubiquitin signaling and congenital diseases 1 1,2 1 Mohammed A. Basar ● David B. Beck ● Achim Werner Received: 16 October 2020 / Revised: 20 November 2020 / Accepted: 24 November 2020 / Published online: 17 December 2020 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2020 Abstract Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate’s stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have fi 1234567890();,: 1234567890();,: identi ed mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs.
    [Show full text]
  • Characterization of the Cellular Network of Ubiquitin Conjugating and Ligating Enzymes Ewa Katarzyna Blaszczak
    Characterization of the cellular network of ubiquitin conjugating and ligating enzymes Ewa Katarzyna Blaszczak To cite this version: Ewa Katarzyna Blaszczak. Characterization of the cellular network of ubiquitin conjugating and ligating enzymes. Cellular Biology. Université Rennes 1, 2015. English. NNT : 2015REN1S116. tel-01547616 HAL Id: tel-01547616 https://tel.archives-ouvertes.fr/tel-01547616 Submitted on 27 Jun 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ANNÉE 2015 THÈSE / UNIVERSITÉ DE RENNES 1 sous le sceau de l’Université Européenne de Bretagne pour le grade de DOCTEUR DE L’UNIVERSITÉ DE RENNES 1 Mention : BIOLOGIE École doctorale Vie-Agro-Santé présentée par Ewa Katarzyna Blaszczak Préparée à l’unité de recherche UMR 6290, IGDR Institut de Génétique et Développement de Rennes Université Rennes 1 Thèse soutenue à Rennes le 26.06.2015 Characterization of devant le jury composé de : Aude ECHALIER-GLAZER the cellular network Maître de conférence University of Leicester / rapporteur of ubiquitin Lionel PINTARD Directeur de recherche
    [Show full text]
  • The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement
    International Journal of Molecular Sciences Review The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement 1, , 2, 3,4 Sara Bachiller * y , Isabel M. Alonso-Bellido y , Luis Miguel Real , Eva María Pérez-Villegas 5 , José Luis Venero 2 , Tomas Deierborg 1 , José Ángel Armengol 5 and Rocío Ruiz 2 1 Experimental Neuroinflammation Laboratory, Department of Experimental Medical Science, Lund University, Sölvegatan 19, 221 84 Lund, Sweden; [email protected] 2 Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla/Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (I.M.A.-B.); [email protected] (J.L.V.); [email protected] (R.R.) 3 Unidad Clínica de Enfermedades Infecciosas, Hospital Universitario de Valme, 41014 Sevilla, Spain; [email protected] 4 Departamento de Especialidades Quirúrgicas, Bioquímica e Inmunología, Facultad de Medicina, 29071 Universidad de Málaga, Spain 5 Departamento de Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, 41013 Sevilla, Spain; [email protected] (E.M.P.-V.); [email protected] (J.Á.A.) * Correspondence: [email protected] These authors contributed equally to the work. y Received: 14 July 2020; Accepted: 31 August 2020; Published: 3 September 2020 Abstract: Neuromuscular disorders (NMDs) affect 1 in 3000 people worldwide. There are more than 150 different types of NMDs, where the common feature is the loss of muscle strength. These disorders are classified according to their neuroanatomical location, as motor neuron diseases, peripheral nerve diseases, neuromuscular junction diseases, and muscle diseases. Over the years, numerous studies have pointed to protein homeostasis as a crucial factor in the development of these fatal diseases.
    [Show full text]
  • UBE2A (HR6A) [GST-Tagged] E2 – Ubiquitin Conjugating Enzyme
    UBE2A (HR6A) [GST-tagged] E2 – Ubiquitin Conjugating Enzyme Alternate Names: HHR6A, HR6A, RAD6A, UBC2, EC 6.3.2.19, Ubiquitin-conjugating enzyme E2A Cat. No. 62-0001-020 Quantity: 20 µg Lot. No. 1384 Storage: -70˚C FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Background Physical Characteristics The enzymes of the ubiquitylation pathway Species: human Protein Sequence: play a pivotal role in a number of cellular MSPILGYWKIKGLVQPTRLLLEYLEEKYEEH processes including the regulated and tar- Source: E. coli expression LYERDEGDKWRNKKFELGLEFPNLPYYIDGD geted proteosomal degradation of sub- VKLTQSMAIIRYIADKHNMLGGCPKER strate proteins. Three classes of enzymes Quantity: 20 µg AEISMLEGAVLDIRYGVSRIAYSKDFETLKVD are involved in the process of ubiquityla- FLSKLPEMLKMFEDRLCHKTYLNGDHVTHP tion; activating enzymes (E1s), conjugating Concentration: 1 mg/ml DFMLYDALDVVLYMDPMCLDAFPKLVCFK enzymes (E2s) and protein ligases (E3s). KRIEAIPQIDKYLKSSKYIAWPLQGWQATFG UBE2A is a member of the E2 conjugating Formulation: 50 mM HEPES pH 7.5, GGDHPPKSDLEVLFQGPLGSPNSRVDSTPAR enzyme family and cloning of the human 150 mM sodium chloride, 2 mM RRLMRDFKRLQEDPPAGVSGAPSENNIMVW gene was first described by Koken et al. dithiothreitol, 10% glycerol NAVIFGPEGTPFEDGTFKLTIEFTEEYPNKPPT (1991). UBE2A shares 70% identity with VRFVSKMFHPNVYADGSICLDILQNRWSPTYD its yeast homologue but lacks the acidic c- Molecular Weight: ~45 kDa VSSILTSIQSLLDEPNPNSPANSQAAQLYQENK terminal domain. The ring finger proteins REYEKRVSAIVEQSWRDC RAD5 and RAD18 interact with UBE2A and Purity: >98% by InstantBlue™ SDS-PAGE Tag (bold text): N-terminal glutathione-S-transferase (GST) other members of the RAD6 pathway (Ul- Protease cleavage site: PreScission™ (LEVLFQtGP) rich and Jentsch, 2000). Phosphorylation of Stability/Storage: 12 months at -70˚C; UBE2A (regular text): Start bold italics (amino acid UBE2A by CDK1 and 2 increases its activ- aliquot as required residues 2-152) Accession number: NP_003327 ity during the G2/M phase of the cell cycle (Sarcevic et al., 2002).
    [Show full text]
  • (USP7) in Histone H2A Ubiquitination
    Characterization of E2E Ubiquitin-Conjugating Enzymes and Ubiquitin-Specific Protease 7 (USP7) in Histone H2A Ubiquitination Hossein Davarinejad A thesis submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Science Graduate Program in Biology York University Toronto, Ontario December 2017 © Hossein Davarinejad, 2017 Abstract Ubiquitin(Ub)-conjugating E2 enzymes play essential roles in ubiquitination of proteins. The UbE2E sub-family members UbE2E1, UbE2E2, and UbE2E3 have N-terminal extensions to the conserved E2 core which contain Ubiquitin-Specific Protease 7 (USP7) binding sequences (P/A/ExxS). Here, we continued our investigations to established that USP7 can interact with E2Es in vitro and in vivo. Our new data indicated that the N-terminal extensions of E2E2 or E2E3 can directly associate with USP7 TRAF domain. We demonstrated that E2E2 or E2E3 are stabilized by USP7 in cells. We also showed that E2Es interact with Ring1B:BMI1, the core components of the Polycomb Repressive Complex 1 (PRC1) and established E2E1 as an in vivo E2 for monoubiquitination of histone H2A on lysine(K) 119. We demonstrated that E2Es can modulate the levels of H2A monoubiquitination in cells and that USP7 may exert an effect on K119-UbH2A levels through regulating E2Es. ii Dedications For my dearest, and departed grandmother. Your memories, love, and grace are eternal. I also dedicate this to Agnieszka, GhoorGhoor, and Charlie for their invaluable support. And for science; may our collective and ever developing understanding of the universe and life lead us to make the world a better and a more peaceful place, for all life is precious.
    [Show full text]
  • Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay
    Copyright by John Francis Anderson 2011 THE ROLE OF SACSIN AS A MOLECULAR CHAPERONE by John Francis Anderson, B.S. Dissertation Presented to the Faculty of the Graduate School of The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas Medical Branch May 2011 Dedication To my wife, Erika R. Anderson. Acknowledgements I am indebted to my mentor, Dr. José M. Barral for his investment in my education. Dr. Barral taught me that curiosity drives scientific investigation and rigorous experiments derive new knowledge. This combination of values is rare to find in a mentor and I am extremely grateful for my experience in his lab. I would like to acknowledge Jason J. Chandler and Paige Spencer for technical assistance. I am especially grateful to Dr. Efrain Siller for daily assistance and discussions on all aspects of this project. I would like to thank Dr. Christian Kaiser providing me a practical education on the design and performance biochemical experiments. I acknowledge Dr. Bernard Brais for generously sharing his time and resources; he provided the rare opportunity to visit an ARSACS patient in her home as well as supplied us with SACS knockout mouse brains. My committee members, Dr. Henry F. Epstein, Dr. Andres F. Oberhauser, Dr. George R Jackson, and Dr. Robert O. Fox and Dr. Darren F. Boehning provided invaluable guidance throughout this work for which I am very grateful. I would like to especially thank Dr. Darren F. Boehning and Dr. Henry F. Epstein for being involved in the details of my project from the beginning of my education at UTMB, through sharing reagents and expertise as well as serving on my committee.
    [Show full text]
  • Structural and Functional Analyses of the Interaction Between the Usp7-Ntd and the E2-Conjugating Enzymes, Ube2e2 and Ube2e3
    STRUCTURAL AND FUNCTIONAL ANALYSES OF THE INTERACTION BETWEEN THE USP7-NTD AND THE E2-CONJUGATING ENZYMES, UBE2E2 AND UBE2E3 LEILA ASHUROV A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGY YORK UNIVERSITY TORONTO, ONTARIO AUGUST 2014 © LEILA ASHUROV, 2014 ABSTRACT Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme that regulates the turnover of proteins in a cell. To date several interacting partners that are involved in various cellular processes have been identified for USP7. Many of the interacting partners of USP7 contain a P/AxxS motif. We have identified two E2 ubiquitin- conjugating enzymes, UbE2E2 and UbE2E3, which contain the P/AxxS motif. Using a co-immunoprecipitation assay we have shown that these E2 proteins interact with USP7 through their P/AxxS motif. Co-crystal structures of the N-terminal domain of USP7 (USP7-NTD) and the E2 peptides reveal that the interactions are formed between USP7- NTD residues 164DWGF167 and the E2 P/AxxS motifs. It has also been established that USP7 may be involved in regulating the cellular levels of UbE2E2. Overall, our findings suggest USP7, a de-ubiquitinating enzyme, is a regulator of ubiquitin-conjugating enzymes, which are involved in the ubiquitination cascade. ii ACKNOWLEDGEMENTS I would like to give my utmost gratitude to my Supervisor Dr. Vivian Saridakis. Thank you for taking me into your lab and providing me with this tremendous opportunity. Your constant support and guidance has allowed me to achieve my goals and complete this project.
    [Show full text]