DOCSLIB.ORG

SUMO E3 Ligase Activity of TRIM Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SUMO E3 Ligase Activity of TRIM Proteins Oncogene (2011) 30, 1108–1116 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE SUMO E3 ligase activity of TRIM proteins Y Chu1 and X Yang Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA SUMOylation governs numerous cellular processes and enzyme E1 (a heterodimer of Aos1/SAE1 and Uba2/ is essential to most eukaryotic life. Despite increasing SAE2), the SUMO-conjugating enzyme E2 (Ubc9) and recognition of the importance of this process, an one of the SUMO ligases or E3s. SUMO E3s confer extremely limited number of small ubiquitin-like modifier SUMOylation specificity and efficiency. However, in (SUMO) protein ligases (E3s) have been identified. Here contrast to the over 600 known ubiquitin E3s (Deshaies we show that at least some members of the functionally and Joazeiro, 2009), only a handful of SUMO E3s diverse tripartite motif (TRIM) superfamily are SUMO have been reported. These include protein inhibitor of E3s. These TRIM proteins bind both the SUMO- activated STAT proteins (PIAS proteins) (Hochstrasser, conjugating enzyme Ubc9 and substrates and strongly 2001; Johnson and Gupta, 2001; Kahyo et al., 2001), the enhance transfer of SUMOs from Ubc9 to these nuclear pore protein RanBP2 (Pichler et al., 2002) and substrates. Among the substrates of TRIM SUMO E3s the polycomb group protein Pc2 (Kagey et al., 2003). are the tumor suppressor p53 and its principal antagonist With the identification of numerous SUMOylation targets Mdm2. The E3 activity depends on the TRIM motif, (Golebiowski et al., 2009), it remains unclear whether a suggesting it to be the first widespread SUMO E3 motif. large number of SUMO E3s also exist and thus, how the Given the large number of TRIM proteins, our results specificity and efficiency of SUMOylation are achieved. may greatly expand the identified SUMO E3s. Further- Tripartite motif-containing (TRIM) proteins form more, TRIM E3 activity may be an important contributor a superfamily in metazoans with B20 members in to SUMOylation specificity and the versatile functions of Caenorhabditis elegans and B65 members in humans TRIM proteins. and mice (Ozato et al., 2008). These proteins are defined Oncogene (2011) 30, 1108–1116; doi:10.1038/onc.2010.462; by the TRIM/RBCC motif of a RING domain, one or published online 25 October 2010 two B-box domains (which, like the RING domain, are zinc-binding domains) and a predicted coiled-coil Keywords: TRIM proteins; SUMO E3 ligase; sumoylation; region. These domains are invariably arranged in the PML; Mdm2; p53 stated order and present at the N-terminal region of these proteins followed by a more variable C-terminal region. TRIM proteins have important roles in a wide range of processes including cell growth, tumor suppres- Introduction sion, DNA damage signaling, senescence, apoptosis, stem cell differentiation and immune responses against Covalent conjugation to small ubiquitin-like modifier viral, and particularly human immunodeficiency virus (SUMO) proteins is a common post-translational infection (Salomoni and Pandolfi, 2002; Dellaire and modification that alters various properties of the target Bazett-Jones, 2004; Nisole et al., 2005; Ozato et al., proteins, including stability, activity, cellular localiza- 2008; Schwamborn et al., 2009). For example, the tion and protein-protein interactions (Johnson, 2004; promyelocytic leukemia protein (PML), also known as Hay, 2005; Geiss-Friedlander and Melchior, 2007). TRIM19, a prototypical TRIM protein, is involved in a Mammalian cells expresses three SUMO proteins chromosomal translocation associated with the vast (SUMO 1–3). SUMO2 and SUMO3 are nearly identical majority of acute promyelocytic leukemia. PML is the in their sequence and function and share about B50% eponymous and main structural component of the PML sequence identity with SUMO1. Similar to the ubiqui- nuclear bodies, whose mechanism of action still remains tination pathway, the SUMO pathway consists of three elusive (Borden, 2002; Bernardi and Pandolfi, 2007). enzymatic steps performed by the SUMO-activating Likewise, TRIM27 (also known as Ret finger protein or RFP), acquires oncogenic activity when it is fused to the Ret receptor tyrosine kinase (Takahashi et al., 1985). Correspondence: Dr X Yang, Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania Other examples include the potent anti-human immu- School of Medicine, 610 BRB II/III, 421 Curie Boulevard, Philadelphia, nodeficiency virus infection mediated by rhesus monkey PA 19096, USA. TRIM5-a (Stremlau et al., 2004), suppression of stem E-mail: [email protected] cell differentiation by TRIM32 (Schwamborn et al., 1Current address: Department of Pediatrics, Columbia University, New York, NY, USA. 2009) and the silencing of viral replication in stem cells Received 14 July 2010; revised 23 August 2010; accepted 25 August 2010; by TRIM28 (Wolf and Goff, 2007). However, despite an published online 25 October 2010 increasing awareness of TRIM proteins’ important SUMO E3 ligase activity Y Chu and X Yang 1109 cellular functions, the biochemical basis for these may be a SUMO E3. We first examined whether PML functions is poorly understood. Certain TRIM proteins stimulates substrate SUMOylation in mammalian cells. exhibit ubiquitin E3 activity, which is attributed to The tumor suppressor p53 is a known SUMOylation the RING domain (Meroni and Diez-Roux, 2005). The substrate (Rodriguez et al., 1999). We expressed p53 and properties of the other motifs with the characteristic the glutathione-S-transferase (GST) fusion of SUMO1 TRIM region remain unknown. In the current study, in PmlÀ/À MEF cells in the presence or absence of PML we show that TRIM proteins are a new class of isoform IV (called PML in this study). This isoform was SUMO E3s. Unlike the other SUMO E3s, the activity of chosen because it can directly bind to p53 (Rodriguez TRIM proteins requires intact RING and B-box et al., 1999). In the presence but not absence of PML, domains. Our results provide a biochemical framework a slower migrating form of p53 was detected with a for understanding SUMOylation and the function of size expected for its conjugation to GST-SUMO1 TRIM proteins. (Figure 1a). In a similar experiment where Flag-tagged SUMO1 was used, p53 conjugation to Flag-SUMO1 was also strongly increased by the coexpressed PML Results (Figure 1a). Conjugation occurred mainly at a pre- viously determined p53 SUMOylation site, Lys386 PML stimulates substrate SUMOylation in mammalian (Rodriguez et al., 1999), as change of this site to Arg cells abolished the SUMO conjugation (Figure 1a). PML is heavily modified by SUMO, and overexpression To confirm that PML stimulates SUMOylation of PML in yeast cells enhanced overall SUMOylation of p53, we expressed p53 and 6xHis-tagged SUMO1 (Kamitani et al., 1998; Muller et al., 2000; Quimby et al., (His-SUMO1), alone or together with PML, in the 2006). These observations led us to reason that PML p53-deficient human lung cancer H1299 cells. The Flag-p53 WT KR Flag-PML -+-+-+-+ Flag-PML(VI) ----+++ GST-SUMO1 ++- - - - -- Flag-PML(IV) - +++++- - Flag-SUMO1 --++--++ p53-GST His-SUMO1 - ++-++ -SUMO1 75 p53- 100 SUMO1 p53-Flag 75 p53 -SUMO1 50 p53 50 250 PML- SUMO1 250 PML- 150 100 PML(IV) 150 SUMO1 75 PML(VI) 100 PML GFP GFP 123456 1 2 3 4 5 6 7 8 HA-p53 + ++ - ++ WCL Ni beads Flag-PML - ++++ + + HA-PML - + ++ + His-SUMO1 + +++ - - -++ His-SUMO1 +++- Flag-SUMO1 -----+ +++ Flag-Mdm2 ++++ +++ p53-His 75 -SUMO1 150 Mdm2- 150 SUMO1 Ni beads 50 100 100 p53- 75 Mdm2 75 SUMO1 75 p53 50 250 PML- 250 PML- 150 SUMO1 150 WCL SUMO1 100 100 PML PML 100 75 75 567 Actin GFP GFP 123456 1234 Figure 1 PML promotes SUMOylation of p53 and Mdm2 in vivo.(a) PmlÀ/À MEF cells were transfected with Flag-PML, p53 and SUMO1 as indicated. Cells were treated with proteasome inhibitor MG132 for 4 h (same below), and whole-cell lysates (WCL) were analyzed by western blot. Molecular weight standards (in kDa) are shown on the left. (b) H1299 cells were transfected with Flag-PML, HA-p53 and His- or Flag-tagged SUMO1 as indicated. Cells were lysed in buffer containing guanidinium-HCl and proteins conjugated to His-SUMO1 were pulled down by Ni2 þ -NTA beads (Ni beads). The bead-bound proteins and WCL were analyzed by western blots. (c) H1299 cells were transfected with PML isoform IV or VI, p53, SUMO1 and GFP as indicated. WCL were analyzed by western blot. (d) PmlÀ/À cells were transfected with HA-PML, Flag-Mdm2 and His-SUMO1 as indicated. Cell lysates were incubated by Ni2 þ -NTA beads. WCL (left) and the bead-bound proteins (right) were analyzed by western blots. Oncogene SUMO E3 ligase activity Y Chu and X Yang 1110 His-SUMO1-conjugated p53 protein was then captured SUMO1 SUMO2 SUMO1 by Ni-NTA beads (nickel-charged affinity resins) under PML +-+ + + - PML +-+ denaturing conditions and analyzed by western blots. Ubc9 - + + - + + Ubc9 -++ 75 150 PML enhanced conjugation of p53 to His-SUMO1 in a 100 p53- SUMO SUMO1 75 dose-dependent manner (Figure 1b, top panel). The -Mdm2 specificity of the Ni-NTA capture was verified by the 100 p53 lack of p53 binding to the beads when His-SUMO1 was 50 50 123 replaced by Flag-SUMO1 (Figure 1b). Furthermore, a 123 456 shorter PML isoform (isoform VI, Figure 3a) that cannot interact with p53, failed to stimulate p53 SUMOylation SUMO1 SUMO2 GST -+- -+- ---+ (Figure 1c). In these experiments, PML was also GST-PML + + - + + - + + ++ - conjugated to SUMO1, as expected (Figures 1a–d). Ubc9 +++ -++ -+++ To examine the generality of PML’s ability to E1 - + + + + + +++ + 75 p53- enhance SUMOylation in vivo, we tested two other SUMO SUMOylation substrates: the ubiquitin ligase Mdm2 p53 and the transcriptional factor c-Jun (Muller et al., 2000), 50 both of which interact with PML (Bernardi et al., 1 2 3 45678910 2004; Salomoni et al., 2005).
Load more

Recommended publications
  • Yeast Genome Gazetteer P35-65
    gazetteer Metabolism 35 tRNA modification mitochondrial transport amino-acid metabolism other tRNA-transcription activities vesicular transport (Golgi network, etc.) nitrogen and sulphur metabolism mRNA synthesis peroxisomal transport nucleotide metabolism mRNA processing (splicing) vacuolar transport phosphate metabolism mRNA processing (5’-end, 3’-end processing extracellular transport carbohydrate metabolism and mRNA degradation) cellular import lipid, fatty-acid and sterol metabolism other mRNA-transcription activities other intracellular-transport activities biosynthesis of vitamins, cofactors and RNA transport prosthetic groups other transcription activities Cellular organization and biogenesis 54 ionic homeostasis organization and biogenesis of cell wall and Protein synthesis 48 plasma membrane Energy 40 ribosomal proteins organization and biogenesis of glycolysis translation (initiation,elongation and cytoskeleton gluconeogenesis termination) organization and biogenesis of endoplasmic pentose-phosphate pathway translational control reticulum and Golgi tricarboxylic-acid pathway tRNA synthetases organization and biogenesis of chromosome respiration other protein-synthesis activities structure fermentation mitochondrial organization and biogenesis metabolism of energy reserves (glycogen Protein destination 49 peroxisomal organization and biogenesis and trehalose) protein folding and stabilization endosomal organization and biogenesis other energy-generation activities protein targeting, sorting and translocation vacuolar and lysosomal
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Identifying SUMO Protease Targets and Investigating E3 Ligase Interactions
    W&M ScholarWorks Dissertations, Theses, and Masters Projects Theses, Dissertations, & Master Projects 2014 Identifying SUMO Protease Targets and Investigating E3 Ligase Interactions Mark Guillotte College of William & Mary - Arts & Sciences Follow this and additional works at: https://scholarworks.wm.edu/etd Part of the Molecular Biology Commons Recommended Citation Guillotte, Mark, "Identifying SUMO Protease Targets and Investigating E3 Ligase Interactions" (2014). Dissertations, Theses, and Masters Projects. Paper 1539626956. https://dx.doi.org/doi:10.21220/s2-wrgj-tz43 This Thesis is brought to you for free and open access by the Theses, Dissertations, & Master Projects at W&M ScholarWorks. It has been accepted for inclusion in Dissertations, Theses, and Masters Projects by an authorized administrator of W&M ScholarWorks. For more information, please contact [email protected]. Identifying SUMO protease targets and investigating E3 ligase interactions Mark Guillotte Baton Rouge, Louisiana Bachelors of Science, Louisiana State University, 2010 A Thesis presented to the Graduate Faculty of the College of William and Mary in Candidacy for the Degree of Master of Science Department of Biology The College of William and Mary January 2014 APPROVAL PAGE This Thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science Mark Guillotte Approved by^he Committee, January 2014 (be Chair Associate Professor Oliver Kerseher, Biology The College of William and Mary ' oVY wG ..G S l1m>. rofessor Lizabeth Allison, Biology The College of William and Mary Professor Diane Shakes, Biology The College of William and Mary ---------- Assistant Professor Shanta Hinton,Biology The College of William and Mary COMPLIANCE PAGE Research approved by Steve Kaattari Protocol number(s): IBC-2012-10-08-8156-opkers Date(s) of approval: 2013-11-02 ABSTRACT Posttranslational modification by the Small Ubiquitin-like Modifier (SUMO) is a pervasive mechanism for controlling protein function.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance
    biomolecules Review Understanding and Exploiting Post-Translational Modifications for Plant Disease Resistance Catherine Gough and Ari Sadanandom * Department of Biosciences, Durham University, Stockton Road, Durham DH1 3LE, UK; [email protected] * Correspondence: [email protected]; Tel.: +44-1913341263 Abstract: Plants are constantly threatened by pathogens, so have evolved complex defence signalling networks to overcome pathogen attacks. Post-translational modifications (PTMs) are fundamental to plant immunity, allowing rapid and dynamic responses at the appropriate time. PTM regulation is essential; pathogen effectors often disrupt PTMs in an attempt to evade immune responses. Here, we cover the mechanisms of disease resistance to pathogens, and how growth is balanced with defence, with a focus on the essential roles of PTMs. Alteration of defence-related PTMs has the potential to fine-tune molecular interactions to produce disease-resistant crops, without trade-offs in growth and fitness. Keywords: post-translational modifications; plant immunity; phosphorylation; ubiquitination; SUMOylation; defence Citation: Gough, C.; Sadanandom, A. 1. Introduction Understanding and Exploiting Plant growth and survival are constantly threatened by biotic stress, including plant Post-Translational Modifications for pathogens consisting of viruses, bacteria, fungi, and chromista. In the context of agriculture, Plant Disease Resistance. Biomolecules crop yield losses due to pathogens are estimated to be around 20% worldwide in staple 2021, 11, 1122. https://doi.org/ crops [1]. The spread of pests and diseases into new environments is increasing: more 10.3390/biom11081122 extreme weather events associated with climate change create favourable environments for food- and water-borne pathogens [2,3]. Academic Editors: Giovanna Serino The significant estimates of crop losses from pathogens highlight the need to de- and Daisuke Todaka velop crops with disease-resistance traits against current and emerging pathogens.
    [Show full text]
  • UBA2 Activates Wnt/ Β-Catenin Signaling Pathway
    UBA2 Activates Wnt/ β-catenin Signaling Pathway during Protection of R28 Retinal Precursor Cells from Hypoxia by Extracellular Vesicles derived from Placental Mesenchymal Stem Cells Kyungmin Koh CHA University Mira Park CHA University Eun Soo Bae CHA University Van-An Duong Gachon University Jong-Moon Park Gachon University Hookeun Lee Gachon University Helen Lew ( [email protected] ) CHA university https://orcid.org/0000-0003-0121-3000 Research Keywords: Exosome, hPMSCs, Optic nerve injury, Retinal precursor cells, UBA2, Wnt/β-catenin DOI: https://doi.org/10.21203/rs.3.rs-33694/v3 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/23 Abstract Background: Stem cell transplantation has been proposed as an alternative treatment for intractable optic nerve disorders characterized by irrecoverable loss of cells. Mesenchymal stem cells, with varying tissue regeneration and recovery capabilities, are being considered for potential cell therapies. To overcome the limitations of cell therapy, we isolated exosomes from human placenta–derived mesenchymal stem cells (hPMSCs), and investigated their therapeutic effects in R28 cells (retinal precursor cells) exposed to CoCl2. Method: After nine hours of exposure to CoCl2, the hypoxic damaged R28 cells were divided into non treatment group (CoCl2+R28 cells) and treatment group (CoCl2+R28 cells treated with exosome). Immunoblot analysis was performed for Pcna, Hif-1α, Vegf, Vimentin, Thy-1, Gap43, Ermn, Neuro≈ament, Wnt3a, β-catenin, phospo-GSK3β, Lef-1, UBA2, Skp1, βTrcp, and ubiquitin. The proteomes of each group were analyzed by liquid chromatography/tandem mass (LC-MS/MS) spectrometry. Differentially expressed proteins (DEPs) were detected by label-free quantiƒcation and the interactions of the proteins were examined through signal transduction pathway and gene ontology analysis.
    [Show full text]
  • Ginkgolic Acid, a Sumoylation Inhibitor, Promotes Adipocyte
    www.nature.com/scientificreports OPEN Ginkgolic acid, a sumoylation inhibitor, promotes adipocyte commitment but suppresses Received: 25 October 2017 Accepted: 15 January 2018 adipocyte terminal diferentiation Published: xx xx xxxx of mouse bone marrow stromal cells Huadie Liu1,2, Jianshuang Li2, Di Lu2, Jie Li1,2, Minmin Liu 3, Yuanzheng He4, Bart O. Williams2, Jiada Li1 & Tao Yang 2 Sumoylation is a post-translational modifcation process having an important infuence in mesenchymal stem cell (MSC) diferentiation. Thus, sumoylation-modulating chemicals might be used to control MSC diferentiation for skeletal tissue engineering. In this work, we studied how the diferentiation of mouse bone marrow stromal cells (mBMSCs) is afected by ginkgolic acid (GA), a potent sumoylation inhibitor also reported to inhibit histone acetylation transferase (HAT). Our results show that GA promoted the diferentiation of mBMSCs into adipocytes when cultured in osteogenic medium. Moreover, mBMSCs pre-treated with GA showed enhanced pre-adipogenic gene expression and were more efciently diferentiated into adipocytes when subsequently cultured in the adipogenic medium. However, when GA was added at a later stage of adipogenesis, adipocyte maturation was markedly inhibited, with a dramatic down-regulation of multiple lipogenesis genes. Moreover, we found that the efects of garcinol, a HAT inhibitor, difered from those of GA in regulating adipocyte commitment and adipocyte maturation of mBMSCs, implying that the GA function in adipogenesis is likely through its activity as a sumoylation inhibitor, not as a HAT inhibitor. Overall, our studies revealed an unprecedented role of GA in MSC diferentiation and provide new mechanistic insights into the use of GA in clinical applications.
    [Show full text]
  • Trim24 Targets Endogenous P53 for Degradation
    Trim24 targets endogenous p53 for degradation Kendra Alltona,b,1, Abhinav K. Jaina,b,1, Hans-Martin Herza, Wen-Wei Tsaia,b, Sung Yun Jungc, Jun Qinc, Andreas Bergmanna, Randy L. Johnsona,b, and Michelle Craig Bartona,b,2 aDepartment of Biochemistry and Molecular Biology, Program in Genes and Development, Graduate School of Biomedical Sciences and bCenter for Stem Cell and Developmental Biology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030; and cDepartment of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030 Edited by Carol L. Prives, Columbia University, New York, NY, and approved May 15, 2009 (received for review December 23, 2008) Numerous studies focus on the tumor suppressor p53 as a protector regulator of p53 suggests that potential therapeutic targets to of genomic stability, mediator of cell cycle arrest and apoptosis, restore p53 functions remain to be identified. and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and Results and Discussion regulation, comes from studies of tumor-derived, cultured cells Creation of a Mouse and Stem Cell Model for p53 Analysis. To where p53 and its regulatory controls may be mutated or dysfunc- facilitate analysis of endogenous p53 in normal cells, we per- tional. To address regulation of endogenous p53 in normal cells, formed gene targeting to create mouse embryonic stem (ES) we created a mouse and stem cell model by knock-in (KI) of a cells and mice (12), which express endogenously regulated p53 tandem-affinity-purification (TAP) epitope at the endogenous protein fused with a C-terminal TAP tag (p53-TAPKI, Fig.
    [Show full text]
  • Insights Into the Role and Mechanism of the AAA+ Adaptor Clps
    Insights into the role and mechanism of the AAA+ adaptor ClpS by Jennifer Yuan Hou Sc.B. Biochemistry Brown University, 2002 SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY JUNE 2009 © 2009 Jennifer Yuan Hou. All Rights Reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of Author:_______________________________________________________ Department of Biology May 22, 2009 Certified by:_____________________________________________________________ Tania A. Baker E. C. Whitehead Professor of Biology Thesis Supervisor Accepted by:_____________________________________________________________ Stephen P. Bell Professor of Biology Co-Chair, Graduate Committee 1 2 Insights into the role and mechanism of the AAA+ adaptor ClpS by Jennifer Yuan Hou Submitted to the Department of Biology on May 22, 2009 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the Massachusetts Institute of Technology ABSTRACT Protein degradation is a vital process in cells for quality control and participation in regulatory pathways. Intracellular ATP-dependent proteases are responsible for regulated degradation and are highly controlled in their function, especially with respect to substrate selectivity. Adaptor proteins that can associate with the proteases add an additional layer of control to substrate selection. Thus, understanding the mechanism and role of adaptor proteins is a critical component to understanding how proteases choose their substrates. In this thesis, I examine the role of the intracellular protease ClpAP and its adaptor ClpS in Escherichia coli.
    [Show full text]
  • UBA2 Promotes Proliferation of Colorectal Cancer
    5552 MOLECULAR MEDICINE REPORTS 18: 5552-5562, 2018 UBA2 promotes proliferation of colorectal cancer PING HE1, XUN SUN2, HONG-JING CHENG1, YA‑BIN ZOU2, QUAN WANG3, CHANG‑LI ZHOU1, WAN-QI LIU1, YUE-MING HAO1 and XIANG-WEI MENG1 Departments of 1Gastroenterology, 2Pathology and 3Gastrointestinal Surgery, Bethune First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China Received December 18, 2017; Accepted August 13, 2018 DOI: 10.3892/mmr.2018.9613 Abstract. Small ubiquitin‑like modifier proteins are involved Introduction in tumorigenesis; however, the potential effects and func- tions of the family member ubiquitin‑like modifier‑activating Colorectal cancer is the third most common cancer world- enzyme 2 (UBA2) on colorectal cancer are not clear. The wide (1); the lifetime risk of developing colorectal cancer is present study aimed to examine the effects of UBA2 on the 4.7% for men and 4.4% for women. Although the mortality proliferation of colorectal cancer cells in vitro and in vivo. The rate from colorectal cancer has been declining for several mRNA and protein expression levels of UBA2 in patients with decades owing to the early diagnosis and improved treatment, colorectal cancer were measured by reverse transcription‑quan- >1 million novel cases are diagnosed each year. Therefore, it titative polymerase chain reaction and immunohistochemistry, is crucial to identify novel biomarkers and therapeutic targets respectively. UBA2 expression levels in colorectal cancer for colorectal cancer to improve the prognosis of the disease. tissues were significantly increased compared with the para- Sumoylation is a transient post‑translational modifica- cancerous normal tissues. The expression of UBA2 was also tion process that is highly regulated by the balance between associated with higher stage colorectal cancer and poor prog- enzyme‑mediated conjugating and deconjugating activities.
    [Show full text]
  • Sumoylation and Phosphorylation Cross-Talk in Hepatocellular Carcinoma
    Review Article SUMOylation and phosphorylation cross-talk in hepatocellular carcinoma Maria Lauda Tomasi, Komal Ramani Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA Contributions: (I) Conception and design: All authors; (II) Administrative support: All authors; (III) provision of study materials or patients: All authors; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Maria Lauda Tomasi, PhD; Komal Ramani, PhD. Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Email: [email protected]; [email protected]. Abstract: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and occurs predominantly in patients with underlying chronic liver disease and cirrhosis. The large spectrum of protein post- translational modification (PTM) includes numerous critical signaling events that occur during neoplastic transformation. PTMs occur to nearly all proteins and increase the functional diversity of proteins. We have reviewed the role of two major PTMs, SUMOylation and phosphorylation, in the altered signaling of key players in HCC. SUMOylation is a PTM that involves addition of a small ubiquitin-like modifiers (SUMO) group to proteins. It is known to regulate protein stability, protein-protein interactions, trafficking and transcriptional activity. The major pathways that are regulated by SUMOylation and may influence HCC are regulation of transcription, cell growth pathways associated with B-cell lymphoma 2 (Bcl-2) and methionine adenosyltransferases (MAT), oxidative stress pathways [nuclear erythroid 2-related factor 2 (Nrf2)], tumor suppressor pathways (p53), hypoxia-inducible signaling [hypoxia-inducible factor-1 (HIF-1)], glucose and lipid metabolism, nuclear factor kappa B (NF-κB) and β-Catenin signaling.
    [Show full text]
  • The Cycle of Protein Engineering: Bioinformatics Design of Two Dimeric Proteins and Computational Design of a Small Globular Domain
    The Cycle of Protein Engineering: Bioinformatics Design of Two Dimeric Proteins and Computational Design of a Small Globular Domain DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Venuka Durani, M.Sc. Graduate Program in Chemistry The Ohio State University 2012 Dissertation Committee: Thomas J. Magliery, Advisor Ross E. Dalbey Karin Musier-Forsyth William C. Ray Copyright by Venuka Durani 2012 Abstract The protein folding problem is an ongoing challenge, and even though there have been significant advances in our understanding of proteins, accurately predicting the effect of amino acid mutations on the structure and stability of a protein remains a challenge. This makes the task of engineering proteins to suit our purposes labor intensive as significant trial and error is involved. In this thesis, we have explored possibilities of better understanding and if possible improving some bioinformatics and computational methods to study proteins and also to engineer them. A significant portion of this thesis is based on bioinformatics approaches involving consensus and correlation analyses of multiple sequence alignments. We have illustrated how consensus and correlation metrics can be calculated and analyzed to explore various aspects of protein structure. Proteins triosephosphate isomerase and Cu, Zn superoxide dismutase were studied using these approaches and we found that a significant amount of information about a protein fold is encoded at the consensus level; however, the effect of amino acid correlations, while subtle, is significant nonetheless and some of the failures of consensus approach can be attributed to broken amino acid correlations.
    [Show full text]