DOCSLIB.ORG

Tumor Susceptibility Gene-101 Regulates Glucocorticoid Receptor Through Disorder-Mediated Allostery

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Tumor Susceptibility Gene-101 Regulates Glucocorticoid Receptor Through Disorder-Mediated Allostery bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931485; this version posted February 3, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. Tumor susceptibility gene-101 regulates glucocorticoid receptor through disorder-mediated allostery Jordan T. White1, James Rives2, Marla E. Tharp1, James O. Wrabl1, E. Brad Thompson1,3, Vincent J. Hilser*1,4 1Department of Biology at Johns Hopkins University, Baltimore, MD 21218; 2Department of Chemistry at Johns Hopkins University; 3Sealy Center for Structural Biology and Molecular Biophysics in the Department of Biochemistry and Molecular Biology at Univ. of Texas Medical Branch, Galveston, TX; 4T. C. Jenkins Department of Biophysics at Johns Hopkins University Running title: TSG101 allosterically regulates GR *To whom correspondence should be addressed: Vincent Hilser: Department of Biology, Johns Hopkins University, Baltimore MD 21218; [email protected]; Tel.(410)-516-6072 Keywords: glucocorticoid receptor, tumor susceptibility gene-101, allostery, allosteric regulation, transcription, transcription coregulator, transcription regulation, thermodynamics, biophysics Abstract Introduction Tumor Susceptibility Gene-101 (TSG101) is Transcription factors (TFs) are multi- involved in endosomal maturation and has been domain, allosteric proteins, in which the binding implicated in the transcriptional regulation of of one effector ligand to its specific domain causes several steroid hormone receptors (SHRs), a conformational change in one or more other although a detailed characterization of such domains, so as to alter transcription. Most TFs regulation has yet to be conducted. Here we contain intrinsically disordered regions (IDRs), directly measure binding of TSG101 to one SHR, which though critical for TF function, are still not glucocorticoid receptor (GR). Using biophysical well understood (1). Generally, TF IDRs consist and cellular assays, we show that the coiled-coil of large ensembles of rapidly interconverting domain of TSG101; 1) binds and folds the conformers, including in many cases, conformers disordered N-terminal domain (NTD) of GR, 2) that contain some degree of structure. By upon binding, improves DNA-binding of GR in definition then, allostery arises due to an effector- vitro, and 3) enhances the transcriptional activity driven shift in the distribution of states such that of GR in vivo. Our findings suggest that TSG101 states that are capable of initiating transcription is a bona fide transcriptional co-regulator of GR. are either promoted or repressed, thus affecting transcriptional activity accordingly (2,3). We have adopted the glucocorticoid receptor (GR) as a model system for our studies because the NTD of GR is known to be intrinsically disordered (ID) 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931485; this version posted February 3, 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. TSG101 allosterically regulates GR and also to be critical for the TF function of the which adopts the canonical zinc-finger binding receptor. motif (17,18). Conversely, we showed that The GR is a 3-domain protein comprised although the NTD of GR appears in its entirety to of (from N to C terminus): N-terminal (NTD), be an IDR, it actually consists of two adjacent, but DNA-binding (DBD), and ligand-binding domains functionally distinct IDRs. The first IDR, which (LBD), where L in this case refers to steroid or we call the functional (or F-domain) includes steroid substitute. However, if the LBD is deleted, residues 99-420. We showed that the F-domain the 2-domain NTD-DBD is constitutively active exists as an equilibrium between a disordered (or due to a potent activation function-1 (AF1) region unfolded) state and a folded ordered state, and that located in its NTD, a fact that makes the GR NTD- the folded state of the F-domain is responsible for DBD amenable to exploring the allosteric impact transcriptional activation. of DNA binding on transcriptional activation, as The second functionally distinct IDR of well as the effects of changes to the NTD on DNA the NTD consists of residues 1-98 (Fig. 1A), and binding (Fig. 1A). We and others have shown that this region serves as a variable-length regulator (or the ID NTD can be made to cooperatively fold R-domain). The R-domain influences both the F- into conformers containing both 2º and 3º domain of the NTD as well as the DBD, providing structure, by use of; 1) protective organic GR with genetically “tunable” energetic osmolytes (4-7), 2) known binding partner frustration (13), whereby DNA binding to the proteins (8-10), 3) phosphorylation of key serines DBD both directly stabilizes (and thus activates) (11,12), and 4) addition of the DBD (13,14). the F-domain, but also indirectly destabilizes Previously, we hypothesized, using a (therefore, repressing) the F-domain through rigorous thermodynamic model, that under a wide stabilization of the R-domain. Thus, our results range of initial energetic conditions, inclusion of revealed that all the GR NTD-DBD isoforms that an IDR in a protein could enhance its allosteric contain the AF1 region (e.g., A, B1, B3 C1, and behavior (15). Specifically, in a 2-domain model C2), with the exception of the C3 isoform, actually in which each domain is capable of binding its consist of three domains; the DBD, the F-domain, own specific ligand, when one or both domains is and variable lengths of the R-domain. Because the ID, binding of ligand to one domain can greatly GR C3 is the only isoform lacking the R-domain influence the probability of the second domain (19), it represents an ideal system to interrogate being in a favorable folded state for binding its the direct impact of changes to the NTD on the ligand. Further, we showed this ensemble DBD, as the absence of any R-domain should allosteric model (EAM) could be applied to greatly simplify interpretation. proteins with multiple domains, allowing us to As a transcriptional cofactor for these probe the role of ID in mediating allosteric control studies we selected the Tumor Susceptibility Gene in numerous domain topologies (16). Indeed, in 101 (TSG101), a protein that is primarily known validating this model, we demonstrated that DNA for its role in the ESCRT-I complex (i.e., binding affinity and transcriptional activity for a Endosomal Sorting Complex Required for series of isoforms of GR could be described in the Transport-I), promoting endosomal maturation context of inter-domain coupling in the GR (13), (19,20). However, it has also been reported to thus, establishing one tenet of the EAM, that bind to the GR, and to possibly act as a cofactor stabilization of an input domain (i.e. the DBD) for GR and other TF’s (Table S1 and (21-25)). The could affect the function of the activation domain. domains of TSG101 that are critical to these Here, we set out to test the reciprocal interactions are shown in Figure 1B. Herein, we prediction of the model; that binding a investigate the binding of TSG101 to GR and the transcriptional cofactor ligand to the NTD could ability of this binding to allosterically affect GR either positively or negatively (depending on the DBD binding to its canonical palindromic DNA sign of the coupling) tune the affinity of the DBD. binding site (i.e., the GR response element To address this question, we utilized the 2-domain (GRE)), as well as influence subsequent GR system described above (13). Importantly, transcriptional activity. within this construct, the DBD is known to be a structured, albeit highly dynamic, globular protein, Results 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.02.931485; this version posted February 3, 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. TSG101 allosterically regulates GR TSG101 binds within the GR NTD Activation (i.e.~120-200) and following (i.e.~240-300) Function-1 (AF1) region residues in the AF1 core. Interestingly, the It has been reported previously that the orientation of the cross-links is suggestive of an coiled-coil region of TSG101 (TSG101cc) binds to inversion in the register of the interaction, the AF1 region of the GR NTD (23). Here we set whereby the GR NTD is aligned parallel with out to establish whether this binding is specific TSG101cc but then doubles back on TSG101cc, and whether it affects the functional coupling we aligning in antiparallel (Fig. 4 inset). Importantly, previously revealed in GR (13). Importantly, we these results are consistent with a model whereby confirmed previous results and extended our the TSG101cc—which is known to form a understanding using quantitative binding studies heterotrimeric coiled-coil in its functional ESCRT and protease protection assays. First, we complex (27)—interacts with the GR NTD in the examined the binding of TSG101cc to the single- two regions before and after the AF1 core residues domain GR C3 NTD and to the two-domain GR (5), thus mimicking the trimeric coiled-coil seen in C3 NTD-DBD (Fig. 2). Our results reveal that the ESCRT complex. We note that this proposed TSG101cc binds to the two constructs with 1:1 interaction model is also consistent with our stoichiometry, with TSG101cc binding more structural thermodynamic analysis of GR using the tightly to the isolated C3 NTD (Kd = 19.6 ± 1.0 COREX algorithm, whereby the two regions of the µM, see Table 1) than to the two-domain C3 GR NTD that interact with TSG101cc are NTD-DBD (Kd = 72.6 ± 14.6 µM).
Load more

Recommended publications
  • Hsa-Mir-21-5P
    Supporting Information An exosomal urinary miRNA signature for early diagnosis of renal fibrosis in Lupus Nephritis 1 2 2 1 1 Cristina Solé , Teresa Moliné , Marta Vidal , Josep Ordi-Ros , Josefina Cortés-Hernández 1Hospital Universitari Vall d’Hebron, Vall d’Hebron Research Institute (VHIR), Lupus Unit, Barcelona, Spain; [email protected] (C.S); [email protected] (J.O-R.); [email protected] (J.C-H.) 2 Hospital Universitari Vall d’Hebron, Department of Renal Pathology, Barcelona, Spain; [email protected] (T.M); [email protected] (M.V). INDEX 1. SI Materials and Methods......................................................................................3 2. SI References…………………………………………………………………………….7 2. SI Figures................................................................................................................8 4. SI Tables................................................................................................................21 2 SI Materials and Methods Patients and samples Patients with biopsy-proven active LN were recruited from the Lupus Unit at Vall d’Hebron Hospital (N=45). All patients fulfilled at least 4 of the American College of rheumatology (ACR) revised classification criteria for SLE [1]. Healthy donors were used as controls (N=20). Urine samples were collected from each patient 1 day before renal biopsy and processed immediately to be stored at -80ºC. Patients with urinary tract infection, diabetes mellitus, pregnancy, malignancy and non-lupus-related renal failure were excluded. In addition, key laboratory measurements were obtained including complement levels (C3 and C4), anti-double-stranded DNA (anti-dsDNA), 24-h proteinuria, blood urea nitrogen (BUN), serum creatinine and the estimated glomerular filtration ratio (eGFR) using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula [2]. SLE disease activity was assessed by the SLE Disease Activity Index 2000 update (SLEDAI-2Ks; range 0–105) [3].
    [Show full text]
  • Combinatorial Avidity Selection of Mosaic Landscape Phages Targeted at Breast Cancer Cells—An Alternative Mechanism of Directed Molecular Evolution
    viruses Article Combinatorial Avidity Selection of Mosaic Landscape Phages Targeted at Breast Cancer Cells—An Alternative Mechanism of Directed Molecular Evolution Valery A. Petrenko 1,* , James W. Gillespie 1 , Hai Xu 1,2, Tiffany O’Dell 1 and Laura M. De Plano 1,3 1 Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, USA 2 National Veterinary Biological Medicine Engineering Research Center, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China 3 Department of Chemical Sciences, Biological, Pharmaceutical and Environmental, University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy * Correspondence: [email protected]; Tel.: +1-334-844-2897 Received: 2 August 2019; Accepted: 22 August 2019; Published: 26 August 2019 Abstract: Low performance of actively targeted nanomedicines required revision of the traditional drug targeting paradigm and stimulated the development of novel phage-programmed, self-navigating drug delivery vehicles. In the proposed smart vehicles, targeting peptides, selected from phage libraries using traditional principles of affinity selection, are substituted for phage proteins discovered through combinatorial avidity selection. Here, we substantiate the potential of combinatorial avidity selection using landscape phage in the discovery of Short Linear Motifs (SLiMs) and their partner domains. We proved an algorithm for analysis of phage populations evolved through multistage screening of landscape phage libraries against the MDA-MB-231 breast cancer cell line. The suggested combinatorial avidity selection model proposes a multistage accumulation of Elementary Binding Units (EBU), or Core Motifs (CorMs), in landscape phage fusion peptides, serving as evolutionary initiators for formation of SLiMs. Combinatorial selection has the potential to harness directed molecular evolution to create novel smart materials with diverse novel, emergent properties.
    [Show full text]
  • Exploring Extracellular Vesicles Biogenesis in Hypothalamic Cells Through a Heavy Isotope Pulse/Trace Proteomic Approach
    cells Article Exploring Extracellular Vesicles Biogenesis in Hypothalamic Cells through a Heavy Isotope Pulse/Trace Proteomic Approach Chee Fan Tan 1,2 , Hui San Teo 2, Jung Eun Park 2, Bamaprasad Dutta 2, Shun Wilford Tse 2, Melvin Khee-Shing Leow 3,4,5 , Walter Wahli 3,6 and Siu Kwan Sze 2,* 1 NTU Institute for Health Technologies, Interdisciplinary Graduate School, Nanyang Technological University, Singapore 637335, Singapore; [email protected] 2 School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore; [email protected] (H.S.T.); [email protected] (J.E.P.); [email protected] (B.D.); [email protected] (S.W.T.) 3 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921, Singapore; [email protected] (M.K.-S.L.); [email protected] (W.W.) 4 Department of Endocrinology, Tan Tock Seng Hospital, Singapore 308433, Singapore 5 Cardiovascular and Metabolic Disorder Program, Duke-NUS Medical School, Singapore 169857, Singapore 6 Center for Integrative Genomics, University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland * Correspondence: [email protected]; Tel.: +65-6514-1006; Fax: +65-6791-3856 Received: 24 March 2020; Accepted: 21 May 2020; Published: 25 May 2020 Abstract: Studies have shown that the process of extracellular vesicles (EVs) secretion and lysosome status are linked. When the lysosome is under stress, the cells would secrete more EVs to maintain cellular homeostasis. However, the process that governs lysosomal activity and EVs secretion remains poorly defined and we postulated that certain proteins essential for EVs biogenesis are constantly synthesized and preferentially sorted to the EVs rather than the lysosome.
    [Show full text]
  • The Role of Tsg101 in the Development of Physiological Cardiac Hypertrophy
    The Role of Tsg101 in the Development of Physiological Cardiac Hypertrophy and Cardio-Protection from Endotoxin-Induced Cardiac Dysfunction A dissertation to be submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Pharmacology and Systems Physiology, College of Medicine By: Kobina Q. Essandoh B.A. in Biochemistry from Cornell College, 2011 Advisor and Committee Chair: Guo-Chang Fan, Ph.D. Abstract In this dissertation, the functional role of Tumor susceptibility gene (Tsg101) in the regulation of physiological cardiac hypertrophy and endotoxin-induced cardiac dysfunction was explored. Development of physiological cardiac hypertrophy has primarily been ascribed to the insulin-like growth factor 1 (IGF-1) and its receptor, IGF-1R, and subsequent activation of the Akt pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiological hypertrophy has not been investigated. Furthermore, cardiac mitochondrial damage and subsequent inflammation are hallmarks of endotoxin-induced myocardial depression. Activation of the Parkin/PINK1 pathway has been shown to promote autophagy of damaged mitochondria (mitophagy) and protect from endotoxin-induced cardiac dysfunction. Tsg101 has been demonstrated to play diverse roles in the cell including virus budding, cytokinesis, transcriptional regulation, endosomal recycling of receptors and activation of autophagic flux. Hence, the first goal of this dissertation was to elucidate the role of Tg101 in endosome-mediated recycling of IGF-1R in physiological cardiac remodeling. The second goal of this dissertation was to investigate whether Tsg101 regulates mitophagy and thus contribute to endotoxin-caused myocardial dysfunction.
    [Show full text]
  • Comparative Analysis of the Ubiquitin-Proteasome System in Homo Sapiens and Saccharomyces Cerevisiae
    Comparative Analysis of the Ubiquitin-proteasome system in Homo sapiens and Saccharomyces cerevisiae Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Hartmut Scheel aus Rheinbach Köln, 2005 Berichterstatter: Prof. Dr. R. Jürgen Dohmen Prof. Dr. Thomas Langer Dr. Kay Hofmann Tag der mündlichen Prüfung: 18.07.2005 Zusammenfassung I Zusammenfassung Das Ubiquitin-Proteasom System (UPS) stellt den wichtigsten Abbauweg für intrazelluläre Proteine in eukaryotischen Zellen dar. Das abzubauende Protein wird zunächst über eine Enzym-Kaskade mit einer kovalent gebundenen Ubiquitinkette markiert. Anschließend wird das konjugierte Substrat vom Proteasom erkannt und proteolytisch gespalten. Ubiquitin besitzt eine Reihe von Homologen, die ebenfalls posttranslational an Proteine gekoppelt werden können, wie z.B. SUMO und NEDD8. Die hierbei verwendeten Aktivierungs- und Konjugations-Kaskaden sind vollständig analog zu der des Ubiquitin- Systems. Es ist charakteristisch für das UPS, daß sich die Vielzahl der daran beteiligten Proteine aus nur wenigen Proteinfamilien rekrutiert, die durch gemeinsame, funktionale Homologiedomänen gekennzeichnet sind. Einige dieser funktionalen Domänen sind auch in den Modifikations-Systemen der Ubiquitin-Homologen zu finden, jedoch verfügen diese Systeme zusätzlich über spezifische Domänentypen. Homologiedomänen lassen sich als mathematische Modelle in Form von Domänen- deskriptoren (Profile) beschreiben. Diese Deskriptoren können wiederum dazu verwendet werden, mit Hilfe geeigneter Verfahren eine gegebene Proteinsequenz auf das Vorliegen von entsprechenden Homologiedomänen zu untersuchen. Da die im UPS involvierten Homologie- domänen fast ausschließlich auf dieses System und seine Analoga beschränkt sind, können domänen-spezifische Profile zur Katalogisierung der UPS-relevanten Proteine einer Spezies verwendet werden. Auf dieser Basis können dann die entsprechenden UPS-Repertoires verschiedener Spezies miteinander verglichen werden.
    [Show full text]
  • T5951dat Anti-TSG101 C-Terminal KAA,PHC 08-06-1
    Anti-TSG101 (C-terminal) produced in rabbit, affinity isolated antibody Catalog Number T5951 Product Description potential co-repressor activity.3 Subcellular localization Anti-TSG101 (C-terminal) is developed in rabbit using a of TSG101 fluctuates in a cell-cycle dependent manner synthetic peptide corresponding to amino acids being localized in the nucleus and Golgi complex during 371-390 located at the C-terminus of human TSG101, interphase and in mitotic spindles and centrosome conjugated to KLH, as immunogen. This sequence is during mitosis.12 identical in rat and mouse. The antibody is affinity- purified using the immunizing peptide immobilized on Reagent agarose. Supplied as a solution in 0.01 M phosphate buffered saline, pH 7.4, containing 15 mM sodium azide. Anti-TSG101 (C-terminal) recognizes TSG101, 46 kDa, by immunoblotting. An additional band of ~105 kDa may Antibody concentration: ~1.5 mg/mL be observed in some preparations of cell/tissue extracts. Staining of the TSG101 band in immunoblotting is Precautions and Disclaimer specifically inhibited with the immunizing peptide. Due to the sodium azide content, a material safety data sheet (MSDS) for this product has been sent to the Tumor susceptibility gene 101 encodes a multi-domain attention of the safety officer of your institution. Consult binding protein (TSG101, 46 kDa), that mediates a the MSDS for information regarding hazards and safe variety of cellular functions including transcriptional handling practices. regulation, ubiquitination, endosomal trafficking, proliferation, and cell survival.1-7 Tsg101 is expressed Storage/Stability in all tissues throughout development and is essential For continuous use, store at 2-8 °C for up to one month.
    [Show full text]
  • Negative Regulation of Cell Growth and Differentiation by TSG101 Through Association with P21cip1͞waf1
    Negative regulation of cell growth and differentiation by TSG101 through association with p21Cip1͞WAF1 Hyesun Oh*, Cristina Mammucari*, Arianna Nenci*, Sara Cabodi*, Stanley N. Cohen†, and G. Paolo Dotto*‡ *Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; and †Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120 Contributed by Stanley N. Cohen, March 4, 2002 TSG101 was discovered in a screen for tumor susceptibility genes variety of nuclear, microtubule, and mitotic spindle abnormal- and has since been shown to have a multiplicity of biological ities (13). Sequence analysis indicates that the TSG101 protein effects. However, the basis for TSG101’s ability to regulate cell contains an amino-terminal region of homology to ubiquitin- growth has not been elucidated. We report here that the TSG101 conjugating enzymes (UBC E2), a proline-rich sequence with the protein binds to the cyclin͞cyclin-dependent kinase (CDK) inhibitor features of a transcription transactivation domain, a leucine (CKI) p21Cip1͞WAF1 and increases stability of the p21 protein in zipper, and a central coiled-coil region (7, 14). Recent experi- HEK293F cells and differentiating primary keratinocytes, suppress- ments have shown that TSG101 has an important role in ing differentiation in a p21-dependent manner. In proliferating ubiquitin-mediated endosomal sorting pathways (15–17). There keratinocytes where the p21 protein is relatively stable, TSG101 also is evidence implicating the TSG101 UBC domain on does not affect the stability or expression of p21 but shows regulation of the p53͞MDM2 feedback control loop in human p21-dependent recruitment to cyclin͞CDK complexes, inhibits cy- tumor cells (18).
    [Show full text]
  • TSGΔ154-1054 Splice Variant Increases TSG101 Oncogenicity by Inhibiting Its E3-Ligase-Mediated Proteasomal Degradation
    www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 7 TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation Huey-Huey Chua,1,* Chiun-Sheng Huang,2,* Pei-Lun Weng1, Te-Huei Yeh3 1 Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan 2 Department of Surgery, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei 10051, Taiwan 3 Department of Otolaryngology, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei 10051, Taiwan * These authors have contributed equally to this work Correspondence to: Te-Huei Yeh, e-mail: [email protected] Keywords: TSG101, alternative splicing, ubiquitination, tumorigenesis, nasopharyngeal carcinoma Received: June 29, 2015 Accepted: January 12, 2016 Published: January 22, 2016 ABSTRACT Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice.
    [Show full text]
  • Resources to Discover and Use Short Linear Motifs in Viral Proteins
    Trends in Biotechnology Review Resources to Discover and Use Short Linear Motifs in Viral Proteins Peter Hraber,1,* Paul E. O’Maille,2 Andrew Silberfarb,3 Katie Davis-Anderson,4 Nicholas Generous,5 Benjamin H. McMahon,1 and Jeanne M. Fair4 Viral proteins evade host immune function by molecular mimicry, often achieved by short linear Highlights motifs (SLiMs) of three to ten consecutive amino acids (AAs). Motif mimicry tolerates mutations, Short linear motifs (SLiMs) are pat- evolves quickly to modify interactions with the host, and enables modular interactions with pro- terns of three to ten consecutive tein complexes. Host cells cannot easily coordinate changes to conserved motif recognition and AAs used by eukaryotic cells for binding interfaces under selective pressure to maintain critical signaling pathways. SLiMs offer tasks that include: signaling, local- potential for use in synthetic biology, such as better immunogens and therapies, but may also ization, degradation, and proteo- present biosecurity challenges. We survey viral uses of SLiMs to mimic host proteins, and infor- lytic cleavage. mation resources available for motif discovery. As the number of examples continues to grow, Viruses use SLiMs to their advan- knowledge management tools are essential to help organize and compare new findings. tage, including interference with antiviral innate immune pathways. How Viruses Do More with Less Viral SLiMs can tolerate mutations, Viruses exploit host cellular processes to replicate, and have developed myriad ways to subvert host evolve quickly to modify host in- immune defenses. Molecular mimicry (see Glossary)isacommonandeffectivestrategy,enablinga teractions, and co-occur in a pathogen to usurp host protein function by resemblance [1,2].
    [Show full text]
  • TSG101 Antibody Cat
    TSG101 Antibody Cat. No.: 28-940 TSG101 Antibody Specifications HOST SPECIES: Rabbit SPECIES REACTIVITY: Dog, Human, Mouse, Rat, Zebrafish Antibody produced in rabbits immunized with a synthetic peptide corresponding a region IMMUNOGEN: of human TSG101. TESTED APPLICATIONS: ELISA, WB TSG101 antibody can be used for detection of TSG101 by ELISA at 1:312500. TSG101 APPLICATIONS: antibody can be used for detection of TSG101 by western blot at 2.5 μg/mL, and HRP conjugated secondary antibody should be diluted 1:50,000 - 100,000. POSITIVE CONTROL: 1) Cat. No. 1211 - HepG2 Cell Lysate PREDICTED MOLECULAR 44 kDa WEIGHT: Properties PURIFICATION: Antibody is purified by protein A chromatography method. CLONALITY: Polyclonal CONJUGATE: Unconjugated PHYSICAL STATE: Liquid September 25, 2021 1 https://www.prosci-inc.com/tsg101-antibody-28-940.html Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% BUFFER: sucrose. CONCENTRATION: batch dependent For short periods of storage (days) store at 4˚C. For longer periods of storage, store STORAGE CONDITIONS: TSG101 antibody at -20˚C. As with any antibody avoid repeat freeze-thaw cycles. Additional Info OFFICIAL SYMBOL: TSG101 ALTERNATE NAMES: TSG101, TSG10, VPS23 ACCESSION NO.: NP_006283 PROTEIN GI NO.: 5454140 GENE ID: 7251 USER NOTE: Optimal dilutions for each application to be determined by the researcher. Background and References TSG101 belongs to a group of apparently inactive homologs of ubiquitin-conjugating enzymes. TSG101 contains a coiled-coil domain that interacts with stathmin, a cytosolic phosphoprotein implicated in tumorigenesis. TSG101 may play a role in cell growth and differentiation and act as a negative growth regulator.
    [Show full text]
  • ESCRT-I Protein Tsg101 Plays a Role in the Post-Macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’S Sarcoma-Associated Herpesvirus
    RESEARCH ARTICLE ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus Binod Kumar, Dipanjan Dutta, Jawed Iqbal, Mairaj Ahmed Ansari, Arunava Roy, Leela Chikoti, Gina Pisano, Mohanan Valiya Veettil, Bala Chandran* a11111 H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, United States Of America * [email protected] Abstract OPEN ACCESS Kaposi's sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface Citation: Kumar B, Dutta D, Iqbal J, Ansari MA, heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins Roy A, Chikoti L, et al. (2016) ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre- Trafficking and Infection of Endothelial Cells by existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitina- Kaposi's Sarcoma-Associated Herpesvirus. PLoS tion of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane Pathog 12(10): e1005960. doi:10.1371/journal. bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human micro- ppat.1005960 vascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Editor: Lindsey Hutt-Fletcher, Louisiana State Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and±III, play a central role in University Health Sciences Center, UNITED STATES clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress.
    [Show full text]
  • Differential Requirements for Alix and ESCRT-III in Cytokinesis and HIV-1 Release
    Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release Jez G. Carlton, Monica Agromayor, and Juan Martin-Serrano* Department of Infectious Diseases, King’s College London School of Medicine, London SE1 9RT, United Kingdom Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved April 23, 2008 (received for review February 28, 2008) The ESCRT machinery functions in topologically equivalent mem- ESCRT-III appears to be the membrane fission machinery brane fission events, namely multivesicular body formation, the recruited by ESCRT-I and Alix (11). Overexpression of ESCRT-III terminal stages of cytokinesis and HIV-1 release. Here, we show subunits arrests viral budding, recapitulating the phenotype of an L that the ESCRT-III-binding protein Alix is recruited to the midbody domain-defective virus (17, 22–24), and mutations in the Bro1 of dividing cells through binding Cep55 via an evolutionarily domain of Alix that abrogate the Alix/ESCRT-III interaction also conserved peptide. Disruption of Cep55/Alix/ESCRT-III interactions abolish the ability of Alix to mediate budding (20, 21) and cell causes formation of aberrant midbodies and cytokinetic failure, division (8). A requirement for ESCRT-III in cell division has been demonstrating an essential role for these proteins in midbody suggested by the arrest of cytokinesis in cells overexpressing morphology and cell division. We also show that the C terminus of YFP-Chmp4 fusion proteins or a catalytically inactive Vps4 (7, 8). Alix encodes a multimerization activity that is essential for its Overall, the anatomy of the Class E VPS pathway is conserved from function in Alix-dependent HIV-1 release and for interaction with yeasts to humans, and ESCRT-III is one of the most conserved Tsg101.
    [Show full text]