UBE2E3 (UBCH9) Sirna Set I Sirna Duplexes Targeted Against Three Exon Regions

Total Page:16

File Type:pdf, Size:1020Kb

UBE2E3 (UBCH9) Sirna Set I Sirna Duplexes Targeted Against Three Exon Regions Catalog # Aliquot Size U219-911-05 3 x 5 nmol U219-911-20 3 x 20 nmol U219-911-50 3 x 50 nmol UBE2E3 (UBCH9) siRNA Set I siRNA duplexes targeted against three exon regions Catalog # U219-911 Lot # Z2109-21 Specificity Formulation UBE2E3 (UBCH9) siRNAs are designed to specifically The siRNAs are supplied as a lyophilized powder and knock-down human UBE2E3 (UBCH9) expression. shipped at room temperature. Product Description UBE2E3 (UBCH9) siRNA is a pool of three individual Reconstitution Protocol synthetic siRNA duplexes designed to knock-down Briefly centrifuge the tubes (maximum RCF 4,000g) to human UBE2E3 (UBCH9) mRNA expression. Each siRNA is collect lyophilized siRNA at the bottom of the tube. 19-25 bases in length. The gene accession number is Resuspend the siRNA in 50 µl of DEPC-treated water NM_006357. (supplied by researcher), which results in a 1x stock solution (10 µM). Gently pipet the solution 3-5 times to mix Gene Aliases and avoid the introduction of bubbles. Optional: aliquot UBCH9; UbcM2 1x stock solutions for storage. Storage and Stability The lyophilized powder is stable for at least 4 weeks at Related Products room temperature. It is recommended that the lyophilized and resuspended siRNAs are stored at or Product Name Catalog Number below -20oC. After resuspension, siRNA stock solutions ≥2 UBE2E3 (UBCH9) Protein U219-30H µM can undergo up to 50 freeze-thaw cycles without UBE2F Protein U220-30H significant degradation. For long-term storage, it is UBE2G1 (UBC7) Protein U221-30H recommended that the siRNA is stored at -70oC. For most UBE2H Protein U223-30H favorable performance, avoid repeated handling and UBE2I Protein U224-30H multiple freeze/thaw cycles. UBE2J1 Protein U225-30G UBE2K (UBC1) Protein U227-30H Scientific Background UBE2L6 (UBCH8) Protein U229-30H UBE2E3 also known as UBCH9 or ubiquitin-conjugating enzyme E2E 3 is a member of the E2 ubiquitin-conjugating enzyme family. Ubiquitination, the covalent attachment of the protein ubiquitin (Ub) to other cellular proteins is an UBE2E3 (UBCH9) siRNA Set I important cellular mechanism for targeting abnormal or siRNA duplexes targeted against three exon regions short-lived proteins for degradation that involves at least three classes of enzymes: ubiquitin-activating enzymes, or Catalog # U219-911 E1s, ubiquitin-conjugating enzymes, or E2s, and ubiquitin- Lot # Z2109-21 protein ligases, or E3s(1). UBE2E3 is highly expressed in Packaging Specifications 2.5 nmol/tube for 3 x 5nmol skeletal muscle (2). UBE2E3 is essential for the proliferation Format Lyophilized powder of RPE-1 cells and is downregulated during RPE layer Stability 1yr at -70oC from date of shipment maturation in the developing mouse eye. These findings Storage & Shipping The lyophilized powder is stable for at indicate that UBE2E3 is a major enzyme in modulating the least 4 weeks at room temperature. It is recommended that the lyophilized balance between RPE cell proliferation and and resuspended siRNAs are stored at differentiation. or below -20oC. After resuspension, siRNA stock solutions ≥2 µM can References undergo up to 50 freeze-thaw cycles 1. Matuschewski, K.et.al: Identification of a novel family without significant degradation. For of ubiquitin-conjugating enzymes with distinct amino- long-term storage, it is recommended that the siRNA is stored at -70oC. For terminal extensions. J. Biol. Chem. 271: 2789-2794, most favorable performance, avoid 1996. repeated handling and multiple 2. Ito, K.et.al: cDNA cloning, characterization, and freeze/thaw cycles. chromosome mapping of UBE2E3 (alias UbcH9), encoding an N-terminally extended human ubiquitin- conjugating enzyme. Cytogenet. Cell Genet. 84: 99- 104, 1999. To place your order, please contact us by phone 1-(604)-232-4600, fax 1-604-232-4601 or by email: [email protected] www.signalchem.com FOR IN VITRO RESEARCH PURPOSES ONLY. NOT INTENDED FOR USE IN HUMAN OR ANIMALS. .
Recommended publications
  • 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]
  • The Ubiquitination Enzymes of Leishmania Mexicana
    The ubiquitination enzymes of Leishmania mexicana Rebecca Jayne Burge Doctor of Philosophy University of York Biology October 2020 Abstract Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. Although 20 deubiquitinating enzymes (DUBs) have been partially characterised in Leishmania mexicana, little is known about the role of E1 ubiquitin-activating (E1), E2 ubiquitin- conjugating (E2) and E3 ubiquitin ligase (E3) enzymes in this parasite. Using bioinformatic methods, 2 E1, 13 E2 and 79 E3 genes were identified in the L. mexicana genome. Subsequently, bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed that the E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for successful promastigote to amastigote differentiation and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, revealed a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a was found to load ubiquitin onto UBC2, allowing UBC2- UEV1 to form K63-linked di-ubiquitin chains in vitro. UBC2 was also shown to cooperate with human E3s RNF8 and BIRC2 in vitro to form non-K63-linked polyubiquitin chains, but association of UBC2 with UEV1 inhibits this ability.
    [Show full text]
  • UBE2I Sirna Set I UBE2I Sirna Set I
    Catalog # Aliquot Size U224-911-05 3 x 5 nmol U224-911-20 3 x 20 nmol U224-911-50 3 x 50 nmol UBE2I siRNA Set I siRNA duplexes targeted against three exon regions Catalog # U224-911 Lot # Z2109-25 Specificity Formulation UBE2I siRNAs are designed to specifically knock-down The siRNAs are supplied as a lyophilized powder and human UBE2I expression. shipped at room temperature. Product Description Reconstitution Protocol UBE2I siRNA is a pool of three individual synthetic siRNA Briefly centrifuge the tubes (maximum RCF 4,000g) to duplexes designed to knock-down human UBE2I mRNA collect lyophilized siRNA at the bottom of the tube. expression. Each siRNA is 19-25 bases in length. The gene Resuspend the siRNA in 50 µl of DEPC-treated water accession number is NM_003345. (supplied by researcher), which results in a 1x stock solution (10 µM). Gently pipet the solution 3-5 times to mix Gene Aliases and avoid the introduction of bubbles. Optional: aliquot C358B7.1; P18; UBC9 1x stock solutions for storage. Storage and Stability Related Products The lyophilized powder is stable for at least 4 weeks at room temperature. It is recommended that the Product Name Catalog Number lyophilized and resuspended siRNAs are stored at or UBE2E3 (UBCH9) Protein U219-30H below -20oC. After resuspension, siRNA stock solutions ≥2 UBE2F Protein U220-30H µM can undergo up to 50 freeze-thaw cycles without UBE2G1 (UBC7) Protein U221-30H significant degradation. For long-term storage, it is UBE2H Protein U223-30H recommended that the siRNA is stored at -70oC. For most UBE2I Protein U224-30H favorable performance, avoid repeated handling and UBE2J1 Protein U225-30G multiple freeze/thaw cycles.
    [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]
  • The Proteasomal Deubiquitinating Enzyme PSMD14 Regulates Macroautophagy by Controlling Golgi-To-ER Retrograde Transport
    Supplementary Materials The proteasomal deubiquitinating enzyme PSMD14 regulates macroautophagy by controlling Golgi-to-ER retrograde transport Bustamante HA., et al. Figure S1. siRNA sequences directed against human PSMD14 used for Validation Stage. Figure S2. Primer pairs sequences used for RT-qPCR. Figure S3. The PSMD14 DUB inhibitor CZM increases the Golgi apparatus area. Immunofluorescence microscopy analysis of the Golgi area in parental H4 cells treated for 4 h either with the vehicle (DMSO; Control) or CZM. The Golgi marker GM130 was used to determine the region of interest in each condition. Statistical significance was determined by Student's t-test. Bars represent the mean ± SEM (n =43 cells). ***P <0.001. Figure S4. CZM causes the accumulation of KDELR1-GFP at the Golgi apparatus. HeLa cells expressing KDELR1-GFP were either left untreated or treated with CZM for 30, 60 or 90 min. Cells were fixed and representative confocal images were acquired. Figure S5. Effect of CZM on proteasome activity. Parental H4 cells were treated either with the vehicle (DMSO; Control), CZM or MG132, for 90 min. Protein extracts were used to measure in vitro the Chymotrypsin-like peptidase activity of the proteasome. The enzymatic activity was quantified according to the cleavage of the fluorogenic substrate Suc-LLVY-AMC to AMC, and normalized to that of control cells. The statistical significance was determined by One-Way ANOVA, followed by Tukey’s test. Bars represent the mean ± SD of biological replicates (n=3). **P <0.01; n.s., not significant. Figure S6. Effect of CZM and MG132 on basal macroautophagy. (A) Immunofluorescence microscopy analysis of the subcellular localization of LC3 in parental H4 cells treated with either with the vehicle (DMSO; Control), CZM for 4 h or MG132 for 6 h.
    [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]
  • The MALDI TOF E2/E3 Ligase Assay As an Universal Tool for Drug Discovery in the Ubiquitin Pathway
    bioRxiv preprint doi: https://doi.org/10.1101/224600; this version posted November 29, 2017. 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. The MALDI TOF E2/E3 ligase assay as an universal tool for drug discovery in the ubiquitin pathway Virginia De Cesare*1, Clare Johnson2, Victoria Barlow2, James Hastie2 Axel Knebel1 and 5 Matthias Trost*1,3 1MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dow St, Dundee, DD1 5EH, Scotland, UK; 2MRC Protein Phosphorylation and Ubiquitylation Unit Reagents and Services, University of Dundee, Dow St, Dundee, DD1 5EH, Scotland, UK; . 3Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle-upon-Tyne, 10 NE2 1HH, UK *To whom correspondence should be addressed: Virginia De Cesare ([email protected]) Matthias Trost ([email protected]) 15 Contact information: V.D.C.: MRC PPU, University of Dundee, Dow St, Dundee, DD1 5EH, Phone: +44 1382 20 85822 M.T.: Newcastle University, Institute for Cell and Molecular Biosciences, Framlington Place, Newcastle-upon-Tyne, NE2 4HH, Phone: +44 191 2087009 Key words: Ubiquitin, E3 ligase, E2 enzyme, MALDI TOF, mass spectrometry, drug 25 discovery, high-throughput, assay, MDM2, HOIP, ITCH 1 bioRxiv preprint doi: https://doi.org/10.1101/224600; this version posted November 29, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Epigenetic Mechanisms Are Involved in the Oncogenic Properties of ZNF518B in Colorectal Cancer
    Epigenetic mechanisms are involved in the oncogenic properties of ZNF518B in colorectal cancer Francisco Gimeno-Valiente, Ángela L. Riffo-Campos, Luis Torres, Noelia Tarazona, Valentina Gambardella, Andrés Cervantes, Gerardo López-Rodas, Luis Franco and Josefa Castillo SUPPLEMENTARY METHODS 1. Selection of genomic sequences for ChIP analysis To select the sequences for ChIP analysis in the five putative target genes, namely, PADI3, ZDHHC2, RGS4, EFNA5 and KAT2B, the genomic region corresponding to the gene was downloaded from Ensembl. Then, zoom was applied to see in detail the promoter, enhancers and regulatory sequences. The details for HCT116 cells were then recovered and the target sequences for factor binding examined. Obviously, there are not data for ZNF518B, but special attention was paid to the target sequences of other zinc-finger containing factors. Finally, the regions that may putatively bind ZNF518B were selected and primers defining amplicons spanning such sequences were searched out. Supplementary Figure S3 gives the location of the amplicons used in each gene. 2. Obtaining the raw data and generating the BAM files for in silico analysis of the effects of EHMT2 and EZH2 silencing The data of siEZH2 (SRR6384524), siG9a (SRR6384526) and siNon-target (SRR6384521) in HCT116 cell line, were downloaded from SRA (Bioproject PRJNA422822, https://www.ncbi. nlm.nih.gov/bioproject/), using SRA-tolkit (https://ncbi.github.io/sra-tools/). All data correspond to RNAseq single end. doBasics = TRUE doAll = FALSE $ fastq-dump -I --split-files SRR6384524 Data quality was checked using the software fastqc (https://www.bioinformatics.babraham. ac.uk /projects/fastqc/). The first low quality removing nucleotides were removed using FASTX- Toolkit (http://hannonlab.cshl.edu/fastxtoolkit/).
    [Show full text]
  • S41467-019-12388-Y.Pdf
    ARTICLE https://doi.org/10.1038/s41467-019-12388-y OPEN A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligases Leo Kiss1,4, Jingwei Zeng 1,4, Claire F. Dickson1,2, Donna L. Mallery1, Ji-Chun Yang 1, Stephen H. McLaughlin 1, Andreas Boland1,3, David Neuhaus1,5 & Leo C. James1,5* 1234567890():,; The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/ Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s. 1 Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. 2Present address: University of New South Wales, Sydney, NSW, Australia. 3Present address: Department of Molecular Biology, Science III, University of Geneva, Geneva, Switzerland. 4These authors contributed equally: Leo Kiss, Jingwei Zeng. 5These authors jointly supervised: David Neuhaus, Leo C.
    [Show full text]
  • Mechanism of Ssph1: a Bacterial Effector Ubiquitin Ligase
    © Copyright 2018 Matthew J. Cook Mechanism of SspH1: A Bacterial Effector Ubiquitin Ligase Hijacks the Eukaryotic Ubiquitylation Pathway Matthew J. Cook A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2018 Reading Committee: Peter S Brzovic, Chair Suzanne Hoppins Ning Zheng Program Authorized to Offer Degree: Biochemistry University of Washington Abstract Mechanism of SspH1: A Bacterial Effector Ubiquitin Ligase Hijacks the Eukaryotic Ubiquitylation Pathway Matthew J. Cook Chair of the Supervisory Committee: Peter S Brzovic Department of Biochemistry Bacterial effector proteins promote the pathogenicity of bacteria by interacting with host cell proteins and modulating signaling pathways in the host cell. Some bacterial effectors hijack the eukaryotic ubiquitylation pathway to promote pathogenicity, despite the absence of the pathway in prokaryotes. One family of bacterial effectors, the IpaH-SspH family of E3 ligases, are unrelated in sequence or structure to eukaryotic E3s. This work elucidates key aspects in the structure and mechanism of one member of the IpaH-SspH family from Salmonella typhimurium, SspH1. Biophysical and biochemical methods were used to examine the structure of SspH1 in solution and the interactions between SspH1 and components of the eukaryotic ubiquitylation pathway, with an emphasis on the mechanisms of ubiquitin transfer from the E2 active site to the SspH1 active site, and from the SspH1 active site to substrate. Important results of this work include 1) a reanalysis of existing crystal structures of IpaH-SspH1 E3 domains leading to a new way of characterizing the structural organization of the SspH1 E3 domain, revealing two independent subdomains.
    [Show full text]
  • HRD1 and UBE2J1 Target Misfolded MHC Class I Heavy Chains for Endoplasmic Reticulum- Associated Degradation
    HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum- associated degradation Marian L. Burr, Florencia Cano, Stanislava Svobodova, Louise H. Boyle, Jessica M. Boname, and Paul J. Lehner1 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom Edited* by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved December 21, 2010 (received for review October 29, 2010) The assembly of MHC class I molecules is governed by stringent orthologs of yeast Hrd1p, HRD1 (Synoviolin) and gp78 (AMFR), endoplasmic reticulum (ER) quality control mechanisms. MHC class I have distinct substrate specificities, exemplifying the increased heavy chains that fail to achieve their native conformation in complexity of mammalian ERAD (6, 7). HRD1 is implicated in the complex with β2-microglobulin (β2m) and peptide are targeted for pathogenesis of rheumatoid arthritis (10), and reported substrates ER-associated degradation. This requires ubiquitination of the MHC include α1-antitrypsin variants (11) and amyloid precursor protein class I heavy chain and its dislocation from the ER to the cytosol for (12). Three E2 ubiquitin-conjugating enzymes function in mam- proteasome-mediated degradation, although the cellular machin- malian ERAD: UBE2J1 and UBE2J2 (yeast Ubc6p orthologs) and ery involved in this process is unknown. Using an siRNA functional UBE2G2 (yeast Ubc7p ortholog) (7, 8, 13). screen in β2m-depleted cells, we identify an essential role for the E3 Many viruses down-regulate cell-surface MHC I to evade im- ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating mune detection (2). The human cytomegalovirus US2 and US11 gene products bind newly synthesized MHC I HCs and initiate enzyme UBE2J1 in the ubiquitination and dislocation of misfolded their rapid dislocation to the cytosol for proteasomal degradation MHC class I heavy chains.
    [Show full text]
  • Ubiquitinating Enzyme Systems Eric Yao, Msc
    TECHNICAL BULLETIN Ubiquitinating Enzyme Systems Eric Yao, MSc. Scientist, Product Management - SignalChem Biotech Inc. Target Ub Recognizing Domain Target A E1 E2 Ub Target P Ub P Ub P E3 Ub Ub Poly-Ub A P P E1 E2 E2 - Interacting Ub P Domain Ub Ub Ub Monomeric 26 S Proteosome Ubiquitin Polymeric Ubiquitin Ub Ub Ub Ub Ub Ub Ub Ub Ub Ub Deconjugation/ Ub Ub Ub Recycling Target Degradation Protein modifications by ubiquitin (Ub) or ubiquitin-like proteins (UBLs) participate in many critical cellular processes such as cell-cycle regulation, DNA repair, oncogenesis, antiviral pathways and most notably, proteasomal degradation of target proteins. Ubiquitination and modification by UBLs share a similar catalytic cascade which requires the sequential action of three classes of enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Recent research has linked dysregulation of the Ub/UBLs modification system to numerous diseases including cancer, immunological disorders and neurodegeneration. Thus, the high substrate specificity provided by combinations of over 30 E2’s and over 600 E3’s makes these enzymes emerging drug targets. In response to a growing market demand, SignalChem has developed an extensive array of products encompassing enzymes, Ub/UBL modifiers and substrates in the ubiquitination, SUMOylation, ISGylation and NEDDylation processes. Using Promega’s AMP-GloTM technology and an optimized assay protocol, we have identified and validated a variety of functional combinations of the enzyme components. With the established protocol, each enzyme in the catalytic cascade has been assessed for their activity towards generation of free AMP. In addition, inhibition profiles of the ubiquitinating enzymes have been obtained using the assay system, further demonstrating their potential to be used in high-throughput screening to identify lead compounds for drug discovery and development programs.
    [Show full text]