DOCSLIB.ORG

The Role of BAG6 in Protein Quality Control

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

The role of BAG6 in protein quality control A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Yee Hui Koay School of Biological Sciences List of contents Page List of tables 5 List of figures 6 List of abbreviations 9 Abstract 13 Declaration 14 Copyright statement 14 Acknowledgement 15 Chapter 1: Introduction 1.1 Protein folding and quality control 16 1.2 Degradation of misfolded proteins 21 1.3 Protein biosynthesis and quality control at the endoplasmic reticulum 24 1.4 BAG6 31 1.4.1 BAG6 structure 32 1.4.2 BAG6 in tail-anchored protein targeting 34 1.4.3 BAG6 in degradation of mislocalised proteins 35 1.4.4 BAG6 triages targeting and degradation 39 1.4.5 BAG6 in endoplasmic reticulum-associated degradation 43 1.5 Aims and objectives of study 46 Chapter 2: Materials and methods 2.1 DH5α competent cells preparation 47 2.2 Plasmid DNA preparation 47 2.3 Plasmid construction 49 2.3.1 Myc-BirA-pcDNA5/FRT/TO 49 2.3.2 BAG6-myc-BirA-pcDNA5/FRT/TO 51 2.3.3 BAG6(∆N)-myc-BirA-pcDNA5/FRT/TO 51 2.3.4 HA3-XBP1u G519C(∆HR2)-pcDNA3.1(+) 52 2.4 Cell culture 52 2 2.5 Stable inducible cell line generation and induction 53 2.6 siRNA transfection 53 2.7 Transient transfection for immunoblotting and immunofluorescence microscopy 54 2.8 Treatment with proteasome and lysosomal protease inhibitors 55 2.9 Endo Hf treatment 55 2.10 Cycloheximide chase 55 2.11 SDS-PAGE and immunoblotting 56 2.12 Immunofluorescence microscopy 58 2.13 Cell cracking 59 2.14 Co-immunoprecipitation 60 2.15 Denaturing immunoprecipitation 60 2.16 BioID 61 2.17 Bioinformatics 62 2.18 Data and statistical analysis 62 Chapter 3: Role of BAG6 and UBR4 in ERAD 3.1 Introduction 64 3.2 BAG6 contributes to degradation of Op91 66 3.3 BAG6 and UBR4 are involved in ERAD of opsin-degron 71 3.4 UBR4 does not associate stably with BAG6 or opsin-degron 73 3.5 Depletion of UBR4 increases opsin-degron ubiquitination 75 3.6 UBR4 knockdown increases association of BAG6 with 80 opsin-degron and RNF126 3.7 Discussion 82 Chapter 4: Novel role of BAG6 in the UPR 4.1 Introduction 87 4.2 BAG6 interacts with XBP1u 90 4.3 BAG6 is required for efficient XBP1u degradation 95 4.4 BAG6 interacts with the C-terminal hydrophobic region of full length XBP1u in order to promote degradation 99 4.5 BAG6 depletion impairs XBP1u ubiquitination 103 3 4.6 Discussion 105 Chapter 5: The use of BioID to identify potential BAG6 substrates and/or interacting factors 5.1 Introduction 111 5.2 Generation and characterisation of BAG6-myc-BirA construct 113 5.3 Optimisation of BioID protocol 117 5.4 The use of transient expression system for efficient biotinylation 120 5.5 Mass spectrometry data analysis 123 5.5.1 Comparison of technical replicates 123 5.5.2 Pre-enrichment analysis of myc-BirA vs BAG6-myc-BirA 125 5.5.3 Comparison of biological replicates 126 5.5.4 Identification of potential BAG6 substrates 128 5.5.5 Identification of potential BAG6 interacting factors 135 5.6 Discussion 138 Chapter 6: Conclusions and future work 6.1 Conclusions 146 6.2 Future work 148 Chapter 7: References 150 Chapter 8: Supplementary data 165 Final word count: 41748 4 List of tables Page Table 2.1 Plasmid constructs used in this study 48 Table 2.2 Standard reaction setup and PCR cycling condition for site-directed mutagenesis using KOD Hot Start DNA Polymerase 50 Table 2.3 PCR cycling condition for subcloning 51 Table 2.4 siRNA sequences used in this study 54 Table 2.5 Primary and secondary antibodies used in immunoblotting 57 Table 2.6 Primary and secondary antibodies used in immunofluorescence microscopy 59 Table 5.1 Mean LFQ intensity of proteins found in both biological replicates and having at least two times higher intensity with BAG6-myc-BirA sample than myc-BirA sample 131 Table 5.2 Hydrophobicity prediction using Kyte and Doolittle scale with window size of 20 and threshold of 0 136 Table 5.3 Potential BAG6-associated protein quality control factors with gene ontology related to ubiquitination pathway 139 5 List of figures Page Figure 1.1 Illustration on a protein folding energy landscape 18 Figure 1.2 The Hsp70 and TRiC/CCT chaperone systems 20 Figure 1.3 The ubiquitination cascade and chain formation 22 Figure 1.4 The three major protein translocation pathways: co- or post-translational translocation dependent on Sec61 and Sec61-independent post-translational translocation 25 Figure 1.5 The mammalian endoplasmic reticulum-associated degradation 28 Figure 1.6 The mammalian unfolded protein response signalling pathways 30 Figure 1.7 Schematic representation of human BAG6 protein domains and its putative binding partners 33 Figure 1.8 The yeast and mammalian guided entry of tail-anchor pathway components 36 Figure 1.9 Model illustrating BAG6 role in protein quality control in the cytosol 37 Figure 1.10 Model illustrating BAG6 role in endoplasmic reticulum pre-emptive quality control 40 Figure 1.11 Current model illustrating BAG6 triages protein targeting and degradation 41 Figure 1.12 Model illustrating BAG6 role in endoplasmic reticulum- associated degradation 44 Figure 3.1 UBR family proteins and the mammalian N-end rule pathway 65 Figure 3.2 BAG6 overexpression stabilises Op91 and causes nuclear re-localisation of Op91 67 Figure 3.3 Degradation of glycosylated form of Op91 is delayed with BAG6 knockdown 70 Figure 3.4 Degradation of opsin-degron is delayed with BAG6 and UBR4 knockdown 72 Figure 3.5 BAG6 and opsin-degron do not co-immunoprecipitate UBR4 74 Figure 3.6 UBR4 knockdown does not significantly alter BAG6 steady state level 76 6 Figure 3.7 UBR4 knockdown increases total cellular ubiquitination and BAG6-associated polyubiquitinated species 78 Figure 3.8 UBR4 knockdown increases opsin-degron ubiquitination 79 Figure 3.9 UBR4 knockdown increases opsin-degron-BAG6 and BAG6- RNF126 interactions 81 Figure 3.10 Proposed model on UBR4 function in ERAD in correlation with BAG6 86 Figure 4.1(A) Schematic representation of human XBP1 protein domains 88 Figure 4.1(B) Model illustrating the splicing event of XBP1 mRNA under ER stress 88 Figure 4.2 BAG6 re-localises and stabilises XBP1u 91 Figure 4.3 BAG6 co-purifies XBP1u with proteasome inhibition 94 Figure 4.4 Proteasome inhibition delays XBP1u degradation 96 Figure 4.5 BAG6 knockdown delays XBP1u degradation 98 Figure 4.6 UBR4 and RNF126 knockdown have no effect on XBP1u degradation 99 Figure 4.7 BAG6 cannot interact with XBP1 variant lacking HR2. XBP1 HR2 mutant is more stable and does not require BAG6 for degradation 101 Figure 4.8 BAG6 knockdown decreases XBP1u ubiquitination 104 Figure 4.9 Proposed model on BAG6-mediated XBP1u degradation 108 Figure 5.1 Model for application of BioID method 112 Figure 5.2 BAG6-myc-BirA re-localises and stabilises Op91 114 Figure 5.3 RIPA buffer extracts more proteins and fewer proteins are being washed away 118 Figure 5.4 Transient overexpression system is used for BioID experiment 121 Figure 5.5 Box plots of LFQ intensity and MS/MS count with respect to the three experiment conditions and technical replicates for BAG6-myc-BirA and BAG6-myc-BirA+Bz samples 124 Figure 5.6 Venn diagrams, histograms and scatter plots of proteins identified from LFQ intensity and MS/MS count of myc-BirA and BAG6-myc-BirA samples 127 7 Figure 5.7(A) Box plot of non-normalised and median-normalised LFQ intensities with respect to the four experiment conditions and two biological replicates except for Bag6(∆N)-myc-BirA with no biological replicate 129 Figure 5.7(B) Venn diagram of proteins identified from myc-BirA sample of two biological replicates 129 Figure 5.7(C) Venn diagram of proteins identified from BAG6-myc-BirA sample of two biological replicates 129 Figure 5.7(D) Dendrogram showing how the experiments cluster 129 Figure 5.8 Flow chart for the identification of potential BAG6 substrates 130 Figure 5.9 Venn diagram of proteins identified from BAG6-myc-BirA and BAG6(∆N)-myc-BirA samples 137 8 List of abbreviations ADP adenosine diphosphate AMP adenosine monophosphate AP-MS affinity purification coupled to mass spectrometry ATE1 arginyl-tRNA--protein transferase 1 ATF4 cyclic AMP-dependent transcription factor ATF-4 ATF6 cyclic AMP-dependent transcription factor ATF-6 ATP adenosine triphosphate BAG6 BCL2-associated athanogene 6 BAT3 HLA-B associated transcript 3 BiP immunoglobulin heavy chain-binding protein BirA biotin ligase CAML calcium-modulating cyclophilin ligand CHIP E3 ubiquitin-protein ligase CHIP DMEM Dulbecco’s Modified Eagle’s Medium EDEM1 ER degradation-enhancing alpha-mannosidase-like protein 1 eIF2α eukaryotic translation initiation factor 2-alpha Endo Hf endoglycosidase Hf ER endoplasmic reticulum ERAD endoplasmic reticulum-associated degradation ER pQC endoplasmic reticulum pre-emptive quality control GET guided entry of tail-anchor gp78 E3 ubiquitin-protein ligase AMFR HECT homologous to the E6-AP carboxyl terminus Hrd1 E3 ubiquitin-protein ligase synoviolin Hsc70 heat shock cognate 71 kDa protein Hsp70 heat shock 70 kDa protein Hsp40 heat shock 40 kDa protein 9 IPOD insoluble protein deposit IRE1 serine/threonine-protein kinase/endoribonuclease IRE1 JUNQ juxta nuclear quality control compartment KCMF1 E3 ubiquitin-protein ligase KCMF1 LC-MS/MS liquid chromatography-tandem mass spectrometry LC3 microtubule-associated proteins 1A/1B light chain 3 LFQ label-free quantification MARCH6 E3 ubiquitin-protein ligase MARCH6 NLS nuclear localisation signal NO nitric oxide Npl4 nuclear protein localization protein 4 homolog NTAN1 protein N-terminal asparagine
Recommended publications
  • The HECT Domain Ubiquitin Ligase HUWE1 Targets Unassembled Soluble Proteins for Degradation

    The HECT Domain Ubiquitin Ligase HUWE1 Targets Unassembled Soluble Proteins for Degradation

    OPEN Citation: Cell Discovery (2016) 2, 16040; doi:10.1038/celldisc.2016.40 ARTICLE www.nature.com/celldisc The HECT domain ubiquitin ligase HUWE1 targets unassembled soluble proteins for degradation Yue Xu1, D Eric Anderson2, Yihong Ye1 1Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA; 2Advanced Mass Spectrometry Core Facility, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA In eukaryotes, many proteins function in multi-subunit complexes that require proper assembly. To maintain complex stoichiometry, cells use the endoplasmic reticulum-associated degradation system to degrade unassembled membrane subunits, but how unassembled soluble proteins are eliminated is undefined. Here we show that degradation of unassembled soluble proteins (referred to as unassembled soluble protein degradation, USPD) requires the ubiquitin selective chaperone p97, its co-factor nuclear protein localization protein 4 (Npl4), and the proteasome. At the ubiquitin ligase level, the previously identified protein quality control ligase UBR1 (ubiquitin protein ligase E3 component n-recognin 1) and the related enzymes only process a subset of unassembled soluble proteins. We identify the homologous to the E6-AP carboxyl terminus (homologous to the E6-AP carboxyl terminus) domain-containing protein HUWE1 as a ubiquitin ligase for substrates bearing unshielded, hydrophobic segments. We used a stable isotope labeling with amino acids-based proteomic approach to identify endogenous HUWE1 substrates. Interestingly, many HUWE1 substrates form multi-protein com- plexes that function in the nucleus although HUWE1 itself is cytoplasmically localized. Inhibition of nuclear entry enhances HUWE1-mediated ubiquitination and degradation, suggesting that USPD occurs primarily in the cytoplasm.
  • Mouse Gm44504 Knockout Project (CRISPR/Cas9)

    Mouse Gm44504 Knockout Project (CRISPR/Cas9)

    https://www.alphaknockout.com Mouse Gm44504 Knockout Project (CRISPR/Cas9) Objective: To create a Gm44504 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Gm44504 gene (NCBI Reference Sequence: NM_001278271 ; Ensembl: ENSMUSG00000015290 ) is located on Mouse chromosome X. 7 exons are identified, with the ATG start codon in exon 4 and the TAG stop codon in exon 7 (Transcript: ENSMUST00000178691). Exon 6 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 6 starts from about 32.91% of the coding region. Exon 6 covers 44.37% of the coding region. The size of effective KO region: ~209 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 6 7 Legends Exon of mouse Gm44504 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 209 bp section of Exon 6 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 209 bp section of Exon 6 is aligned with itself to determine if there are tandem repeats.
  • Genome-Wide Transcriptional Sequencing Identifies Novel Mutations in Metabolic Genes in Human Hepatocellular Carcinoma DAOUD M

    Genome-Wide Transcriptional Sequencing Identifies Novel Mutations in Metabolic Genes in Human Hepatocellular Carcinoma DAOUD M

    CANCER GENOMICS & PROTEOMICS 11 : 1-12 (2014) Genome-wide Transcriptional Sequencing Identifies Novel Mutations in Metabolic Genes in Human Hepatocellular Carcinoma DAOUD M. MEERZAMAN 1,2 , CHUNHUA YAN 1, QING-RONG CHEN 1, MICHAEL N. EDMONSON 1, CARL F. SCHAEFER 1, ROBERT J. CLIFFORD 2, BARBARA K. DUNN 3, LI DONG 2, RICHARD P. FINNEY 1, CONSTANCE M. CULTRARO 2, YING HU1, ZHIHUI YANG 2, CU V. NGUYEN 1, JENNY M. KELLEY 2, SHUANG CAI 2, HONGEN ZHANG 2, JINGHUI ZHANG 1,4 , REBECCA WILSON 2, LAUREN MESSMER 2, YOUNG-HWA CHUNG 5, JEONG A. KIM 5, NEUNG HWA PARK 6, MYUNG-SOO LYU 6, IL HAN SONG 7, GEORGE KOMATSOULIS 1 and KENNETH H. BUETOW 1,2 1Center for Bioinformatics and Information Technology, National Cancer Institute, Rockville, MD, U.S.A.; 2Laboratory of Population Genetics, National Cancer Institute, National Cancer Institute, Bethesda, MD, U.S.A.; 3Basic Prevention Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD, U.S.A; 4Department of Biotechnology/Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, U.S.A.; 5Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea; 6Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea; 7Department of Internal Medicine, College of Medicine, Dankook University, Cheon-An, Korea Abstract . We report on next-generation transcriptome Worldwide, liver cancer is the fifth most common cancer and sequencing results of three human hepatocellular carcinoma the third most common cause of cancer-related mortality (1). tumor/tumor-adjacent pairs.
  • QRICH1 Variants in Ververi-Brady Syndrome—Delineation of the Genotypic and Phenotypic Spectrum

    QRICH1 Variants in Ververi-Brady Syndrome—Delineation of the Genotypic and Phenotypic Spectrum

    Received: 23 June 2020 Revised: 21 September 2020 Accepted: 27 September 2020 DOI: 10.1111/cge.13853 SHORT REPORT QRICH1 variants in Ververi-Brady syndrome—delineation of the genotypic and phenotypic spectrum Melanie Föhrenbach1 |RamiAbouJamra2 | Arndt Borkhardt3 | Triantafyllia Brozou3 | Petra Muschke4 | Bernt Popp2,5 |LindaK.Rey1 |JörgSchaper6 |HaraldSurowy1 | Martin Zenker4 | Christiane Zweier5 | Dagmar Wieczorek1 |SilkeRedler1 1Institute of Human Genetics, Heinrich-Heine- University, Düsseldorf, Germany Abstract 2Institute of Human Genetics, University Ververi-Brady syndrome (VBS, # 617982) is a rare developmental disorder, and loss- Medical Center Leipzig, Leipzig, Germany of-function variants in QRICH1 were implicated in its etiology. Furthermore, a recog- 3Department of Pediatric Oncology, Hematology and Clinical Immunology, nizable phenotype was proposed comprising delayed speech, learning difficulties and Heinrich-Heine-University, Düsseldorf, dysmorphic signs. Here, we present four unrelated individuals with one known non- Germany sense variant (c.1954C > T; p.[Arg652*]) and three novel de novo QRICH1 variants, 4Institute of Human Genetics, University Hospital of Magdeburg, Otto-von-Guericke- respectively. These included two frameshift mutations (c.832_833del; p.(Ser278Leufs*25), University, Magdeburg, Germany c.1812_1813delTG; p.(Glu605Glyfs*25)) and interestingly one missense mutation 5Institute of Human Genetics, Friedrich- (c.2207G > A; p.[Ser736Asn]), expanding the mutational spectrum. Enlargement of Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany the cohort by these four individuals contributes to the delineation of the VBS phenotype 6 Department of Diagnostic and Interventional and suggests expressive speech delay, moderate motor delay, learning difficulties/mild Radiology, Heinrich-Heine-University, Düsseldorf, Germany ID, mild microcephaly, short stature and notable social behavior deficits as clinical hall- marks. In addition, one patient presented with nephroblastoma.
  • Bag6 Complex Contains a Minimal Tail-Anchor–Targeting Module and a Mock BAG Domain

    Bag6 Complex Contains a Minimal Tail-Anchor–Targeting Module and a Mock BAG Domain

    Bag6 complex contains a minimal tail-anchor–targeting module and a mock BAG domain Jee-Young Mocka, Justin William Chartrona,Ma’ayan Zaslavera,YueXub,YihongYeb, and William Melvon Clemons Jr.a,1 aDivision of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125; and bLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Gregory A. Petsko, Weill Cornell Medical College, New York, NY, and approved December 1, 2014 (received for review February 12, 2014) BCL2-associated athanogene cochaperone 6 (Bag6) plays a central analogous yeast complex contains two proteins, Get4 and Get5/ role in cellular homeostasis in a diverse array of processes and is Mdy2, which are homologs of the mammalian proteins TRC35 part of the heterotrimeric Bag6 complex, which also includes and Ubl4A, respectively. In yeast, these two proteins form ubiquitin-like 4A (Ubl4A) and transmembrane domain recognition a heterotetramer that regulates the handoff of the TA protein complex 35 (TRC35). This complex recently has been shown to be from the cochaperone small, glutamine-rich, tetratricopeptide important in the TRC pathway, the mislocalized protein degrada- repeat protein 2 (Sgt2) [small glutamine-rich tetratricopeptide tion pathway, and the endoplasmic reticulum-associated degrada- repeat-containing protein (SGTA) in mammals] to the delivery tion pathway. Here we define the architecture of the Bag6 factor Get3 (TRC40 in mammals) (19–22). It is expected that the complex, demonstrating that both TRC35 and Ubl4A have distinct mammalian homologs, along with Bag6, play a similar role (23– C-terminal binding sites on Bag6 defining a minimal Bag6 complex.
  • Call ERN ITHACA QRICH1

    Call ERN ITHACA QRICH1

    DepartmentRigshospitalet of Clinical Genetics Copenhagen University Hospital - Rigshospitalet QRICH1 Zeynep Tümer Kennedy Center Department of Genetics QRICH1-associated developmental delay and intellectual disability in 36 unrelated individuals ANNOUNCEMENT 23. june 2020 Targeted gene(s)/phenotype under study : QRICH1 Abstract : De novo pathogenic variants of QRICH1 (Glutamine-rich protein 1, OMIM #617387) has recently been described in five patients (Ververi et al. 2018; Lui et al. 2019). The variants have been associated with developmental delay and intellectual disability, mild facial dysmorphism and chondrodysplasia in some cases. Through Gene Matcher (Genematcher.org) we have now identified 19 further patients with QRICH1-variants. Currently, we are in the process of defining the phenotype-genotype spectrum of QRICH1- related disorders in the patient cohort (a total of 24 including the published cases) and preparing a manuscript. We welcome further cases to this study to reach a better understanding of this rare disorder. We aim to close inclusion of further cases 15th July 2020. Coordinating clinician/researcher: Zeynep Tümer CURRENT STATUS ➢ The current number of unpublished indivials are now 27 – two of the patients are through ITHACA collaboration (Prof. Favier’s group) ➢ 4 new patients reported meanwhile (Föhrenbach et al. 2020). ➢ TOTAL number of individuals will be 36 (including 9 published) QRICH1 has 11 isoforms and all encode the same 776 amino acid protein. • glutamine (Q) rich region • CARD domain (Caspase activation
  • Exome Sequencing of 457 Autism Families Recruited Online Provides Evidence for Autism Risk Genes

    Exome Sequencing of 457 Autism Families Recruited Online Provides Evidence for Autism Risk Genes

    www.nature.com/npjgenmed ARTICLE OPEN Exome sequencing of 457 autism families recruited online provides evidence for autism risk genes Pamela Feliciano1, Xueya Zhou 2, Irina Astrovskaya 1, Tychele N. Turner 3, Tianyun Wang3, Leo Brueggeman4, Rebecca Barnard5, Alexander Hsieh 2, LeeAnne Green Snyder1, Donna M. Muzny6, Aniko Sabo6, The SPARK Consortium, Richard A. Gibbs6, Evan E. Eichler 3,7, Brian J. O’Roak 5, Jacob J. Michaelson 4, Natalia Volfovsky1, Yufeng Shen 2 and Wendy K. Chung1,8 Autism spectrum disorder (ASD) is a genetically heterogeneous condition, caused by a combination of rare de novo and inherited variants as well as common variants in at least several hundred genes. However, significantly larger sample sizes are needed to identify the complete set of genetic risk factors. We conducted a pilot study for SPARK (SPARKForAutism.org) of 457 families with ASD, all consented online. Whole exome sequencing (WES) and genotyping data were generated for each family using DNA from saliva. We identified variants in genes and loci that are clinically recognized causes or significant contributors to ASD in 10.4% of families without previous genetic findings. In addition, we identified variants that are possibly associated with ASD in an additional 3.4% of families. A meta-analysis using the TADA framework at a false discovery rate (FDR) of 0.1 provides statistical support for 26 ASD risk genes. While most of these genes are already known ASD risk genes, BRSK2 has the strongest statistical support and reaches genome-wide significance as a risk gene for ASD (p-value = 2.3e−06).
  • Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching

    Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching

    Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching Yu Zhaoa, Yuting Lina, Honghong Zhanga, Adriana Mañasa, Wenwen Tangb, Yuzhu Zhangc, Dianqing Wub, Anning Linc, and Jialing Xianga,1 aDepartment of Biology, Illinois Institute of Technology, Chicago, IL 60616; bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520; and cBen May Department for Cancer Research, University of Chicago, Chicago, IL 60637 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 1, 2015 (received for review May 6, 2015) The serine-threonine kinase Akt is a key regulator of cell prolifer- Results ation and survival, glucose metabolism, cell mobility, and tumor- Ubl4A-Deficient Mice Display Increased Neonatal Mortality and a igenesis. Activation of Akt by extracellular stimuli such as insulin Defect in Liver Glycogen Synthesis. To better understand the bi- centers on the interaction of Akt with PIP3 on the plasma mem- ological functions of Ubl4A, we generated Ubl4A-deficient mice brane, where it is subsequently phosphorylated and activated (Fig. S1). Ubl4A knockout (KO) mice were viable but displayed by upstream protein kinases. However, it is not known how Akt increased neonatal mortality (occurring mostly within 24 h after is recruited to the plasma membrane upon stimulation. Here we A report that ubiquitin-like protein 4A (Ubl4A) plays a crucial role in birth) compared with their wild type (WT) littermates (Fig. 1 ). insulin-induced Akt plasma membrane translocation. Ubl4A knock- We noticed that some Ubl4A KO pups displayed signs of cyanosis A Inset out newborn mice have defective Akt-dependent glycogen syn- before death (Fig.
  • Lipopolysaccharide Treatment Induces Genome-Wide Pre-Mrna Splicing

    Lipopolysaccharide Treatment Induces Genome-Wide Pre-Mrna Splicing

    The Author(s) BMC Genomics 2016, 17(Suppl 7):509 DOI 10.1186/s12864-016-2898-5 RESEARCH Open Access Lipopolysaccharide treatment induces genome-wide pre-mRNA splicing pattern changes in mouse bone marrow stromal stem cells Ao Zhou1,2, Meng Li3,BoHe3, Weixing Feng3, Fei Huang1, Bing Xu4,6, A. Keith Dunker1, Curt Balch5, Baiyan Li6, Yunlong Liu1,4 and Yue Wang4* From The International Conference on Intelligent Biology and Medicine (ICIBM) 2015 Indianapolis, IN, USA. 13-15 November 2015 Abstract Background: Lipopolysaccharide (LPS) is a gram-negative bacterial antigen that triggers a series of cellular responses. LPS pre-conditioning was previously shown to improve the therapeutic efficacy of bone marrow stromal cells/bone-marrow derived mesenchymal stem cells (BMSCs) for repairing ischemic, injured tissue. Results: In this study, we systematically evaluated the effects of LPS treatment on genome-wide splicing pattern changes in mouse BMSCs by comparing transcriptome sequencing data from control vs. LPS-treated samples, revealing 197 exons whose BMSC splicing patterns were altered by LPS. Functional analysis of these alternatively spliced genes demonstrated significant enrichment of phosphoproteins, zinc finger proteins, and proteins undergoing acetylation. Additional bioinformatics analysis strongly suggest that LPS-induced alternatively spliced exons could have major effects on protein functions by disrupting key protein functional domains, protein-protein interactions, and post-translational modifications. Conclusion: Although it is still to be determined whether such proteome modifications improve BMSC therapeutic efficacy, our comprehensive splicing characterizations provide greater understanding of the intracellular mechanisms that underlie the therapeutic potential of BMSCs. Keywords: Alternative splicing, Lipopolysaccharide, Mesenchymal stem cells Background developmental pathways, and other processes associated Alternative splicing (AS) is important for gene regulation with multicellular organisms.
  • Supporting Information

    Supporting Information

    Supporting Information Friedman et al. 10.1073/pnas.0812446106 SI Results and Discussion intronic miR genes in these protein-coding genes. Because in General Phenotype of Dicer-PCKO Mice. Dicer-PCKO mice had many many cases the exact borders of the protein-coding genes are defects in additional to inner ear defects. Many of them died unknown, we searched for miR genes up to 10 kb from the around birth, and although they were born at a similar size to hosting-gene ends. Out of the 488 mouse miR genes included in their littermate heterozygote siblings, after a few weeks the miRBase release 12.0, 192 mouse miR genes were found as surviving mutants were smaller than their heterozygote siblings located inside (distance 0) or in the vicinity of the protein-coding (see Fig. 1A) and exhibited typical defects, which enabled their genes that are expressed in the P2 cochlear and vestibular SE identification even before genotyping, including typical alopecia (Table S2). Some coding genes include huge clusters of miRNAs (in particular on the nape of the neck), partially closed eyelids (e.g., Sfmbt2). Other genes listed in Table S2 as coding genes are [supporting information (SI) Fig. S1 A and C], eye defects, and actually predicted, as their transcript was detected in cells, but weakness of the rear legs that were twisted backwards (data not the predicted encoded protein has not been identified yet, and shown). However, while all of the mutant mice tested exhibited some of them may be noncoding RNAs. Only a single protein- similar deafness and stereocilia malformation in inner ear HCs, coding gene that is differentially expressed in the cochlear and other defects were variable in their severity.
  • Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching

    Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching

    Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching Yu Zhaoa, Yuting Lina, Honghong Zhanga, Adriana Mañasa, Wenwen Tangb, Yuzhu Zhangc, Dianqing Wub, Anning Linc, and Jialing Xianga,1 aDepartment of Biology, Illinois Institute of Technology, Chicago, IL 60616; bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520; and cBen May Department for Cancer Research, University of Chicago, Chicago, IL 60637 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 1, 2015 (received for review May 6, 2015) The serine-threonine kinase Akt is a key regulator of cell prolifer- Results ation and survival, glucose metabolism, cell mobility, and tumor- Ubl4A-Deficient Mice Display Increased Neonatal Mortality and a igenesis. Activation of Akt by extracellular stimuli such as insulin Defect in Liver Glycogen Synthesis. To better understand the bi- centers on the interaction of Akt with PIP3 on the plasma mem- ological functions of Ubl4A, we generated Ubl4A-deficient mice brane, where it is subsequently phosphorylated and activated (Fig. S1). Ubl4A knockout (KO) mice were viable but displayed by upstream protein kinases. However, it is not known how Akt increased neonatal mortality (occurring mostly within 24 h after is recruited to the plasma membrane upon stimulation. Here we A report that ubiquitin-like protein 4A (Ubl4A) plays a crucial role in birth) compared with their wild type (WT) littermates (Fig. 1 ). insulin-induced Akt plasma membrane translocation. Ubl4A knock- We noticed that some Ubl4A KO pups displayed signs of cyanosis A Inset out newborn mice have defective Akt-dependent glycogen syn- before death (Fig.
  • PRODUCT SPECIFICATION Prest Antigen QRICH1 Product Datasheet

    PRODUCT SPECIFICATION Prest Antigen QRICH1 Product Datasheet

    PrEST Antigen QRICH1 Product Datasheet PrEST Antigen PRODUCT SPECIFICATION Product Name PrEST Antigen QRICH1 Product Number APrEST79533 Gene Description glutamine-rich 1 Alternative Gene FLJ20259 Names Corresponding Anti-QRICH1 (HPA037678) Antibodies Description Recombinant protein fragment of Human QRICH1 Amino Acid Sequence Recombinant Protein Epitope Signature Tag (PrEST) antigen sequence: MNNSLENTISFEEYIRVKARSVPQHRMKEFLDSLASKGPEALQEFQQTAT TTMVYQQGGNCIYTDSTEVAGSLLELACPVTTSVQPQTQQEQQIQV Fusion Tag N-terminal His6ABP (ABP = Albumin Binding Protein derived from Streptococcal Protein G) Expression Host E. coli Purification IMAC purification Predicted MW 28 kDa including tags Usage Suitable as control in WB and preadsorption assays using indicated corresponding antibodies. Purity >80% by SDS-PAGE and Coomassie blue staining Buffer PBS and 1M Urea, pH 7.4. Unit Size 100 µl Concentration Lot dependent Storage Upon delivery store at -20°C. Avoid repeated freeze/thaw cycles. Notes Gently mix before use. Optimal concentrations and conditions for each application should be determined by the user. Product of Sweden. For research use only. Not intended for pharmaceutical development, diagnostic, therapeutic or any in vivo use. No products from Atlas Antibodies may be resold, modified for resale or used to manufacture commercial products without prior written approval from Atlas Antibodies AB. Warranty: The products supplied by Atlas Antibodies are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this warranty is limited to one year from date of sales for products used, handled and stored according to Atlas Antibodies AB's instructions. Atlas Antibodies AB's sole liability is limited to replacement of the product or refund of the purchase price.