Ubiquitin E3 Ligase Mediated Regulation of HMG-Coa Reductase
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Ubiquitin E3 ligase mediated regulation of HMG-CoA reductase Sam Menzies Cambridge Institute of Medical Research Pembroke College University of Cambridge October 2017 This dissertation is submitted for the degree of Doctor of Philosophy Preface Preface This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration with others, except as declared in the Acknowledgements and specified in the text. It is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma or other qualification at the University of Cambridge or any other University or similar institution except as declared in the Preface and specified in the text. This dissertation contains less than 60,000 words. The work described in this dissertation has resulted in the following publication: Timms RT*, Menzies SA*, Tchasovnikarova IA*, Christensen LC*, Williamson JC, Antrobus R, Dougan G, Eligaard L, Lehner PJ (2016) Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens. Nat Commun. 10;7:11786 (*equal contribution) Sam Menzies October 2017 Summary Summary Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules. The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity. 5 Abbreviations Abbreviations ACAT2 acyl-CoA:cholesterol acyltransferase 2 ApoB apolipoprotein B100 ATP adenosine triphosphate AUP1 ancient ubiquitous protein 1 Β2M β2-microglobulin BAG6 BCL2 associated athanogene 6 BiP Immunoglobulin heavy chain binding protein B&W bind and wash Cas9 CRISPR-associated system 9 Cdc48p cell division cycle 48 CFTR cystic fibrosis transmembrane conductance regulator CHIP C-terminus of HSP70-interacting protein CoA co-enzyme A COPII coat protein complex II CRISPR clustered regularly interspaced palindromic repeats CUE coupling of ubiquitin to ER degradation Der1p degradation in the ER 1 Derlin Der1-like protein DGAT2 diacylglycerol acyltransferase 2 DMEM Dulbecco’s modified eagle medium Doa10p degradation of Matα2-10 protein dsRNA double-stranded RNA DUOX1 dual oxidase 1 ECL enhanced chemiluminescence substrate EDEM ER degradation enhancer mannosidase α-like protein EMC ER membrane complex ER endoplasmic reticulum 7 Abbreviations ERAD ER-associated degradation ERManI ER α1,2-mannosidase I ERLIN ER lipid raft associated FACS fluorescence-activated cell sorting FCS fetal calf serum GALNT11 polypeptide N-acetylgalctosaminyltransferase 11 GFP green fluorescent protein Gp78 glycoprotein 78 gRNA guide RNA HCMV human cytomegalovirus HECT homologous to E6 associated protein C-terminus Herp homocysteine/ER stress-inducible protein HLA human leukocyte antigen HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA HMGCR HMG-CoA reductase HO-1 heme oxygenase-1 Hrd1 HMG-CoA degradation 1 HRP horseradish peroxidase IAA iodoacetamide IDOL inducible degrader of LDLR IMDM Iscove’s Modified Dulbecco’s Medium Insig insulin induced gene Kar2p karyogamie 2 LC-MS liquid chromatography-mass spectrometry LDL low density lipoprotein LDLR LDL receptor LXR liver-X receptor MARCH6 membrane-associated RING-CH protein 6 MEF mouse embryonic fibroblast MHC-I major histocompatibility complex class I mRNA messenger RNA 8 Abbreviations NHEJ non-homologous end joining NK natural killer NPC Niemann-Pick type C OS9 osteosarcoma amplified 9 OST oligosaccharide transferase p97/VCP 97 kDa protein/valosin-containing protein PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCSK9 proprotein convertase substilisin/kexin type 9 PCR polymerase chain reaction PDI protein disulphide isomerase PMSF phenylmethanesulphonylfluoride PVDF polyvinylidene fluoride Rad radiation mutant RFP2 RING-finger protein 2 RING really interesting new gene RISC RNA-induced silencing complex RNAi RNA interference RNF RING finger protein ROS reactive oxygen species Rpn regulatory particle non-ATPase SCAP SREBP cleavage-activating protein SCF Skp-cullin 1-F box SDS sodium dodecyl sulphate Sec24 secretory 24 Sec61 secretory 61 SEL1L suppressor of lin-12, C. elegans (sel-1)-like SFFV spleen focus-forming virus shRNA small hairpin RNA siRNA small interfering RNA SPP signal peptide peptidase 9 Abbreviations SREBP sterol regulatory binding element protein SSD sterol sensing domain TBS Tris-buffered saline TCRα T-cell receptor chain alpha TECR trans-2,3-enoyl CoA reductase TMEM transmembrane protein TRC8 translocated in renal carcinoma, chromosome 8 TRIM tripartite motif containing protein Trx thioredoxin-like domain Ub ubiquitin UBA ubiquitin-association domain UBAC2 UBA containing protein 2 UBC ubiquitin-conjugating UBE2 ubiquitin-conjugating enzyme UBL ubiquitin like UBX ubiquitin-regulatory X domain UBXD UBX domain containing protein Ufd ubiquitin fusion degradation UGT1 UDP glucose: glucose glucosyl-transferase 1 UIM ubiquitin-interaction motif UPS ubiquitin proteasome system UROD uroporphyrinogen decarboxylase US2 HCMV protein unique short region 2 US11 HCMV protein unique short region 11 VIM VCP-intracting motif VIMP VCP interacting membrane protein WT wild-type w/v weight per volume XTP3-B XTP3-transactivated protein B Yos9p yeast OS9 homologous protein 10 Table of Contents Table of Contents Summary……………………………………………….…………………………………………………………………………5 Abbreviations……………………………………………….………………………………………………………………….7 Table of Contents……………………………………………….…………………………………………………………..11 Chapter 1: Introduction……………………………………………….…………………………………..…………….19 1.1 The ubiquitin system………………………………………………………………………….……………. 19 1.1.1 E2 ubiquitin-conjugating enzymes………………………………………………………20 1.1.2 HECT and RING E3 ubiquitin ligases…………………………………………………….21 1.1.3 ER-associated degradation (ERAD)……………………………………………………..21 1.1.4 S. cerevisiae ERAD E3 ligase complexes………………………………………………22 1.1.5 Mammalian ERAD E3 ligase complexes……………………..…………………….…23 1.1.6 ERAD substrate recognition………………………………………………………………..26 1.1.7 Oxidoreductases…………………………………………………………………………………28 1.1.8 Retrotranslocation and membrane extraction…………………………………...28 1.1.9 Degradation by the proteasome…………………………………………………………30 1.2 Cholesterol homeostasis……………………………………..………………………………………......31 1.2.1 Transcriptional control of cholesterol homeostasis………………………..…..31 1.2.2 Regulation of cholesterol homeostasis by ERAD……………..……………….…35 1.2.3 Cholesterol uptake……………………………………………………………..……………...43 1.3 Forward genetic screens…………………………………………………………………………..…….…44 1.3.1 Forward genetic screens in model organisms……………………………………..44 1.3.2 Forward genetic screens in human cells……………………………………………..45 1.3.3 Retroviral gene-trap mutagenesis screens in near-haploid human cells……………………………………………………………………………………………………………..47 1.3.4 Genome-wide CRISPR/Cas9 knockout screens…………………………….………49 1.4 Aims of this thesis……………………………………………………………………………………………..51 Chapter 2: Materials and Methods………………………………………………………………………………….52 2.1 Materials…………………………………………………………………………………………………………..52 11 Table of Contents 2.1.1 Buffers………………………………………………………………………………………………..52