Functional Analysis of Cereblon, an Evolutionary Conserved Substrate Receptor of the Cullin4-RING E3 Ubiquitin Ligase, in Mammalian Cells and in Drosophila melanogaster ヒト Cereblon タンパク質と そのキイロショウジョウバエ相同体の機能解析 February 2017 Satoru WAKABAYASHI 若林 慧 Functional Analysis of Cereblon, an Evolutionary Conserved Substrate Receptor of the Cullin4-RING E3 Ubiquitin Ligase, in Mammalian Cells and in Drosophila melanogaster ヒト Cereblon タンパク質と そのキイロショウジョウバエ相同体の機能解析 February 2017 Waseda University Graduate School of Advanced Science and Engineering Department of Advanced Science and Engineering Research on Life Science and Medical Biosciences Satoru WAKABAYASHI 若林 慧 Table of contents Chapter 1. Introduction .............................................................................................................. 6 1.1. Human Cereblon gene was identified as a candidate gene for onset of an intellectual disability.................................................................................................................................... 6 1.2. The gene's product CRBN contains two evolutionary conserved domains ....................... 7 1.3. CRBN is a substrate receptor of the Cullin4-RING E3 ubiquitin ligase complex ........... 10 1.3.1. The ubiquitin system ................................................................................................ 10 1.3.2. The Cullin4-RING E3 ubiquitin ligases ................................................................... 12 1.3.3. Endogenous substrates of the CRBN-containing Cullin4-RING E3 ubiquitin ligase ............................................................................................................................................ 15 1.3.3.1. Large conductance Ca2+ and voltage-activated K+ channels ................ 15 1.3.3.2. CLC-1 Cl- channels ............................................................................... 15 1.3.3.3. MEIS2 .................................................................................................... 16 1.3.3.4. Glutamine synthetase ............................................................................. 16 1.3.4. Thalidomide and its analogs alter functions of CRBN ............................................. 17 1.3.4.1. Thalidomide and the immunomodulatory drugs ................................... 17 1.3.4.2. Identification of CRBN as the primary target of thalidomide ............... 18 1.3.4.3. CRBN mediates therapeutic effects of IMiDs against multiple myeloma ............................................................................................................................ 18 1.3.4.4. CRBN mediates therapeutic effects of lenalidomide against 1 myelodysplastic syndrome with deletion of chromosome 5q ............................ 19 1.3.5. Ubiquitin-independent functions of CRBN .............................................................. 20 1.3.5.1. CRBN regulates metabolism via interaction with AMP-activated protein kinase .................................................................................................................. 20 1.3.5.2. CRBN mediates CD147-MCT1 complex maturation ........................... 20 1.3.5.3. CRBN expression in mitochondria results in enhanced resistance against oxidative stress ................................................................................................... 21 1.3.5.4. Regulatory functions of CRBN in immune cells ................................... 21 1.4. Aim of the thesis .............................................................................................................. 22 Chapter 2. Human CRBN localizes to aggresome and shows cytoprotective effect against proteasomal dysfunction ........................................................................................................... 23 2.1. Introduction ...................................................................................................................... 23 2.2. Materials and methods ..................................................................................................... 26 2.2.1. Reagents ................................................................................................................... 26 2.2.2. Antibodies ................................................................................................................. 26 2.2.3. Molecular cloning ..................................................................................................... 27 2.2.4. Cell culture ............................................................................................................... 27 2.2.5. Transfection and reagents treatment ......................................................................... 27 2.2.6. Protein extraction ...................................................................................................... 28 2.2.7. Western blotting ........................................................................................................ 28 2.2.8. Immunofluorescent microscopy and image acquisition ........................................... 28 2 2.2.9. Cytotoxicity assay .................................................................................................... 29 2.3. Results .............................................................................................................................. 30 2.3.1. CRBN is localized at aggresomes under proteasomal inhibition ............................. 30 2.3.2. A microtubule destabilizer and pomalidomide affects CRBN's localization at aggresomes ......................................................................................................................... 32 2.3.3. CRBN forms ubiquitin-positive, high-molecular weight aggregates under proteasomal inhibition ........................................................................................................ 35 2.3.4. CRBN shows cytoprotective effect against proteasomal inhibition ......................... 38 2.4. Discussion ........................................................................................................................ 41 Chapter 3. Characterization of Ohgata, the Drosophila ortholog of human CRBN ........... 43 3.1. Introduction ...................................................................................................................... 43 3.2. Materials and methods ..................................................................................................... 45 3.2.1. Antibodies ................................................................................................................. 45 3.2.2. cDNA ........................................................................................................................ 46 3.2.3. Molecular cloning ..................................................................................................... 46 3.2.4. Generating OHGT-specific antibodies...................................................................... 47 3.2.5. Fly works .................................................................................................................. 50 3.2.6. Generating an ohgata mutant alleles using the CRISPR-Cas9 genome editing technology .......................................................................................................................... 52 3.2.7. Phenotypic analyses .................................................................................................. 55 3.2.8. Developmental timing assay ..................................................................................... 55 3 3.2.9. Immunohistochemistry and image acquisition ......................................................... 56 3.2.10. Clonal analysis ........................................................................................................ 56 3.2.11. Protein extraction .................................................................................................... 56 3.2.12. Western blotting ...................................................................................................... 57 3.2.13. Generating tagged OHGT expression vectors ........................................................ 57 3.2.14. Cell culture and transfection ................................................................................... 57 3.2.15. Fractionation of S2 cells ......................................................................................... 58 3.2.16. Co-immunoprecipitation ......................................................................................... 58 3.2.17. In vivo ubiquitination .............................................................................................. 58 3.2.18. mRNA extraction .................................................................................................... 59 3.2.19. cDNA synthesis ...................................................................................................... 59 3.2.20. Quantitative Real-time PCR ................................................................................... 60 3.2.21. In silico analyses ..................................................................................................... 61 3.2.22. Statistics .................................................................................................................. 62 3.3. Results .............................................................................................................................. 63 3.3.1. The Drosophila