새로운 프로테아좀 조절 단백질에 관한 연구

Abstract

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 8. 정용근.The proteasome is a large protein complex that degrades diverse proteins in ubiquitine-proteasome system (UPS). Numerous substrates which play roles in many signaling to maintain homeostasis are known to be degraded by the complicated degradation processes. In addition, aberrant regulation in UPS and of this complex is associated with various diseases such as cancer, disorder of immune response and neurodegenerative disease. However, it is not known whether and how this elaborate machinery is regulated by diverse cellular signaling. Thus, discovery of novel proteasome regulators is important to understand UPS-associated cellular function and the pathogenesis of various diseases related to proteasome malfunction. To identify new proteasome modulators regulating the proteasome activity, a cell-based functional screening was established using Degron-GFP and a collection of cDNA library. In this study, I have isolated iRhom1 as a stimulator of proteasome activity from genome-wide functional screening using cDNA expression and an unstable GFP-degron. Expression level of iRhom1 regulated enzymatic activity and assembly of proteasome complexes. iRhom1 expression was induced by endoplasmic reticulum (ER) stressors, leading to the enhancement of proteasome activity, especially in ER-containing microsomes. iRhom1 interacted with PAC1 and PAC2, the 20S proteasome assembly chaperones, affecting their protein stability by dimerization of them. In addition, iRhom1 deficiency in D. melanogaster accelerated the rough-eye phenotype of mutant Huntingtin, while transgenic flies expressing either human iRhom1 or Drosophila iRhom showed rescue of the rough-eye phenotype. S5b was previously identified as a proteasome-assembly chaperone in yeast and a negative regulator of 26S proteasome in mammalian. Although regulation of GRK2 is considered as one of cell death mediators in neuronal cells, the regulation of GRK2 expression is not known. Here, I show that GRK2 is regulated by S5b in neuronal cells and mouse model. GRK2 is down-regulated in the cortex and hippocampus of S5b transgenic mice, a chronic inflammation model and also reduced by S5b expression in HT22 mouse hippocampal cells. Conversely, knockdown of S5b expression increases GRK2 level through increasing the stability of GRK2 protein, independent of its ability to impair proteasome activity. GRK2 and GRK2 K220R, a kinase dead mutant, similarly interacts with S5b in the mouse cortex and HT22 cells through its C-terminal domain, and this domain also decreases GRK2 level. Membrane targeting of GRK2 is affected by S5b expression, as assessed with immunocytochemistry, fractionation, and surface biotinylation assays. In addition, neurotoxic effect of S5b is suppressed by overexpression of GRK2 but not by GRK2 K220R. Thus, S5b may exert its toxic effect through down-regulation of GRK2, a neurotoxic mediator, in neuronal cells, showing an aberrant role of S5b as a negative regulator of GRK2 in neuronal cell death. In addition, Psmd5/S5b knockout mouse was successfully generated by the Cas9/CRISPR-mediated Psmd5/S5b knockout cassette and show enhanced proteasome activity compared to aged matched littermates. Together, S5b plays a diverse role in the regulation of proteasome activity under pathologic condition and in neuronal cell death through GRK2. In conclusion, I suggest a novel stress signaling pathway responsible for proteasome regulation and critical role of S5b in neuronal cell death independent of its inhibitory function of proteasome.ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .i CONTENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v LIST OF FIGURES AND TABLES. . . . . . . . . . . . . .ix ABBREVIATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii CHAPTER I. iRhom1 regulates proteasome activity via PAC 1/2 under ER stress I-1. Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 I-2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . .3 I-3. Materials and Methods . . . . . . . . . . . . . . .6 Cell culture and transfection. . . . . . . . . . . . . . . . . . . . . .6 Generation of stable cell line. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Genome-wide functional screening. . . . . . . . . . . . . . . . . . . . . .6 Plasmid construction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Antibodies and western blotting. . . . . . . . . . . . . . . . . . . . . . . . .7 Assays for proteasome activities. . . . . .. . . . . . . . . . . . . . . . . . . . . .8 Reverse transcriptase-PCR . . . . . . . . . . . . . . . . . . .8 Subcellular fractionation. . . . . . . . . . . . . . . . . . . . . . . . . .9 Glycerol gradient analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Immunoprecipitation assay. . . . . . . . . . . . . . . . . . . . . . . . . . . .10 Immunocytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Native gel analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 Filter trap assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 Drosophila genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 I-4. RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 iRhom1 isolated by functional screening enhances proteasome activity . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .13 iRhom1 affects the assembly of proteasome complexes. . . . . .. . . .15 iRhom1 regulates microsomal proteasome activity in response to ER stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16 iRhom1 increases protein stability and dimerization of PAC1 and PAC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19 iRhom1 relieves mutant Huntingtin aggregation in cells and Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 I-5. DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77 I-6. REFERENCES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82 CHAPTER II. S5b induces neuronal cell death via downregulation of GRK2 II-1. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94 II-2. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . .96 II-3. Materials and Methods . . . . . . . . . . . . . . .98 Antibodies and sh- or si- RNA construction . . . . . . . . . . . . . . . . .98 Cell Culture and DNA Transfection . . . . . . . . . . . . . . . . . . . . . . . .98 SDS-PAGE and Immunoblot Analysis. . . . . . . . . . . . . . . . . . . . . .98 Immunoprecipitation and Immunohistochemisty . . . . . . . . . . . .99 Subcellular Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Biotinylation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 II-4. RESULTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 101 GRK2 level is regulated by S5b in HT22 cells and the brain of S5b transgenic mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101 S5b interacts with GRK2 through its C-terminus. . . . . . . . . . . . . . 102 S5b impairs the targeting of GRK2 to the plasma membrane. . . . . .103 S5b affects neuronal cell death probably via down-regulation of GRK2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105 Generation of Psmd5/S5b knockout mice with enhanced proteasome Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .105 II-5. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130 II-6. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135 ABSTRACT IN KOREAN/국문 초록. . . . . . . . . . .143 LIST OF FIGURES Figure I-1. Stimulatory effect of iRhom1 overexpression on proteasome activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Figure I-2. Ectopic expression of iRhom1 reduces degron (GFPU) and elevates proteasome catalytic activity . . . . . . . . . . . . . . . . . . . . . .25 Figure I-3. Effects of cDNAs encoding polytopic membrane proteins on proteasome activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27 Figure I-4. Downregulation of iRhom1 reduces proteasome activity and increases the accumulation of ubiquitin-conjugates. . . . . . . . . .29 Figure I-5. Ectopic expression of iRhom1 increases catalytic activity of proteasome and reduces ub-conjugation . . . . . . . . . . . . . . . . . . ...31 Figure I-6. Ectopic expression of iRhom1 increases proteasome assembly in native gel and reduces MG132 induced ub-conjugation. . . . . . .33 Figure I-7. Overexpression effects of the Rhomboid protein family and their activity-dead mutants on proteasome activity. . . . . . . . . . . . . . . .35 Figure I-8. iRhom1 does not affect RNA or protein levels of proteasome subunit . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 Figure I-9. Downregulation of iRhom1 impairs the assembly of proteasome complexes by native gel analysis. . . . . . . . . . . . . . . . . . . . . .39 Figure I-10. Knockdown of iRhom1 expression impairs the assembly of proteasome complexes in a fractionation assay . . . . . . . . . . . . .41 Figure I-11. Ectopic expression of iRhom1 does not increase protein levels of proteasome subunit but only elevates proteasome activity in fractionation assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43 Figure I-12. iRhom1 localizes in the ER of HeLa and HEK293T cells. . . . ...45 Figure I-13. iRhom1 regulates proteasome activity in the microsomal fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47 Figure I-14. iRhom1 regulates proteasome assembly in the microsomal Fractions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49 Figure I-15. iRhom1 is increased by ER stress . . . . . . . . . . . . . . . . . . . . . . . . . .51 Figure I-16. Increase in iRhom1 expression by stress signals . . . . . . .53 Figure I-17. Knockdown of iRhom1 expression impairs ER stress-induced activation and assembly of proteasomes in the microsomal fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..55 Figure I-18. The amounts of PAC1 and PAC2 proteins are decreased by iRhom1-knockdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57 Figure I-19. iRhom1 enhances the stability of PAC1 protein. . . . . . . . . . . . . .59 Figure I-20. iRhom1 regulates the stability of PAC1 and PAC2 proteins. . . .61 Figure I-21. iRhom1 affects the interaction between PAC1 and PAC2. . . . . .63 Figure I-22. ER stress increases PAC1/PAC2 dimerization in an iRhom1- dependent manner. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65 Figure I-23. Expression level of iRhom1 modulates the aggregation of mutant Huntingtin in cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 Figure I-24. Ectopic expression of PAC1 and PAC2 elevates proteasome Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 Figure I-25. Expression level of iRhom1 modulates the aggregation of the rough-eye phenotype in a fly model expressing Htt120Q. . . . .71 Figure I-26. Overexpression of drosophila iRhom or human iRhom1 in drosophila eye shows mild disturbance in eye development and increases proteasome activity. . . . . . . . . . . . . . . . . . . . . . . . . . . .73 Figure I-27. Schematic diagram showing the proposed role of iRhom1 in proteasome activation under ER stress. . . . . . . . . . . . . . . . . . . . .75 Figure II-1. S5b overexpression downregulates GRK2 in the cortex and hippocampus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107 Figure II-2. Ectopic expression level of S5b reduces GRK2. . . . . . . . . . . . . .109 Figure II-3. Knockdown of S5b expression increases GRK2 at post- translational level. . . . . . . . . . . . . . . . . . . . . . . . . . . . .111 Figure II-4. S5b interacts with GRK2 via S5b C-terminal domain. . . . . . . 113 Figure II-5. Regulation in the translocation of GRK2 from cytosol to plasma membrane by S5b expression. . . . . . . . . . . . . . . . . . . . . . . . . .115 Figure II-6. S5b recruits membrane GRK2 into cytosol. . . . . . . . . . . . . . . . . .117 Figure II-7. Ectopic expression of S5b induces apoptosis in HT22 cell line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 119 Figure II-8. GRK2 activation suppresses S5b overexpression induced cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Figure II-9 Generation of PSMD5/S5b knockout mice . . . . . . . . . . . . . . . . .123 Figure II-10. S5b expression levels were determined in th tissue of WT and PSMD5/S5b deficient mouse . . . . . . . . . . . . . . . . . . . . . . . . . . .125 Figure II-11. Elevated proteasome activity in S5b knockout mouse . . . . . 127 Figure II-12. Proposed model for the role of GRK2 in S5b-mediated neuronal cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129Docto