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研究生： 陳柏樺
Chen, Bo-Hua
論文名稱： Hsc70/Stub1對氧化壓力下過氧化體清除機制之探討
Hsc70/Stub1 drives individual turnover of oxidatively-stressed peroxisomes
指導教授： 楊維元
Yang, Wei Yuan
呂平江
Lyu, Ping-Chiang
口試委員: 陳光超
Chen, Guang-Chao
顏雪琪
Yen, Hsueh-Chi Sherry
陳瑞華
Chen, Ruey-Hwa
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 92
中文關鍵詞： 氧化壓力 、過氧化體 、胞器品質控管 、抗壓蛋白 、分子監護子 、細胞自噬
外文關鍵詞： peroxisomes, pexophagy, organelle, Heat shock protein 70, Hsp70, roGFP2, Stub1
相關次數： 點閱：70  下載：0 
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    • 中文摘要
    過氧化體參與非常長鏈脂肪酸與支鏈脂肪酸的氧化降解，此降解過程在過氧化體內產生活性氧化物質，因此過氧化體特別容易受到氧化損傷。細胞中很可能存在過氧化體的品質控管機制但目前並未被清楚了解。我們藉由光在活細胞中選定部分的過氧化體內產生氧化壓力進而發現細胞會選擇性地清除在氧化壓力下的過氧化體，此清除過程需要泛素。細胞需要藉由抗壓蛋白分子監護子70 (Heat shock protein 70s)將E3泛素連結酶Stub1帶到氧化壓力下的過氧化體而讓Stub1進行泛素化與促進細胞自噬清除機制來清除氧化壓力下的過氧化體。 我們也人為地將E3泛素連結酶Stub1標靶到健康的過氧化體上就足以讓Stub1對過氧化體進行泛素化與促進過氧化體細胞自噬的能力。我們發現其中細胞自噬銜接蛋白p62在此過氧化體細胞自噬的重要性，E3泛素連結酶Stub1可能一部分透過徵集p62到氧化壓力下的過氧化體上而促進氧化壓力下之過氧化體被細胞自噬機制清除。我們發現造成遺傳性小腦性運動失調(autosomal recessive cerebellar ataxia, ARCA)相關的突變型Stub1無法促進此氧化壓力下的過氧化體細胞自噬。降低細胞Stub1的表現，隨著時間會增加細胞中高氧化壓力的過氧化體之比例。因此在遺傳性小腦性運動失調疾病(autosomal recessive cerebellar ataxia, ARCA)中，我們的發現為不正常的過氧化體品質控管參與了疾病的發展提供了可能性。

    Abstract

    Peroxisomes carry out beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. The control of peroxisome quality should exist but is not well-understood. By using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we found that cells can individually turnover ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70s mediated the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology And U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to drive their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes was sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. Our data indicated that p62 is one of the pivotal autophagic adaptors for stub1 to recruit autophagosome marker LC3. We further determined that Stub1 mutants found in Ataxia patients were defective in their abilities to trigger pexophagy. Stub1-depletion increased the fraction of oxidative peroxisomes in cells. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.

    Table of contents 中文摘要........................................................¬1 Abstract............................................................2 Table of contents.....................................................3 Introduction..........................................................9 1. Reactive Oxygen Species (ROS).......................................9 2. ROS regulation in peroxisomes.......................................10 2.1. ROS producing in peroxisomes..................................10 2.2. ROS scavenging in peroxisomes.................................11 2.3. Peroxisomes are essential organelles for cell metabolism..............12 3. Peroxisome quality control..........................................15 4. Hsp70/Stub1 in protein quality control system.......................... 16 4.1 Hsp70 chaperones............................................ 16 4.2 Stub1, the Hsp70 chaperone-dependent E3 ligase.................... 17 4.2.1 Stub1 in ubiquitination system.............................. 17 4.2.2 Hsp70 and chaperone-assisted selective autophagy (CASA)....... 19 Results.............................................................. 22 Stressing peroxisomes with ROS through peroxisome-targeted KillerRed...... 22 Ubiquitin-dependent pexophagy on ROS-stressed peroxisomes...............22 Stub1 translocated onto ROS-stressed peroxisomes........................24 Heat shock protein 70s mediated Stub1 translocation onto ROS-stressed peroxisomes...................................................... 26 Depleting Stub1 results in cellular accumulation of impaired peroxisomes.......27 Ataxia-related Stub1 mutants are defective in their ability to signal ubiquitin-dependent pexophagy............................................... 28 Discussion........................................................... 29 Materials and methods................................................ 31 Plasmids......................................................... 31 RNA interference.................................................. 32 Cell culture conditions...............................................33 SDS-PAGE and Western blot..........................................33 Quantifying peroxisomal redox potential................................34 Quantification of peroxisomal EGFP-LC3B following damage...............35 Monitoring turnover of damaged peroxisomes............................35 Targeting Cry2-mCherry-Stub1 to healthy peroxisomes.....................36 Immunofluorescence assay for LED-illuminated NIH3T3 cells on confocal dish.............................................................36 Figures............................................................. 38 Fig. 1. The schematic of how we trigger ROS-generation specifically within the peroxisomal lumen. .................................................38 Fig. 2. Light illumination specifically generated ROS within the peroxisome lumen. ...........................................................39 Fig. 3. The fluorescence of EGFP-PTS1 was not affected by 559 nm illumination. ......................................................40 Fig. 4. ROS-stressed peroxisomes accumulated ubiquitination. ...............41 Fig. 5. ROS-stressed peroxisomes accumulated autophagy adaptor p62. ........42 Fig. 6. Autophagic membrane marker LC3 colocalized with ROS-stressed peroxisomes. ......................................................43 Fig. 7. Entering acidic environments was demonstrated by the loss of EGFP signal from PMP34-EGFP-TagBFP2 labeled and ROS-stressed peroxisomes. ........44 Fig. 8. ROS-stressed peroxisomes colocalized with lysosomes indicated by Lamp1-mGFP. ...........................................................45 Fig. 9. Turnover of ROS-stressed peroxisomes required functional lysosomes. .......................................................46 Fig. 10. Reporter construct EGFP-Stub1 translocated onto ROS-stressed peroxisomes. ......................................................47 Fig. 11. EGFP-Stub1 did not translocate onto ROS-stressed mitochondria. ......48 Fig. 12. Scheme to target Stub1 onto non-stressed peroxisomes. ..............49 Fig. 13. Targeting Stub1 onto non-stressed peroxisomes by CRY2-CIBN dimerization induced EGFP-LC3B accumulation. .........................50 Fig. 14. Targeting CRY2-mCherry-Stub1 onto peroxisomes induced peroxisomal ubiquitination. .....................................................51 Fig. 15. Targeting CRY2-mCherry alone onto peroxisomes did not trigger peroxisomal ubiquitination. ..........................................52 Fig. 16. Targeting E3 ligase dead CRY2-mCherry-Stub1 H261Q to peroxisomes did not result in LC3B translocation. .......................................53 Fig. 17. Overexpressing Stub1 ligase dead mutant H261Q blocks cellular turnover of ROS-stressed peroxisomes. ........................................54 Fig. 18. Stub1 is required for EGFP-LC3B accumulation onto ROS-stressed peroxisomes. ......................................................55 Fig. 19. Depleting NIH3T3 cellular p62 delayed LC3 accumulation onto ROS-stressed peroxisomes. ...............................................56 Fig. 20. Knockdown of Stub1 delayed p62 accumulation onto ROS-stressed peroxisomes. ......................................................57 Fig. 21. Stub1 K31A mutant was unable to translocate onto ROS-stressed peroxisomes. ......................................................58 Fig. 22. EGEP-Hsp70 accumulated onto ROS-stressed peroxisomes. ..........59 Fig. 23. EGFP-Hsc70 similarly accumulated onto ROS-stressed peroxisomes. ...60 Fig. 24. Co-chaperone reporter construct EGFP-Hsp40 accumulated on ROS-stressed peroxisomes. ...............................................61 Fig. 25. Binding to Stub1 is not required for EGFP-Hsp70 translocation. ........62 Fig. 26. Efficiencies of the Hsp70 and Hsc70 siRNAs utilized in this paper. ......63 Fig. 27. Hsc70/Hsp70 are required for EGFP-LC3B accumulation onto ROS-stressed peroxisomes. ......................................................64 Fig. 28. Hsp70 inhibitor PES-Cl blocks EGFP-Hsp70 translocation onto ROS-stressed peroxisomes. ...............................................65 Fig. 29. Hsp70 inhibitor PES-Cl blocks EGFP-Hsc70 translocation onto ROS-stressed peroxisomes. ...............................................66 Fig. 30. Hsp70 inhibitor PES-Cl prohibits EGFP-Stub1 translocation onto ROS-stressed peroxisomes. ...............................................67 Fig. 31. PES-Cl inhibited EGFP-LC3B translocation onto ROS-stressed peroxisomes. ......................................................68 Fig. 32. Probing redox states of each peroxisome by a reporter construct roGFP2-PTS1. ...........................................................69 Fig. 33. Depletion of Stub1 increased the number and fraction of high redox state peroxisomes in cell. .................................................70 Fig. 34. Depletion of Stub1 increased the fraction of high redox state peroxisomes in cell with time. ...................................................71 Fig. 35. Peroxisome quality is compromised by depleting endogenous Hsc70/Hsp70. .....................................................72 Fig. 36. Table: Testing whether Ataxia-related Stub1 mutants translocate onto ROS-stressed peroxisomes................................................73 Fig. 37. Overexpressing Ataxia-related Stub1 mutants diminishes LC3B recruitment onto ROS-stressed peroxisomes. .......................................74 Fig. 38. Endogenous Stub1 translocated onto ROS-stressed peroxisomes. ......75 Fig. 39. Endogenous ubiquitin accumulated onto ROS-stressed peroxisomes. ....76 Fig. 40. Endogenous autophagy adaptor p62 accumulated onto ROS-stressed peroxisomes. ......................................................77 Fig. 41. Endogenous autophagosome marker LC3B accumulated onto ROS-stressed peroxisomes. ......................................................78 Fig. 42. EGFP-Stub1 was translocated onto peroxisomes continuously ROS-stressed by LED illumination. ...............................................79 Fig. 43. Peroxisomes continuously ROS-stressed by LED illumination accumulated EGFP-p62. .......................................................80 Fig. 44. EGFP-LC3B was translocated onto peroxisomes continuously ROS-stressed by LED illumination. ...............................................81 Fig. 45. EGFP-Stub1 was translocated onto peroxisomes ROS-stressed by illuminating the peroxisome-localized TMR HaloTag ligand. ................82 Fig. 46. TMR HaloTag-PTS1 targeting accompanied by continuous LED illumination successfully induced peroxisome damage indicated by EGFP-Ub accumulation. .....................................................83 Fig. 47. Endogenous ROS caused peroxisome damage indicated by translocated EGFP-Stub1. ......................................................84 Acknowledgements....................................................85 Abbreviations........................................................85 References...........................................................86

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