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研究生:羅凱倫
研究生(外文):Karen Gutierrez Rosal
論文名稱:CYP11A1 重塑粒線體皺褶的機制
論文名稱(外文):Investigating the mechanism of CYP11A1-dependent mitochondria crista remodeling
指導教授:鍾邦柱 博士
指導教授(外文):Bon-chu Chung, PhD
口試委員:李秀敏 博士史有伶 博士李宜靜 博士張繼堯 博士
口試委員(外文):Hsou-min Li, PhDYu-Ling Shih, Ph.D.Yi-Ching Lee, PhDChi-Yao Chang, PhD
口試日期:2022-03-28
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:英文
論文頁數:80
中文關鍵詞:CYP11A1
外文關鍵詞:CYP11A1
相關次數:
  • 被引用被引用:0
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摘要

CYP11A1 是一位於線粒體內膜的酵素,催化類固醇合成的第一步。作為類固醇生成細胞的標記基因,CYP11A1 的多寡可作為類固醇生成細胞分化的程度。此外,在完全分化的類固醇生成細胞中粒線體型態為管泡狀嵴。然而,CYP11A1 在參與粒線體結構變化和類固醇生成細胞分化的機制尚未被研究清楚。
我們設計了非甾體生成的猴腎 COS1 細胞,在強力黴素誘導後表達 CYP11A1,並研究此蛋白質對細胞的粒線體型態的影響。我們也找出了在CYP11A1 結構中主要負責粒線體形態變化的區域。並透過搜索 CYP11A1 的協同蛋白,研究協同蛋白對CYP11A1塑造線粒體形態中的影響。最後,並測試當 CYP11A1 過度表達時,對線粒體接觸位點和嵴組織型態系統的作用。
我們發現當 CYP11A1 過表達,會導致線粒體中管狀囊狀嵴的形成。更進一步的確定位於CYP11A1蛋白質中的A'-螺旋,胺基酸序列57-68, 是主要讓粒線體重塑成嵴型態的關鍵結構域。我們鑑定熱休克蛋白 60 (Hsp60)為 CYP11A1 相互作用蛋白,並發現 Hsp60 是 CYP11A1累積和嵴重塑所必需的。最後,我們發現當 CYP11A1 過表達時,會造成粒線體亞群MIC10的接觸位點和嵴組織系統減少。
總結來說,CYP11A1 參與了類固醇生成細胞中線粒體的管狀囊嵴形成。在此蛋白質中A'-螺旋主要負責形成管狀囊狀嵴和整合蛋白質到膜中。 而Hsp60是CYP11A1的協同蛋白質,幫助 CYP11A1 累積在類固醇生成細胞中。 CYP11A1 的累積會導致 粒線體亞群MIC10 複合物的減少並改變粒腺體結構。

Abstract

The enzyme CYP11A1 (P450scc) is located in the inner mitochondria that catalyzes the first step of steroid production. CYP11A1 is a marker gene for steroid-producing cells. Its abundance characterizes the extent of steroid cell differentiation. In addition, fully differentiated steroidogenic cells have specialized mitochondria with tubulovesicular crista structure. However, the role of CYP11A1 in mitochondrial structure changes and steroid-producing cell differentiation is unknown.
To examine changes in mitochondria crista structure, I used stable clones of COS1 cells overexpressing CYP11A1 when induced by doxycycline. I also determined the specific domains of CYP11A1 that trigger crista remodeling. Furthermore, I checked CYP11A1-interacting proteins and their role in shaping mitochondria cristae. Lastly, I determined the effect of CYP11A1 overexpression on the amount of mitochondrial contact site and cristae organizing system.
My results showed that overexpression of CYP11A1 triggers crista remodeling from lamellar to tubulovesicular type. I also found that the A’- helix of CYP11A1 located at amino acids #57-68 is sufficient for membrane anchorage and crista remodeling. I also identified HSPD1 (HSP60) as an interacting protein with CYP11A1 that is required for its accumulation and function in crista remodeling. Finally, my results showed that when CYP11A1 was overexpressed, one of the subunits of the mitochondrial contact site and cristae organizing system, the MIC10 complex, was reduced.
In conclusion, I found that CYP11A1 remodels mitochondria crista structure and its A’- helix domain is sufficient for crista remodeling and membrane insertion. Also, I found that HSPD1 is required for CYP11A1 accumulation and function for crista remodeling. Finally, the CYP11A1 accumulation resulted to the reduction of MIC10 complex and changes mitochondria cristae.

Table of Contents

Acknowledgments I
Table of Contents III
摘要 VIII
Abstract IX
1. Introduction 1
1.1 Background of the Study 1
1.1.1 Overview of Steroidogenesis 1
1.1.2 Electron transport complexes and their specific functions 1
1.1.3 Structure of cytochrome P450 (CYP11A1) 2
1.1.4 Maintenance of mitochondria cristae 2
1.2 Objective of the Study 3
2. Materials and Methods 4
2.1 RNA isolation, plasmid, and cloning 4
2.2 Cell culture, transient transfection, generation of stable clones 5
2.3 Immunofluorescence 6
2.4 Enzymatic activity by ELISA Assay 6
2.5 Protein extraction, membrane protein extraction 7
2.6 Affinity chromatography and immunoprecipitation 7
2.7 Mass Spectrometry Analysis 8
2.8 Mitochondria isolation from cells 9
2.9 Transmission Electron Microscopy 10
2.10 HSPD1 (Hsp60) knockdown 10
2.11 Polyacrylamide gel electrophoresis and Western Blot 11
2.12 Measurement of mitochondria oxygen consumption rate and membrane potential 11
2.13 Quantification and Statistical analysis 12
3. Results 14
3.1 Cristae remodeling by CYP11A1 14
3.2 Cristae remodeling by zebrafish Cyp11a1 and Cyp11a2 14
3.3 Identification of regions in CYP11A1 for membrane anchorage and cristae remodeling 15
3.4 Metabolic activity of stable clones of CYP11A1 16
3.5 Identification of CYP11A1-interacting proteins 17
3.6 Hsp60 knockdown and its effect on mitochondria cristae 18
3.7 Mechanism of cristae remodeling by CYP11A1 in steroid-producing cells 19
4. Discussion 20
5. Conclusion 23
6. References 24
7. List of Tables 28
7.1 Table 1. Key resources used in this study 27
7.2 Table 2. CYP11A1 interacting proteins identified by mass spectrometry 34
8. Figure Legends 36
9. List of Figures 41
9.1 Introductory Figures 42
9.1.1 Introductory Figure I1. Flowchart of steroid production 42
9.2 .1 Introductory Figure I2. Differentiation of adrenal glands showing increased amount of CYP11A1 43
9.3.1 Introductory Figure I3. Differentiation of human placental cells from cytotrophoblast to syncytiotrophoblast. Flowchart of steroid production 44
9.4.1 Introductory Figure I4. Flowchart of steroid production 45
9.5.1 Introductory Figure I5. Flowchart of steroid production 46
9.6.1 Introductory Figure I6. Flowchart of steroid production 47
9.7.1 Introductory Figure I7. Flowchart of steroid production 48
9.2 Figures 49
9.2.1 Figure 1. Stable clones of COS1 overexpressing CYP11A1 49
9.2.3 Figure 2. Mitochondria cristae structure of stable clones of COS1 overexpressing CYP11A1 50
9.2.3 Figure 3. Localization of CYP11A1 homologues in zebrafish 51
9.2.4 Figure 4. Cristae remodeling by human CYP11A1 and zebrafish Cyp11a1 and Cyp11a2 52
9.2.5 Figure 5. Domains of CYP11A1 53
9.2.6 Figure 6. Localization CYP11A1 fragments and full length fused with EGFP in the mitochondria of transfected COS1 cells 54
9.2.7 Figure 7. Partitioning of EGFP-fused CYP11A1 fragments and full length by alkaline buffer extraction 55
9.2.8 Figure 8. Localization of CYP11A1 fragments in stable clones of COS1 56
9.2.7 Figure 9. Mitochondria cristae structure of COS1 cells transfected with CYP11A1 57
9.2.10 Figure 10. Components of electron transport complex from stable clones of CYP11A1 with or without induction of CYP11A1 58
9.2.11 Figure 11. Mitochondrial respiration activity in stable clone overexpressing CYP11A1 59
9.2.12 Figure 12. Measurement of mitochondrial membrane potential in stable clones of CYP11A1 by TMRM staining 60
9.2.13 Figure 13. BN-PAGE and immunoblot detection of CYP11A1 (SCC) complexes isolated from mitochondria of stable clones of CYP11A1 61
9.2.14 Figure 14. Mass spectra analysis of stable clone overexpressing CYP11A1 62
9.2.15 Figure 15. Detection of interacting proteins with CYP11A1 63
9.2.16 Figure 16. Depletion of Hsp60 and observation of mitochondria cristae 64
9.2.17 Figure 17. Detection and quantification of MIC10 complex in stable clones overexpressing CYP11A1 65
9.2.18 Figure 18. Schematic diagram of cristae remodeling by CYP11A1 in steroidogenic cells 66
10. Appendices 67
10.1 Appendix Table 68
10.1.1 Appendix Table AT1. Components of 1X PBS buffer H 7.4 68
10.1.2 Appendix Table AT2. Components of lysis buffer 68
10.1.3 Appendix Figure AT3. Components of 1X TBS 68
10.1.4 Appendix Table AT4. Components of sample buffer 68
10.1.5 Components of dark cathode buffer pH 6.8 for BN- PAGE 68
10.1.6 Appendix Tabel AT6. Components of light cathode buffer pH 6.8 for BN- PAGE 68
10.1.7 Appendix Table AT7. Components of anode buffer pH 6.8 for BN- PAGE 68
10.2 Appendix Figure 69
10.2.1 Appendix Figure A1. Uncropped immunoblot blot showing stable clones (overexpressing CYP11A1 when induced with doxycycline (doxy) 69
10.2.2 Appendix A2. Uncropped immunohistochemistry images of zebrafish Cyp11a1-EGFP and Cyp11a2-EGFP transfected in COS1 cells showing localization of GFP in mitochondria 70
10.2.3 Appendix Figure A3. Uncropped immunohistochemistry images of CYP11A1-GFP fragments transfected in COS1 cells showing localization of GFP in mitochondria 71
10.2.4 Appendix Figure A4. Uncropped immunohistochemistry images of EGFP-fused CYP11A1 signal peptide (AA #1-39) and the anchoring region A’-helix (39+A’-GFP) showing its localization in the mitochondria 72
10.2.5 Appendix Figure A5. Uncropped immunoblots showing partitioning of EGFP-fused CYP11A1 fragments after alkaline buffer extraction and ultracentrifugation 73
10.2.6 Appendix Figure A6. Uncropped immunoblot showing partitioning of EGFP-fused CYP11A1 signal peptide (AA #1-39) and anchoring region A’- helix 74
10.2.7 Appendix Figure A7. Uncropped immunoblot showing partitioning of EGFP-fused CYP11A1 fragments in stable clones (SC) of COS1 cells 75
10.2.8 Appendix Figure A8. Uncropped immunoblots showing the components of electron transport complex from stable clones of CYP11A1 with or without induction of CYP11A1 76
10.2.9 Appendix Figure A9. Uncropped immunoblot showing CYP11A1 (SCC) and Hsp60 complexes from BN-PAGE and Western blot 77
10.2.10 Appendix Figure A10. Uncropped images of verification of CYP11A1 (SCC) and Hsp60 interaction 78
10.2.11 Appendix Figure A11. Uncropped immunoblot showing depletion of Hsp60 by siRNA (H60) 79
10.2.12 Appendix Figure A12. Uncropped immunoblots showing MIC10 and SCC amount 80


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