在動物細胞中研究CAP2 和多甲氧基黃酮於粒線體融合及分裂中扮演之角色

簡易檢索 / 詳目顯示

回結果列表

研究生：	林思廷 Lin, Szu-Ting
論文名稱：	在動物細胞中研究CAP2 和多甲氧基黃酮於粒線體融合及分裂中扮演之角色 Study of the role of CAP2 and Polymethoxylated flavones on mitochondria fusion and fission in mammalian cells
指導教授：	張壯榮 Chang, Chuang-Rung
口試委員:	高茂傑羅至佑兵岳忻
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	60
中文關鍵詞：	粒線體、甲氧基黃酮、天然物、融合與分裂
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

粒線體參與許多細胞生理代謝的機制，如ATP的生成，鈣離子濃度以及細胞凋亡的調控。粒線體功能不正常時會導致疾病產生。粒線體不斷的進行分裂和融合，除調控粒線體形態並維持正常功能。許多研究發現神經退化性疾病細胞中粒線體形態因動態平衡調控不同而與正常細胞不同。因此在這篇論文中，我從兩個方面來研究影響粒線體動態平衡的機制。在分子機制方面，主要是針對之前實驗室中發現會與粒線體分裂Drp1互相結合的cyclase-associated protein 2(CAP2)。CAP2是一個在演化上高度保留的蛋白，它在調控actin dynamic中扮演重要的角色，也參與Ras/cAMP signaling Pathway。Ras/cAMP signaling Pathway會影響調控Drp1的活性的蛋白激酶A(PKA)。故推測CAP2在動物細胞中會參與粒線體形態調控的機制。我利用轉殖的方式將載體或是RNAi送入在 HeLa cells及SH-SY5Y cells來大量表現或是抑制CAP2，並用共軛焦顯微鏡來觀察粒線體形態的改變。實驗結果證實CAP2會影響融合與分裂的平衡而導致片段狀或網狀的粒線體。在論文第二部分則是探討天然藥材成份對粒線體形態調控的影響。中藥藥材中使用的乾燥柑橘屬果皮中常見多甲氧基黃酮(PMF)，尤其是川陳皮素(Nobiletin)及橘皮素(Tangeretin)。目前已知多甲氧基黃酮能抗發炎，抗癌症以及抗粥狀動脈硬化。研究發現也發現多甲氧基黃酮以及5號碳去甲氧基黃酮會參與癌細胞的細胞凋亡，粒線體動態平衡調控與細胞凋亡有密切關聯。為探討多甲氧基黃酮以及5號碳去甲氧基黃酮對於粒線體之影響，我分別使用不同濃度的川陳皮素、橘皮素和5號碳去甲氧基的川陳皮素、橘皮素來檢測其對HeLa cell的細胞存活率以及粒線體形態的影響。實驗結果發現PMFs濃度越高細胞存活率越低，且在10 μM濃度下會影響粒腺體的動態平衡。這些橘皮萃取物具有治療因粒線體導致神經元退化疾病的天然藥物之潛力。

Mitochondria are dynamic organelles that involved in ATP synthesis, calcium concentration regulations and apoptosis. Aberrant mitochondrial morphology caused by abnormal equivalence of fusion and fission in were found in neurodegenerative patients. The aim of my thesis is to study the regulation of mitochondria dynamics. In the first part, I focused on the protein that interacts with mitochondria fission Drp1 called CAP2 (cyclase associated protein 2). CAP2 is a highly conserved protein that plays critical roles in regulating acting dynamics and Ras/cAMP signaling pathway. I manipulated CAP2 expression levels by transfecting either overexpression vectors or RNAi and applied confocal microscope to monitor mitochondrial morphology. My experiments found that overexpression of CAP2 caused mitochondria fragmentation and inhibition of CAP2 lead to elongation. These results indicated that CAP2 does involved in regulating mitochondria dynamics. The second part of my thesis is focus on the effects of nature products on mitochondria dynamics. The dehydrated citrus peels used in traditional Chinese medicine contain the most common flavones: polymethoxylated flavones (PMFs). Both nobiletin and tangeretin are abundant PMFs in citrus peels. Many researches had demonstrated that PMF and 5-OH PMFs involved in anti-inflammatory, antioxidants, antithrombotic and anticarcinogenic processes and affected apoptosis. In my experiments, the survival rates of Hela cells treated with different concentrations of nobiletin, tangeretin, 5-OH nobiletin and 5-OH tangeretin were dosage dependent. In addition, 10 μM PMFs treatments altered mitochondrial dynsmics in HeLa cells. Our results of PMFs suggested these molecules may be therapeutically useful in treating the neurodegenerative disorders caused by mitochondrial fusion and fission regulation defects.

CONTENTS
中文摘要    ii
ABSTRACT    iii
誌謝    v
CONTENTS    vii
LIST OF FIGURES    ix
LIST OF TABLES    xi
ABBREVIATION    xiii
Chapter 1    General introduction    1
1.1   Structures and functions of mitochondria    1
1.2   Mitochondrion and neurodegenerative diseases    2
1.3   Mitochondrial dynamics, function and diseases    3
1.4   Goal of this study    4
Chapter 2    Material and methods     6
2.1   Plasmids extraction     6
2.2   Cells and Cell culture     6
2.3    Cell lines Transfection    7
2.4    Antibody purification     7
2.5    SDS PAGE and Western blotting     8
2.6    Drug resistance assay and cell viability assay     9
2.7    Fixation     9
2.8    Immunofluorescent Cell Staining     9
2.9    Statistics     9
Chapter 3  The role of CAP2 in mitochondria fusion and fission regulation.     11
3.1    Introduction     11
3.2    Results     13
3.2.1 Verifications of antibodies that can recognize CAP2     13
3.2.2 Overexpression CAP2 in SH-SY5Y cells causes mitochondrial elongation…………………………………………………………… 15
3.2.3 Knockdown CAP2 by RNAi in CAP2-overexpressed in SH-SY5Y cells causes mitochondrial fragmentation………………………………… 16
3.2.4 Overexpression CAP2 in HeLa cells caused mitochondria elongation. …………………………………………………………...17
3.2.5 Knockdown CAP2 by RNAi in CAP2-overexpressed HeLa cells caused mitochondrial fragmentation…………………………………………18
3.2.6 Discussion……………………………………………………………...20
Chapter 4    The role of Polymethoxylated flavones (PMFs) on mitochondrial
fusion and fission regulation..………………………………………23
4.1    Introduction    23
4.2    Results    25
4.2.1 Survival of the PMF treatment effect is dosage dependent……………25
4.2.2 Mitochondrial morphology in Hela cells is affected after PMFs and
5-OH PMFstreatments………………………………………………….26
4.2.3  Discussion…………………………………………………………….27
REFERENCE     29

 
LIST OF FIGURES
Figure 1  Mitochondria dynamic is regulated by fission and fusion processes.     35
Figure 2  cAMP pathway regulates PKA activity to phosphorylate Drp1 at Ser637 and downregulate the GTPase activity in mammalian cells.    36
Figure 3  Srv2/CAP function domains    37
Figure 4  Ras/cAMP/ adenyl cyclase pathway in yeast.    38
Figure5  Evaluating CAP2 antibody, using 3809 #3 and 3809 #4 serum in western blot with SH-SY5Y total cell lysate.    39
Figure 6  Reduing serum vo;ume and increasing Tween 20 to improve Western blot background.    40
Figure 7  Diagram of antibody purification    41
Figure 8  Different fraction samples taken out during antibody purification on
SDS-PAGE.    42
Figure 9  Evaluation of purified antibody.    43
Figure 10 Evaluating purified antibodies by Odyssey® Infrared Imaging system.    44
Figure 11 Evaluating 3810 #3 and purified 3810 #3 for Western blotting.    45
Figure 12 Using anti-c Myc for detecting CAP2 in overexpressed cells    46
Figure 13 Anti yeast C-terminal Srv2 can be used to detect CAP2 in mammalian cells    47
Figure 14 CAP2 overexpress in SH-SY5Y cells    48
Figure15 Alteration of CAP2 expression affects mitochondrial morphology in SH-SY5Y cells.    49
Figure 16  Identifying optimized ratio of Lipofectamine™ 2000 to CAP2 RNAi by Western blot for SH-SY5Y cells.    50
Figure 17  CAP2 RNAi can inhibit overexpressed CAP2 in SH-SY5Y cells    51
Figure 18  CAP2 RNAi can inhibit overexpressed CAP2 in HeLa cells.    52
Figure 19 CAP2 expression levels affects mitochondrial morphology
in HeLa cells.    53
Figure 20  The structure of PMFs and 5-OH PMFs.    54
Figure 21  Monitoring cell survival after treated with PMFs by MTT assay    55
Figure 22 PMFs and 5-OH PMFs treated cells affect mitochondrial morphology in 10 μM..    56
Figure 23 Quantitative data of classifications demonstrated that 10 μM PMFs and 5-OH PMFs treated cells affect mitochondrial morphology.    57

 LIST OF TABLES

Table 1    Transfection programs.     58
Table 2    Antibodies used in Western blot     59
Table 3    The CAP protein peptide as antigen to product the specific antibody    60

                                

Büeler, H. (2009). Impaired mitochondrial dynamics and function in the pathogenesis of Parkinson's disease. Experimental Neurology 218, 235-246.
Bereiter-Hahn, J., and Vöth, M. (1994). Dynamics of mitochondria in living cells: Shape changes, dislocations, fusion, and fission of mitochondria. Microscopy Research and Technique 27, 198-219.
Bocco, A., Cuvelier, M.-E., Richard, H., and Berset, C. (1998). Antioxidant Activity and Phenolic Composition of Citrus Peel and Seed Extracts. Journal of Agricultural and Food Chemistry 46, 2123-2129.
Chang, C.-R., and Blackstone, C. (2007). Cyclic AMP-dependent Protein Kinase Phosphorylation of Drp1 Regulates Its GTPase Activity and Mitochondrial Morphology. Journal of Biological Chemistry 282, 21583-21587.
Chen, H., and Chan, D.C. (2009). Mitochondrial dynamics--fusion, fission, movement, and mitophagy--in neurodegenerative diseases. Human Molecular Genetics 18, R169-176.
Cho, D.-H., Nakamura, T., Fang, J., Cieplak, P., Godzik, A., Gu, Z., and Lipton, S.A. (2009). S-Nitrosylation of Drp1 Mediates β-Amyloid-Related Mitochondrial Fission and Neuronal Injury. Science 324, 102-105.
Deng, H., Dodson, M.W., Huang, H., and Guo, M. (2008). The Parkinson's disease genes pink1 and parkin promote mitochondrial fission and/or inhibit fusion in Drosophila. Proceedings of the National Academy of Sciences 105, 14503-14508.
Figueroa-Romero, C., Iñiguez-Lluhí, J.A., Stadler, J., Chang, C.-R., Arnoult, D., Keller, P.J., Hong, Y., Blackstone, C., and Feldman, E.L. (2009). SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. The FASEB Journal 23, 3917-3927.
Gourlay, C.W., and Ayscough, K.R. (2005). Identification of an upstream regulatory pathway controlling actin-mediated apoptosis in yeast. Journal of Cell Science 118, 2119-2132.
Gourlay, C.W., and Ayscough, K.R. (2006). Actin-Induced Hyperactivation of the Ras Signaling Pathway Leads to Apoptosis in Saccharomyces cerevisiae. Molecular and Cellular Biology 26, 6487-6501.
Griparic, L., van der Wel, N.N., Orozco, I.J., Peters, P.J., and van der Bliek, A.M. (2004). Loss of the Intermembrane Space Protein Mgm1/OPA1 Induces Swelling and Localized Constrictions along the Lengths of Mitochondria. Journal of Biological Chemistry 279, 18792-18798.
Gunter, T.E., Yule, D.I., Gunter, K.K., Eliseev, R.A., and Salter, J.D. (2004). Calcium and mitochondria. FEBS Letters 567, 96-102.
Hirai, K., Aliev, G., Nunomura, A., Fujioka, H., Russell, R.L., Atwood, C.S., Johnson, A.B., Kress, Y., Vinters, H.V., Tabaton, M., et al. (2001). Mitochondrial Abnormalities in Alzheimer's Disease. The Journal of Neuroscience 21, 3017-3023.
Karbowski, M., Neutzner, A., and Youle, R.J. (2007). The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division. The Journal of Cell Biology 178, 71-84.
Kroemer, G., and Reed, J.C. (2000). Mitochondrial control of cell death. Nature Medicine 6, 513-519.
Li, S., Lo, C.-Y., and Ho, C.-T. (2006). Hydroxylated Polymethoxyflavones and Methylated Flavonoids in Sweet Orange (Citrus sinensis) Peel. Journal of Agricultural and Food Chemistry 54, 4176-4185.
Lin, M.T., and Beal, M.F. (2006). Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787-795.
Liu, Y., Peterson, D.A., Kimura, H., and Schubert, D. (1997). Mechanism of Cellular 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction. Journal of Neurochemistry 69, 581-593.
Manczak, M., Anekonda, T.S., Henson, E., Park, B.S., Quinn, J., and Reddy, P.H. (2006). Mitochondria are a direct site of Aβ accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Human Molecular Genetics 15, 1437-1449.
Manthey, J.A., Cesar, T.B., Jackson, E., and Mertens-Talcott, S. (2010). Pharmacokinetic Study of Nobiletin and Tangeretin in Rat Serum by High-Performance Liquid Chromatography−Electrospray Ionization−Mass Spectrometry. Journal of Agricultural and Food Chemistry 59, 145-151.
Manthey, J.A., and Grohmann, K. (2001). Phenols in Citrus Peel Byproducts. Concentrations of Hydroxycinnamates and Polymethoxylated Flavones in Citrus Peel Molasses. Journal of Agricultural and Food Chemistry 49, 3268-3273.
Manthey, J.A., and Guthrie, N. (2002). Antiproliferative Activities of Citrus Flavonoids against Six Human Cancer Cell Lines. Journal of Agricultural and Food Chemistry 50, 5837-5843.
Michaeli, T., Bloom, T.J., Martins, T., Loughney, K., Ferguson, K., Riggs, M., Rodgers, L., Beavo, J.A., and Wigler, M. (1993). Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase-deficient Saccharomyces cerevisiae. Journal of Biological Chemistry 268, 12925-12932.
Nakagawa, Y., Ikegami, H., Yamato, E., Takekawa, K., Fujisawa, T., Hamada, Y., Ueda, H., Uchigata, Y., Miki, T., Kumahara, Y., et al. (1995). A New Mitochondrial DNA Mutation Associated with Non-Insulin-Dependent Diabetes Mellitus. Biochemical and Biophysical Research Communications 209, 664-668.
Nicholls, D.G., and Budd, S.L. (2000). Mitochondria and Neuronal Survival. Physiological Reviews 80, 315-360.
Okamoto, K., and Shaw, J.M. (2005). Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes. In Annual Review of Genetics, pp. 503-536.
Orrenius, S., Zhivotovsky, B., and Nicotera, P. (2003). Regulation of cell death: the calcium-apoptosis link. Nature Reviews Molecular Cell Biology 4, 552-565.
Otera, H., Wang, C., Cleland, M.M., Setoguchi, K., Yokota, S., Youle, R.J., and Mihara, K. (2010). Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells. The Journal of Cell Biology 191, 1141-1158.
Peche, V., Shekar, S., Leichter, M., Korte, H., Schröder, R., Schleicher, M., Holak, T., Clemen, C., Ramanath-Y, B., Pfitzer, G., et al. (2007). CAP2, cyclase-associated protein 2, is a dual compartment protein. Cellular and Molecular Life Sciences 64, 2702-2715.
Pinton, P., Giorgi, C., Siviero, R., Zecchini, E., and Rizzuto, R. (2008). Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene 27, 6407-6418.
Pozzan, T., and Rizzuto, R. (2000). High tide of calcium in mitochondria. Nat Cell Biol 2, E25-E27.
Schapira, A.H.V. (2006). Mitochondrial disease. The Lancet 368, 70-82.
Sergeev, I.N., Li, S., Colby, J., Ho, C.-T., and Dushenkov, S. (2006). Polymethoxylated flavones induce Ca2+-mediated apoptosis in breast cancer cells. Life Sciences 80, 245-253.
Sergeev, I.N., Li, S., Ho, C.-T., Rawson, N.E., and Dushenkov, S. (2009). Polymethoxyflavones Activate Ca2+-Dependent Apoptotic Targets in Adipocytes. Journal of Agricultural and Food Chemistry 57, 5771-5776.
Smirnova, E., Shurland, D.-L., Ryazantsev, S.N., and van der Bliek, A.M. (1998). A Human Dynamin-related Protein Controls the Distribution of Mitochondria. The Journal of Cell Biology 143, 351-358.
Song, Z., Ghochani, M., McCaffery, J.M., Frey, T.G., and Chan, D.C. (2009). Mitofusins and OPA1 Mediate Sequential Steps in Mitochondrial Membrane Fusion. Molecular and Cellular Biology 20, 3525-3532.
Su, B., Wang, X., Zheng, L., Perry, G., Smith, M.A., and Zhu, X. (2010). Abnormal mitochondrial dynamics and neurodegenerative diseases. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1802, 135-142.
Tillement, L., Lecanu, L., and Papadopoulos, V. (2011). Alzheimer's disease: Effects of [beta]-amyloid on mitochondria. Mitochondrion 11, 13-21.
Trimmer, P.A., Swerdlow, R.H., Parks, J.K., Keeney, P., Bennett, J.P., Miller, S.W., Davis, R.E., and Parker, W.D. (2000). Abnormal Mitochondrial Morphology in Sporadic Parkinson's and Alzheimer's Disease Cybrid Cell Lines. Experimental Neurology 162, 37-50.
Wallace, D.C., Shoffner, J.M., Trounce, I., Brown, M.D., Ballinger, S.W., Corral-Debrinski, M., Horton, T., Jun, A.S., and Lott, M.T. (1995). Mitochondrial DNA mutations in human degenerative diseases and aging. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1271, 141-151.
Wang, C., Zhou, G.-L., Vedantam, S., Li, P., and Field, J. (2008). Mitochondrial shuttling of CAP1 promotes actin- and cofilin-dependent apoptosis. Journal of Cell Science 121, 2913-2920.
Wang, X., Su, B., Lee, H.-g., Li, X., Perry, G., Smith, M.A., and Zhu, X. (2009). Impaired Balance of Mitochondrial Fission and Fusion in Alzheimer's Disease. The Journal of Neuroscience 29, 9090-9103.
Whitman, S.C., Kurowska, E.M., Manthey, J.A., and Daugherty, A. (2005). Nobiletin, a citrus flavonoid isolated from tangerines, selectively inhibits class A scavenger receptor-mediated metabolism of acetylated LDL by mouse macrophages. Atherosclerosis 178, 25-32.
Willoughby, D., Wong, W., Schaack, J., Scott, J.D., and Cooper, D.M.F. (2006). An anchored PKA and PDE4 complex regulates subplasmalemmal cAMP dynamics. EMBO J 25, 2051-2061.
Winklhofer, K.F., and Haass, C. (2010). Mitochondrial dysfunction in Parkinson's disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1802, 29-44.
Zhu, P.-P., Patterson, A., Stadler, J., Seeburg, D.P., Sheng, M., and Blackstone, C. (2004). Intra- and Intermolecular Domain Interactions of the C-terminal GTPase Effector Domain of the Multimeric Dynamin-like GTPase Drp1. Journal of Biological Chemistry 279, 35967-35974.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文