系統識別號 U0026-0812200915214959
論文名稱(中文) 轉化生長因子beta引起之運輸蛋白TRAPPC6A凝聚與細胞凋亡
論文名稱(英文) TGF-beta induced aggregation of trafficking protein TRAPPC6A and apoptosis
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 張景雲
研究生(英文) Jean-Yun Chang
學號 t1696114
學位類別 碩士
語文別 英文
論文頁數 58頁
口試委員 口試委員-徐麗君
中文關鍵字 含雙色胺酸功能區氧化還原酶(WOX1/ WWOX)  運輸蛋白顆粒複合體6A(TRAPPC6A/ TPC6A)  轉化生長因子beta(TGF-beta)  神經退化  蛋白質凝聚  細胞凋亡  阿茲海默症  運輸蛋白 
英文關鍵字 TPC6A  apoptosis  Alzheimer's disease  protein aggregation  neurodegeneration  TRAPPC6A  TGF-beta  trafficking protein  WWOX  WOX1 
中文摘要 蛋白質不正常凝聚會造成神經退化疾病如阿茲海默症。在此,我們以TGF-beta1刺激細胞,分離出一個截斷型(truncated)運輸蛋白顆粒複合體6A(trafficking protein particle complex 6A),簡稱TPC6Adelta,其可與腫瘤抑制蛋白含雙色胺酸功能區氧化還原酶WOX1(又名WWOX或FOR)結合。與原生型(wild type)相比 (22 kDa),TPC6Adelta缺少了胺基酸29至42的部份。TPC6A為運輸蛋白顆粒(TRAPP)複合體中的一個次單元體,此複合體在酵母菌中參與內質網至高基氏體間的小泡運輸。相對地,哺乳類的TPC6A會傾向位於細胞核中及核周圍。利用共同免疫沉澱法,我們證實內生型(endogenous)WOX1與TPC6A有交互作用。由螢光共振能量轉移(FRET)分析中也發現,TPC6Adelta會與WOX1 C端D3功能區以及短鏈酒精去氫酶/還原酶功能區(ADH/SDR domain)結合。長期的轉化生長因子beta(TGF-beta)刺激會使TPC6Adelta與WOX1的結合降低,最終造成TPC6Adelta的凝聚。值得注意的是,TPC6A的凝聚物質能在阿茲海默症(Alzheimer’s disease, AD)病患及轉殖基因AD老鼠模式的海馬迴中發現。TPC6A凝聚發生在較年輕(60±13歲,n=11)的未失智族群之比率比AD患者(81±10歲,n=27)更多,暗示TPC6A可能是一個AD發展的指標。縮時顯微技術(time-lapse microscopy)顯示TGF-beta引起細胞內TPC6Adelta的凝聚,此凝聚現象可能會導致Abeta的產生以及神經細胞死亡。WOX1與TPC6Adelta抵制彼此造成的細胞凋亡,暗示WOX1抑制TPC6Adelta在細胞中的凝聚。總結以上結果,我們發現一個新的AD致病機轉,其包含TGF-beta所引發的TPC6A凝聚現象。在機理上,TGF-beta引發TPC6A與WOX1的分離,進而造成TPC6A凝聚,Abeta沉積與斑塊形成。
英文摘要 Protein aggregation is central to the development of neurodegeneration such as Alzheimer’s disease. Here, we isolated a TGF-beta1-induced trafficking protein particle complex 6A, designated TPC6Adelta, which binds tumor suppressor WOX1 (also named WWOX or FOR). Compared to the wild type (22 kDa), TPC6Adelta has an N-terminal truncation of amino acid 29-42. TPC6A is a component of transport protein particle (TRAPP) complex in vesicle trafficking from ER to the Golgi complex in yeast. In contrast, mammalian TPC6A mainly localizes in the nuclei and at the perinuclear area. By co-immunoprecipitation, endogenous WOX1 was shown to interact with TPC6A. By FRET analysis, TPC6Adelta physically bound to the C-terminal tail (D3 domain) and ADH/SDR domain of WOX1. Prolonged stimulation of cells with TGF-beta1 reduced the binding of TPC6Adelta and WOX1, which ultimately resulted in TPC6Adelta aggregation. Notably, plaque-like TPC6A aggregates were found in the hippocami of patients with Alzheimer’s disease (AD) and transgenic AD mice model. Most importantly, TPC6A aggregates (> 0.2 micrometer in diameter) were more abundant in the younger nondemented human populations (60±13.3 years old, n=11) than in the older AD patients (81±9.7 years old, n=27), suggesting TPC6A is a likely marker for AD development. Time-lapse microscopy revealed that TGF-beta induced aggregation of intracellular TPC6Adelta. The aggregation may lead to Abeta superproduction and neuronal death. WOX1 and TPC6Adelta nullified each other in causing apoptosis, implying that WOX1 prevents aggregation of TPC6Adelta in cells. Together, here we discovered a novel mechanism for the pathogenesis of AD, which involves TGF-beta-induced TPC6A aggregation. Mechanistically, TGF-beta induces dissociation TPC6A from WOX1, followed by TPC6A aggregation, Abeta deposition and plaque formation.
論文目次 中文摘要........I
Table of contents......IV
Index of figures.......VI
Goals of this study.....1
WW domain-containing oxidoreductase WOX1........1
TRAPP complexes: in vivo aggregation and potential role in neurodegeneration....6
TGF-beta and Alzheimer’s disease (AD).....8
Materials and methods...10
Cell Lines......10
cDNA Expression Constructs and Transient Expression.....10
Construction of Full-length TPC6A cDNA..11
Antibody Production in Rabbit...12
Co-immunoprecipitation and Western Blotting.....13
Immunofluorescence, Förster/Fluorescence Resonance Energy Transfer (FRET) analysis, and Time-lapse Fluorescence Microscopy......13
Filter Retardation Assay........15
Cell Cycle Analysis.....16
Ectopic TPC6A and WOX1 are colocalized at perinuclear area......17
WOX1 physically interacts with TPC6A....17
Dissociation of TPC6Adelta/WOX1 complexes leads to TPC6Adelta aggregation.......19
TGF-beta induces the aggregation of TPC6Adelta in vitro........19
TPC6A aggregates or plaques are present in the hippocampi of APP/PS2-transgenic mice....20
Aggregation of TPC6A is present in hippocampi of postmortem AD and nondemented controls..20
TPC6Adelta induces Abeta superproduction upon TGF-beta1 stimulation.......21
TPC6A and WOX1 counteract each other in inducing apoptosis......22
A postulated model for TPC6A aggregation and neuronal death.....23
WOX1 may act as a chaperone.....24
TGF-beta-induced proteins undergo self-aggregation in the presence of TGF-beta........25
Mammalian TPC6A may have additional functions...26
參考文獻 Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., et al. (2000). Inflammation and Alzheimer's disease. Neurobiol Aging, 21(3), 383-421.

Aqeilan, R. I., Donati, V., Gaudio, E., Nicoloso, M. S., Sundvall, M., Korhonen, A., et al. (2007). Association of Wwox with ErbB4 in breast cancer. Cancer Res, 67(19), 9330-9336.

Aqeilan, R. I., Hagan, J. P., Aqeilan, H. A., Pichiorri, F., Fong, L. Y., & Croce, C. M. (2007). Inactivation of the Wwox gene accelerates forestomach tumor progression in vivo. Cancer Res, 67(12), 5606-5610.

Aqeilan, R. I., Hagan, J. P., de Bruin, A., Rawahneh, M., Salah, Z., Gaudio, E., et al. (2009). Targeted ablation of the WW domain-containing oxidoreductase tumor suppressor leads to impaired steroidogenesis. Endocrinology, 150(3), 1530-1535.

Aqeilan, R. I., Hassan, M. Q., de Bruin, A., Hagan, J. P., Volinia, S., Palumbo, T., et al. (2008). The WWOX tumor suppressor is essential for postnatal survival and normal bone metabolism. J Biol Chem, 283(31), 21629-21639.

Aqeilan, R. I., Kuroki, T., Pekarsky, Y., Albagha, O., Trapasso, F., Baffa, R., et al. (2004). Loss of WWOX expression in gastric carcinoma. Clin Cancer Res, 10(9), 3053-3058.

Aqeilan, R. I., Palamarchuk, A., Weigel, R. J., Herrero, J. J., Pekarsky, Y., & Croce, C. M. (2004). Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor. Cancer Res, 64(22), 8256-8261.

Aqeilan, R. I., Pekarsky, Y., Herrero, J. J., Palamarchuk, A., Letofsky, J., Druck, T., et al. (2004). Functional association between Wwox tumor suppressor protein and p73, a p53 homolog. Proc Natl Acad Sci U S A, 101(13), 4401-4406.

Aqeilan, R. I., Trapasso, F., Hussain, S., Costinean, S., Marshall, D., Pekarsky, Y., et al. (2007). Targeted deletion of Wwox reveals a tumor suppressor function. Proc Natl Acad Sci U S A, 104(10), 3949-3954.

Balch, W. E., Morimoto, R. I., Dillin, A., & Kelly, J. W. (2008). Adapting proteostasis for disease intervention. Science, 319(5865), 916-919.
Bednarek, A. K., Keck-Waggoner, C. L., Daniel, R. L., Laflin, K. J., Bergsagel, P. L., Kiguchi, K., et al. (2001). WWOX, the FRA16D gene, behaves as a suppressor of tumor growth. Cancer Res, 61(22), 8068-8073.

Bukau, B., Weissman, J., & Horwich, A. (2006). Molecular chaperones and protein quality control. Cell, 125(3), 443-451.

Cai, Y., Chin, H. F., Lazarova, D., Menon, S., Fu, C., Cai, H., et al. (2008). The structural basis for activation of the Rab Ypt1p by the TRAPP membrane-tethering complexes. Cell, 133(7), 1202-1213.

Chang, N. S., Doherty, J., & Ensign, A. (2003). JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis. J Biol Chem, 278(11), 9195-9202.

Chang, N. S., Doherty, J., Ensign, A., Lewis, J., Heath, J., Schultz, L., et al. (2003). Molecular mechanisms underlying WOX1 activation during apoptotic and stress responses. Biochem Pharmacol, 66(8), 1347-1354.

Chang, N. S., Doherty, J., Ensign, A., Schultz, L., Hsu, L. J., & Hong, Q. (2005). WOX1 is essential for tumor necrosis factor-, UV light-, staurosporine-, and p53-mediated cell death, and its tyrosine 33-phosphorylated form binds and stabilizes serine 46-phosphorylated p53. J Biol Chem, 280(52), 43100-43108.

Chang, N. S., Hsu, L. J., Lin, Y. S., Lai, F. J., & Sheu, H. M. (2007). WW domain-containing oxidoreductase: a candidate tumor suppressor. Trends Mol Med, 13(1), 12-22.

Chang, N. S., Mattison, J., Cao, H., Pratt, N., Zhao, Y., & Lee, C. (1998). Cloning and characterization of a novel transforming growth factor-beta1-induced TIAF1 protein that inhibits tumor necrosis factor cytotoxicity. Biochem Biophys Res Commun, 253(3), 743-749.

Chang, N. S., Pratt, N., Heath, J., Schultz, L., Sleve, D., Carey, G. B., et al. (2001). Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity. J Biol Chem, 276(5), 3361-3370.

Chang, N. S., Schultz, L., Hsu, L. J., Lewis, J., Su, M., & Sze, C. I. (2005). 17beta-Estradiol upregulates and activates WOX1/WWOXv1 and WOX2/WWOXv2 in vitro: potential role in cancerous progression of breast and prostate to a premetastatic state in vivo. Oncogene, 24(4), 714-723.

Chen, S. T., Chuang, J. I., Wang, J. P., Tsai, M. S., Li, H., & Chang, N. S. (2004). Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system. Neuroscience, 124(4), 831-839.

Cole, G. M., Morihara, T., Lim, G. P., Yang, F., Begum, A., & Frautschy, S. A. (2004). NSAID and antioxidant prevention of Alzheimer's disease: lessons from in vitro and animal models. Ann N Y Acad Sci, 1035, 68-84.

Deuerling, E., & Bukau, B. (2004). Chaperone-assisted folding of newly synthesized proteins in the cytosol. Crit Rev Biochem Mol Biol, 39(5-6), 261-277.

Ethell, I. M., Hagihara, K., Miura, Y., Irie, F., & Yamaguchi, Y. (2000). Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines. J Cell Biol, 151(1), 53-68.

Fabbri, M., Iliopoulos, D., Trapasso, F., Aqeilan, R. I., Cimmino, A., Zanesi, N., et al. (2005). WWOX gene restoration prevents lung cancer growth in vitro and in vivo. Proc Natl Acad Sci U S A, 102(43), 15611-15616.

Finch, C. E. (2003). Neurons, glia, and plasticity in normal brain aging. Neurobiol Aging, 24 Suppl 1, S123-127; discussion S131.

Gaudio, E., Palamarchuk, A., Palumbo, T., Trapasso, F., Pekarsky, Y., Croce, C. M., et al. (2006). Physical association with WWOX suppresses c-Jun transcriptional activity. Cancer Res, 66(24), 11585-11589.

Ghosh, A. K., Steele, R., & Ray, R. B. (2003). Modulation of human luteinizing hormone beta gene transcription by MIP-2A. J Biol Chem, 278(26), 24033-24038.

Gourley, C., Paige, A. J., Taylor, K. J., Ward, C., Kuske, B., Zhang, J., et al. (2009). WWOX Gene Expression Abolishes Ovarian Cancer Tumorigenicity In vivo and Decreases Attachment to Fibronectin via Integrin {alpha}3. Cancer Res.

Gray, C. W., & Patel, A. J. (1993). Regulation of beta-amyloid precursor protein isoform mRNAs by transforming growth factor-beta 1 and interleukin-1 beta in astrocytes. Brain Res Mol Brain Res, 19(3), 251-256.

Gwynn, B., Smith, R. S., Rowe, L. B., Taylor, B. A., & Peters, L. L. (2006). A mouse TRAPP-related protein is involved in pigmentation. Genomics, 88(2), 196-203.

Harris-White, M. E., Chu, T., Balverde, Z., Sigel, J. J., Flanders, K. C., & Frautschy, S. A. (1998). Effects of transforming growth factor-beta (isoforms 1-3) on amyloid-beta deposition, inflammation, and cell targeting in organotypic hippocampal slice cultures. J Neurosci, 18(24), 10366-10374.

Hartl, F. U., & Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 295(5561), 1852-1858.

Hong, Q., Hsu, L. J., Schultz, L., Pratt, N., Mattison, J., & Chang, N. S. (2007). Zfra affects TNF-mediated cell death by interacting with death domain protein TRADD and negatively regulates the activation of NF-kappaB, JNK1, p53 and WOX1 during stress response. BMC Mol Biol, 8, 50.

Hsu, L. J., Hong, Q., Schultz, L., Kuo, E., Lin, S. R., Lee, M. H., et al. (2008). Zfra is an inhibitor of Bcl-2 expression and cytochrome c release from the mitochondria. Cell Signal, 20(7), 1303-1312.

Hu, W. H., Pendergast, J. S., Mo, X. M., Brambilla, R., Bracchi-Ricard, V., Li, F., et al. (2005). NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation. J Biol Chem, 280(32), 29233-29241.

Ishii, H., Vecchione, A., Furukawa, Y., Sutheesophon, K., Han, S. Y., Druck, T., et al. (2003). Expression of FRA16D/WWOX and FRA3B/FHIT genes in hematopoietic malignancies. Mol Cancer Res, 1(13), 940-947.

Jin, C., Ge, L., Ding, X., Chen, Y., Zhu, H., Ward, T., et al. (2006). PKA-mediated protein phosphorylation regulates ezrin-WWOX interaction. Biochem Biophys Res Commun, 341(3), 784-791.

Jones, S., Newman, C., Liu, F., & Segev, N. (2000). The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32. Mol Biol Cell, 11(12), 4403-4411.

Keil, U., Hauptmann, S., Bonert, A., Scherping, I., Eckert, A., & Muller, W. E. (2006). Mitochondrial dysfunction induced by disease relevant AbetaPP and tau protein mutations. J Alzheimers Dis, 9(2), 139-146.

Kim, Y. G., Raunser, S., Munger, C., Wagner, J., Song, Y. L., Cygler, M., et al. (2006). The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering. Cell, 127(4), 817-830.

Kummel, D., Muller, J. J., Roske, Y., Henke, N., & Heinemann, U. (2006). Structure of the Bet3-Tpc6B core of TRAPP: two Tpc6 paralogs form trimeric complexes with Bet3 and Mum2. J Mol Biol, 361(1), 22-32.

Kummel, D., Muller, J. J., Roske, Y., Misselwitz, R., Bussow, K., & Heinemann, U. (2005). The structure of the TRAPP subunit TPC6 suggests a model for a TRAPP subcomplex. EMBO Rep, 6(8), 787-793.

Kummel, D., Oeckinghaus, A., Wang, C., Krappmann, D., & Heinemann, U. (2008). Distinct isocomplexes of the TRAPP trafficking factor coexist inside human cells. FEBS Lett, 582(27), 3729-3733.

Lai, F. J., Cheng, C. L., Chen, S. T., Wu, C. H., Hsu, L. J., Lee, J. Y., et al. (2005). WOX1 is essential for UVB irradiation-induced apoptosis and down-regulated via translational blockade in UVB-induced cutaneous squamous cell carcinoma in vivo. Clin Cancer Res, 11(16), 5769-5777.

Lee, M.H., Lin, S.R., Chang, N.S. (2009). TGF-beta1-induced TIAF1 self-association leads to apoptosis. In Alzheimer's & Parkinson's Disease: Advances, Concepts & New Challenges, Medimond International Proceedings (in press).

Ludes-Meyers, J. H., Kil, H., Bednarek, A. K., Drake, J., Bedford, M. T., & Aldaz, C. M. (2004). WWOX binds the specific proline-rich ligand PPXY: identification of candidate interacting proteins. Oncogene, 23(29), 5049-5055.

Luo, J., Lang, J. A., & Miller, M. W. (1998). Transforming growth factor beta1 regulates the expression of cyclooxygenase in cultured cortical astrocytes and neurons. J Neurochem, 71(2), 526-534.

Monning, U., Sandbrink, R., Banati, R. B., Masters, C. L., & Beyreuther, K. (1994). Transforming growth factor beta mediates increase of mature transmembrane amyloid precursor protein in microglial cells. FEBS Lett, 342(3), 267-272.

Morimoto, R. I. (2008). Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes Dev, 22(11), 1427-1438.

Nakayama, S., Semba, S., Maeda, N., Aqeilan, R. I., Huebner, K., & Yokozaki, H. (2008). Role of the WWOX gene, encompassing fragile region FRA16D, in suppression of pancreatic carcinoma cells. Cancer Sci, 99(7), 1370-1376.

Nakayama, S., Semba, S., Maeda, N., Matsushita, M., Kuroda, Y., & Yokozaki, H. (2009). Hypermethylation-mediated reduction of WWOX expression in intraductal papillary mucinous neoplasms of the pancreas. Br J Cancer, 100(9), 1438-1443.

Nottingham, R. M., & Pfeffer, S. R. (2008). Team effort by TRAPP forces a nucleotide fumble. Cell, 133(7), 1141-1143.

Pluciennik, E., Kusinska, R., Potemski, P., Kubiak, R., Kordek, R., & Bednarek, A. K. (2006). WWOX--the FRA16D cancer gene: expression correlation with breast cancer progression and prognosis. Eur J Surg Oncol, 32(2), 153-157.

Qin, H. R., Iliopoulos, D., Semba, S., Fabbri, M., Druck, T., Volinia, S., et al. (2006). A role for the WWOX gene in prostate cancer. Cancer Res, 66(13), 6477-6481.

Ramos, D., Abba, M., Lopez-Guerrero, J. A., Rubio, J., Solsona, E., Almenar, S., et al. (2008). Low levels of WWOX protein immunoexpression correlate with tumour grade and a less favourable outcome in patients with urinary bladder tumours. Histopathology, 52(7), 831-839.

Ron, D., & Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol, 8(7), 519-529.

Sacher, M., Barrowman, J., Wang, W., Horecka, J., Zhang, Y., Pypaert, M., et al. (2001). TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport. Mol Cell, 7(2), 433-442.

Sacher, M., Jiang, Y., Barrowman, J., Scarpa, A., Burston, J., Zhang, L., et al. (1998). TRAPP, a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion. Embo J, 17(9), 2494-2503.

Sacher, M., Kim, Y. G., Lavie, A., Oh, B. H., & Segev, N. (2008). The TRAPP complex: insights into its architecture and function. Traffic, 9(12), 2032-2042.

Schmued, L. C., Albertson, C., & Slikker, W., Jr. (1997). Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res, 751(1), 37-46.

Schultz, L., Khera, S., Sleve, D., Heath, J., & Chang, N. S. (2004). TIAF1 and p53 functionally interact in mediating apoptosis and silencing of TIAF1 abolishes nuclear translocation of serine 15-phosphorylated p53. DNA Cell Biol, 23(1), 67-74.

Sudol, M., Recinos, C. C., Abraczinskas, J., Humbert, J., & Farooq, A. (2005). WW or WoW: the WW domains in a union of bliss. IUBMB Life, 57(12), 773-778.

Sze, C. I., Su, M., Pugazhenthi, S., Jambal, P., Hsu, L. J., Heath, J., et al. (2004). Down-regulation of WW domain-containing oxidoreductase induces Tau phosphorylation in vitro. A potential role in Alzheimer's disease. J Biol Chem, 279(29), 30498-30506.

van der Wal E.A., Gomez–Pinilla F., Cotman C.W. (1993). Transforming growth factor-beta 1 is in plaques in Alzheimer and Down pathologies. Neuroreport, 4, 69-72.

Wang, M., Gu, J., Wang, Y., & Gong, B. (2009). Loss of WWOX expression in human extrahepatic cholangiocarcinoma. J Cancer Res Clin Oncol, 135(1), 39-44.

Wang, X., Chao, L., Jin, G., Ma, G., Zang, Y., & Sun, J. (2009). Association between CpG island methylation of the WWOX gene and its expression in breast cancers. Tumour Biol, 30(1), 8-14.

Watanabe, A., Hippo, Y., Taniguchi, H., Iwanari, H., Yashiro, M., Hirakawa, K., et al. (2003). An opposing view on WWOX protein function as a tumor suppressor. Cancer Res, 63(24), 8629-8633.
Wyss-Coray, T., Lin, C., von Euw, D., Masliah, E., Mucke, L., & Lacombe, P. (2000). Alzheimer's disease-like cerebrovascular pathology in transforming growth factor-beta 1 transgenic mice and functional metabolic correlates. Ann N Y Acad Sci, 903, 317-323.

Wyss-Coray, T., Lin, C., Yan, F., Yu, G. Q., Rohde, M., McConlogue, L., et al. (2001). TGF-beta1 promotes microglial amyloid-beta clearance and reduces plaque burden in transgenic mice. Nat Med, 7(5), 612-618.

Wyss-Coray, T., Masliah, E., Mallory, M., McConlogue, L., Johnson-Wood, K., Lin, C., et al. (1997). Amyloidogenic role of cytokine TGF-beta1 in transgenic mice and in Alzheimer's disease. Nature, 389(6651), 603-606.
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