腫瘤易感基因TSG101具有許多生理功能包括囊泡傳輸，核受體轉錄活性及細胞生長分化之調控等，然而調控TSG101功能之細胞訊息路徑仍不清楚，實驗室過去的研究結果顯示TSG101為磷酸化蛋白可被PKC及GSK3β所磷酸化，因此本論文進一步以in vitro激酶磷酸化作用結合MALDI-TOF質譜儀分析及胜肽陣列分析等方法，探討TSG101蛋白上之磷酸化位點，結果顯示PKC磷酸化TSG101蛋白上的胺基酸 S13、 S48、S103 、S367和T383， 而GSK3β磷酸化位於TSG101蛋白胺基酸序列的S182 和 S212。而TSG101的
coiled-coil功能區,則含有2個 CKⅡ可能的磷酸化位點，本實驗之結果顯示TSG101蛋白的生物功能可能受到這些激酶之調控。此外，蛋白質proteasome降解系統為一個重要維持細胞內蛋白正常功能運作的系統，其與細胞的生長、分化及細胞週期之訊息傳遞等之調控有關。此系統運作異常與神經退化性疾病及癌症之發生息息相關，此系統之抑制劑有些已成功應用在這些疾病之治療上。在本論文研究中，已成功建立以GFP為基礎的蛋白質泛素化作用報導質體(pUb-X-GFP)永久轉染細胞株，將可用於探討身為不活化型E2之TSG101，在proteasome蛋白降解過程中所扮演的角色，並可做為新proteasome抑制劑篩選之細胞平台。

TSG101 exhibits multiple functions, including vesicular trafficking, cell growth, differentiation, and transcriptional regulation. However, the cellular signaling that regulates TSG101 functions remains unexplored. Our previous result indicates that TSG101 can be phosphorylated by PKC and GSK3β. In this thesis, we further investigate the detail phosphorylated amino acid residues by using in vitro kinase assay in conjunction with MALDI-TOF and peptide array analysis. The results indicate that S13, S48, S103 and S367, T383 residues could be phosphorylated by PKC, S182 and S212 by GSK3β.
Coiled-coil domain of TSG101, which contains 2 consensus CKⅡ phosphorylation sites,could be phosphorylated by CKⅡ. These results indicate the functions of TSG101 might be regulated by these kinases.
Proteasome mediated protein degradation is important to maintain proper cellular functions including cell growth, differentiation and signaling associated with cell cycle control. Impaired function of this system has been implicated in human diseases associated with neurodegeneration and cancer. The inhibitor of proteasome has been successfully used in treatment of these related diseases. In this thesis, we successfully established Ub-X-GFP reporter cell lines, which could be used in the future study on the functional role of TSG101, an E2 variant, in the proteasome mediated degradation pathway. Furthermore, these cell lines will serve a useful cellular platform for screening new proteasome inhibitors.

中文摘要………………………………………………………..1
英文摘要………………………………………………………..2
背景介紹………………………………………………………..4
實驗目的……………………………………………………….10
材料與方法…………………………………………………….12
結果…………………………………………………………….27
討論…………………………………………………………….32
參考文獻………………………………………………………..36
圖表……………………………………………………………..41
附錄……………………………………………………………..57

Byrne JA, Simms LA, Little MH, Algar EM, Smith PJ.1993. Three non-overlapping regions of chromosome arm 11p allele loss identified in infantile tumors of adrenal and liver. Genes Chromosomes Cancer. 18:104-111.

Carney, M.E., G.L. Maxwell, J.M. Lancaster, C. Gumbs, J. Marks, A. Berchuck, and P.A. Futreal. 1998. Aberrant splicing of the TSG101 tumor suppressor gene in human breast and ovarian cancers. J Soc Gynecol Investig. 5:281-285.

Chang, J.G., T.H. Su, H.J. Wei, J.C. Wang, Y.J. Chen, C.P. Chang, and C.J. Jeng. 1999. Analysis of TSG101 tumour susceptibility gene transcripts in cervical and endometrial cancers. Br J Cancer. 79:445-450.

Chen, Y.J., P.H. Chen, S.Y. Lin, and J.G. Chang. 1999. Analysis of aberrant transcription of TSG101 in hepatocellular carcinomas. Eur J Cancer. 35:302-308.

Colgrove, R.C., A. Millet, G.R. Bauer, J. Pitt, and S.L. Welles. 2005. Gag-p6 Tsg101 binding site duplications in maternal-infant HIV infection. AIDS Res Hum Retroviruses. 21:191-199.

Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG. 2000. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome- dependent proteolysis in living cell .Nat Biotechnol.18:538-543.

Fearon ER, Feinberg AP, Hamilton SH, Vogelstein B. 1985. Loss of genes on the short arm of chromosome 11 in bladder cancer. Nature. 318:377-380

Gayther SA, Barski P, Batley SJ, Li L, de Foy KA, Cohen SN, Ponder BA, Caldas C.1997. Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours. Oncogene.15:2119-2126

Hampl, M., J. Hampl, J. Plaschke, G. Fitze, G. Schackert, H.D. Saeger, and H.K. Schackert. 1998. Evidence that TSG101 aberrant transcripts are PCR artifacts. Biochem Biophys Res Commun. 248:753-760.

Johnson, M.C., J.L. Spidel, D. Ako-Adjei, J.W. Wills, and V.M. Vogt. 2005. The C-terminal half of TSG101 blocks Rous sarcoma virus budding and sequesters Gag into unique nonendosomal structures. J Virol. 79:3775-3786.

Krempler A, Henry MD, Triplett AA, Wagner KU. 2002.Targeted deletion of the Tsg101 gene results in cell cycle arrest at G1/S and p53-independent cell death. J Biol Chem. 277:43216-43223.

Lee, M.P., and A.P. Feinberg. 1997. Aberrant splicing but not mutations of TSG101 in human breast cancer. Cancer Res. 57:3131-3134.

Li, L., X. Li, U. Francke, and S.N. Cohen. 1997. The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer. Cell. 88:143-154.

Li, L., J. Liao, J. Ruland, T.W. Mak, and S.N. Cohen. 2001. TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control. Proc Natl Acad Sci U S A. 98:1619-1624.

Lin, P.M., T.C. Liu, J.G. Chang, T.P. Chen, and S.F. Lin. 1998a. Aberrant TSG101 transcripts in acute myeloid leukaemia. Br J Haematol. 102:753-758.

Lin, S.Y., Y.J. Chen, and J.G. Chang. 1998b. Multiple truncated transcripts of TSG101 in gastrointestinal cancers. J Gastroenterol Hepatol. 13:1111-1114.

Lindsten K, Dantuma NP. 2003 .Monitoring the ubiquitin/proteasome system in conformational diseases. Ageing Res Rev 2:433-449.
Lindsten K, Menendez-Benito V, Masucci MG, Dantuma NP. 2003 A transgenic mouse model of the ubiquitin/proteasome system. Nat Biotechnol. 21:897-902.Lo, Y.F., T.C. Chen, S.C. Chen, and C.C. Chao. 2000. Aberrant expression of TSG101 in Taiwan Chinese breast cancer. Breast Cancer Res Treat. 60:259-266.

Masuhara, M., K. Nagao, M. Nishikawa, T. Kimura, and T. Nakano. 2003. Enhanced degradation of MDM2 by a nuclear envelope component, mouse germ cell-less. Biochem Biophys Res Commun. 308:927-932.
Menendez-Benito V, Heessen S, Dantuma NP. 2005 Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates.Methods Enzymol.399:490-511.

Moyret-Lalle, C., C. Duriez, J. Van Kerckhove, C. Gilbert, Q. Wang, and A. Puisieux. 2001. p53 induction prevents accumulation of aberrant transcripts in cancer cells. Cancer Res. 61:486-488.

Myers, E.L., and J.F. Allen. 2002. Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 gag polyprotein and results in increased levels of ubiquitinated gag. J Virol. 76:11226-11235.

Neefjes J, Dantuma NP. 2004. Fluorescent probes for proteolysis: tools for drug discovery. Nat Rev Drug Discov. 3:58-69.

Oh, H., C. Mammucari, A. Nenci, S. Cabodi, S.N. Cohen, and G.P. Dotto. 2002. Negative regulation of cell growth and differentiation by TSG101 through association with p21(Cip1/WAF1). Proc Natl Acad Sci U S A. 99:5430-5435.

Ruland, J., C. Sirard, A. Elia, D. MacPherson, A. Wakeham, L. Li, J.L. de la Pompa, S.N. Cohen, and T.W. Mak. 2001. p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc Natl Acad Sci U S A. 98:1859-1864.

Salomons FA, Verhoef LG, Dantuma NP. 2005 Fluorescent reporters for the ubiquitin-proteasome system.Essays Biochem.41:113-128.

Segura-Morales, C., C. Pescia, C. Chatellard-Causse, R. Sadoul, E. Bertrand, and E. Basyuk. 2005. Tsg101 and Alix interact with murine leukemia virus Gag and cooperate with Nedd4 ubiquitin ligases during budding. J Biol Chem. 280:27004-27012.

Steiner, P., D.M. Barnes, W.H. Harris, and R.A. Weinberg. 1997. Absence of rearrangements in the tumour susceptibility gene TSG101 in human breast cancer. Nat Genet. 16:332-333.

Sun Z, Pan J, Bubley G, Balk SP.1997. Frequent abnormalities of TSG101 transcripts in human prostate cancer. Oncogene. 15:3121-3125

Sun Z, Pan J, Hope WX, Cohen SN, Balk SP. 1999.Tumor susceptibility gene 101 protein represses androgen receptor transactivation and interacts with p300. Cancer.86:689-696.

Sundquist, W.I., H.L. Schubert, B.N. Kelly, G.C. Hill, J.M. Holton, and C.P. Hill. 2004. Ubiquitin recognition by the human TSG101 protein. Mol Cell. 13:783-789.

Tran YK, Newsham IF. 1996 .High-density marker analysis of 11p15.5 in non-small cell lung carcinomas reveals allelic deletion of one shared and one distinct region when compared to breast carcinomas. Cancer Res.56:2916-2921.

Trink, B., S.I. Pai, S.L. Spunt, V. Raman, P. Cairns, J. Jen, E. Gabrielson, S. Sukumar, and D. Sidransky. 1998. Absence of TSG101 transcript abnormalities in human cancers. Oncogene. 16:2815-2818.

Turpin E, Dalle B, de Roquancourt A, Plassa LF, Marty M, Janin A, Beuzard Y, de The H. 1999. Stress-induced aberrant splicing of TSG101: association to high tumor grade and p53 status in breast cancers. Oncogene.18:7834-7837

VerPlank, L., F. Bouamr, T.J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C.A. Carter. 2001. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci U S A. 98:7724-7729.

Wagner, K.U., A. Krempler, Y. Qi, K. Park, M.D. Henry, A.A. Triplett, G. Riedlinger, I.E. Rucker, and L. Hennighausen. 2003. Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues. Mol Cell Biol. 23:150-162.

Weitzel JN, Patel J, Smith DM, Goodman A, Safaii H, Ball HG. 1994.Molecular genetic changes associated with ovarian cancer. Gynecol Oncol. 55:245-252.

Xie W, Li L, Cohen SN.1998Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency.. Proc Natl Acad Sci U S A.95:1595-1600.

Zhong Q, Chen CF, Chen Y, Chen PL, Lee WH. 1997. Identification of cellular TSG101 protein in multiple human breast cancer cell lines. Cancer Res. 57:4225-4228.

Zhu, G., R. Gilchrist, N. Borley, H.W. Chng, M. Morgan, J.F. Marshall, R.S. Camplejohn, G.H. Muir, and I.R. Hart. 2004. Reduction of TSG101 protein has a negative impact on tumor cell growth. Int J Cancer. 109:541-547.