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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林能裕(Neng-Yu Lin) | |
| dc.contributor.author | Jie-Ming Lin | en |
| dc.contributor.author | 林潔明 | zh_TW |
| dc.date.accessioned | 2021-06-08T03:07:29Z | - |
| dc.date.copyright | 2020-09-01 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-19 | |
| dc.identifier.citation | Amaravadi, R. K., Yu, D., Lum, J. J., Bui, T., Christophorou, M. A., Evan, G. I., ... Thompson, C. B. (2007). Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. The Journal of clinical investigation, 117(2), 326-336 Axe, E. L., Walker, S. A., Manifava, M., Chandra, P., Roderick, H. L., Habermann, A., ... Ktistakis, N. T. (2008). Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of cell biology, 182(4), 685-701 Bennett, E. P., Mandel, U., Clausen, H., Gerken, T. A., Fritz, T. A., Tabak, L. A. (2012). Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family. Glycobiology, 22(6), 736-756. Cazet, A., Julien, S., Bobowski, M., Burchell, J., Delannoy, P. (2010). Tumour-associated carbohydrate antigens in breast cancer. Breast cancer research, 12(3), 204. Chia, J., Tay, F., Bard, F. (2019). The GalNAc-T activation (GALA) pathway: drivers and markers. PloS one, 14(3), e0214118. Chung, C. Y. S., Shin, H. R., Berdan, C. A., Ford, B., Ward, C. C., Olzmann, J. A., ... Nomura, D. K. (2019). Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition. Nature chemical biology, 15(8), 776-785 Ding, Z. B., Hui, B., Shi, Y. H., Zhou, J., Peng, Y. F., Gu, C. Y., ... Song, K. (2011). Autophagy activation in hepatocellular carcinoma contributes to the tolerance of oxaliplatin via reactive oxygen species modulation. Clinical Cancer Research, 17(19), 6229-6238. Eiselé, J. L., Tello, D., Osinaga, E., Roseto, A., Alzari, P. M. (1993). Crystallization and preliminary crystallographic analysis of a tetrameric isolectin from Vicia villosa, specific for the Tn antigen. Journal of molecular biology, 230(2), 670-672. Fahie, K., Zachara, N. E. (2016). Molecular functions of glycoconjugates in autophagy. Journal of molecular biology, 428(16), 3305-3324. Fanger, M. W., Smyth, D. G. (1972). The oligosaccharide units of rabbit immunoglobulin G. Multiple carbohydrate attachment sites. Biochemical Journal, 127(5), 757-765. Fu, J., Gerhardt, H., McDaniel, J. M., Xia, B., Liu, X., Ivanciu, L., ... Nye, E. (2008). Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice. The Journal of clinical investigation, 118(11), 3725-3737 Galluzzi, L., Maiuri, M. C., Vitale, I., Zischka, H., Castedo, M., Zitvogel, L., Kroemer, G. (2007). Cell death modalities: classification and pathophysiological implications. Cell death and differentiation, 14(7), 1237. Gill, D. J., Clausen, H., Bard, F. (2011). Location, location, location: new insights into O-GalNAc protein glycosylation. Trends in cell biology, 21(3), 149-158. Glick, D., Barth, S., Macleod, K. F. (2010). Autophagy: cellular and molecular mechanisms. The Journal of pathology, 221(1), 3-12 HENNEBICQ-REIG, S., LESUFFLEUR, T., CAPON, C., BOLOS, C. D., KIM, I., MOREAU, O., ... AUBERT, J. P. (1998). Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-α-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion. Biochemical Journal, 334(1), 283-295. Homa, F., Hollander, T., Lehman, D. J., Thomsen, D. R., Elhammer, A. P. (1993). Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase. Journal of Biological Chemistry, 268(17), 12609-12616. Hu, Q., Hou, Y. C., Huang, J., Fang, J. Y., Xiong, H. (2017). Itraconazole induces apoptosis and cell cycle arrest via inhibiting Hedgehog signaling in gastric cancer cells. Journal of Experimental Clinical Cancer Research, 36(1), 1-11. Jo, Y. K., Park, N. Y., Park, S. J., Kim, B. G., Shin, J. H., Jo, D. S., ... Kim, S. Y. (2016). O-GlcNAcylation of ATG4B positively regulates autophagy by increasing its hydroxylase activity. Oncotarget, 7(35), 57186. Ju, T., Cummings, R. D. (2002). A unique molecular chaperone Cosmc required for activity of the mammalian core 1 β3-galactosyltransferase. Proceedings of the national academy of sciences, 99(26), 16613-16618. Ju, T., Otto, V. I., Cummings, R. D. (2011). The Tn antigen—structural simplicity and biological complexity. Angewandte Chemie International Edition, 50(8), 1770-1791 Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., ... Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. The EMBO journal, 19(21), 5720-5728. Kalra, A. V., Campbell, R. B. (2007). Mucin impedes cytotoxic effect of 5-FU against growth of human pancreatic cancer cells: overcoming cellular barriers for therapeutic gain. British journal of cancer, 97(7), 910-918 Kauffman, K. J., Yu, S., Jin, J., Mugo, B., Nguyen, N., O'Brien, A., ... Melia, T. J. (2018). Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases. Autophagy, 14(6), 992-1010. Kimmelman, A. C. (2011). The dynamic nature of autophagy in cancer. Genes development, 25(19), 1999-2010. Kimura, S., Noda, T., Yoshimori, T. (2007). Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy, 3(5), 452-460. Kirkin, V., McEwan, D. G., Novak, I., Dikic, I. (2009). A role for ubiquitin in selective autophagy. Molecular cell, 34(3), 259-269. Klionsky, D. J. (2007). Autophagy: from phenomenology to molecular understanding in less than a decade. Nature reviews Molecular cell biology, 8(11), 931-937 Klionsky, D. J., Abdelmohsen, K., Abe, A., Abedin, M. J., Abeliovich, H., Acevedo Arozena, A., ... Adhihetty, P. J. (2016). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 12(1), 1-222. Kondo-Okamoto, N., Noda, N. N., Suzuki, S. W., Nakatogawa, H., Takahashi, I., Matsunami, M., ... Okamoto, K. (2012). Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy. Journal of Biological Chemistry, 287(13), 10631-10638. Lee, P. C., Chen, S. T., Kuo, T. C., Lin, T. C., Lin, M. C., Huang, J., ... Huang, M. C. (2020). C1GALT1 is associated with poor survival and promotes soluble Ephrin A1-mediated cell migration through activation of EPHA2 in gastric cancer. Oncogene, 39(13), 2724-2740. Levy, J. M. M., Thorburn, A. (2011). Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacology therapeutics, 131(1), 130-141. Liang, G., Liu, M., Wang, Q., Shen, Y., Mei, H., Li, D., Liu, W. (2017). Itraconazole exerts its anti-melanoma effect by suppressing Hedgehog, Wnt, and PI3K/mTOR signaling pathways. Oncotarget, 8(17), 28510. Lin, M. C., Chien, P. H., Wu, H. Y., Chen, S. T., Juan, H. F., Lou, P. J., Huang, M. C. (2018). C1GALT1 predicts poor prognosis and is a potential therapeutic target in head and neck cancer. Oncogene, 37(43), 5780-5793. Mauthe, M., Orhon, I., Rocchi, C., Zhou, X., Luhr, M., Hijlkema, K. J., ... Reggiori, F. (2018). Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy, 14(8), 1435-1455. Perez-Vilar, J., Eckhardt, A. E., Hill, R. L. (1996). Porcine submaxillary mucin forms disulfide-bonded dimers between its carboxyl-terminal domains. Journal of Biological Chemistry, 271(16), 9845-9850. Piller, V., Piller, F., CARTRON, J. P. (1990). Comparison of the carbohydrate‐binding specificities of seven N‐acetyl‐D‐galactosamine‐recognizing lectins. European journal of biochemistry, 191(2), 461-466. Puri, K. D., Gopalakrishnan, B., Surolia, A. (1992). Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4). FEBS letters, 312(2-3), 208-212. Redmann, M., Benavides, G. A., Berryhill, T. F., Wani, W. Y., Ouyang, X., Johnson, M. S., ... Zhang, J. (2017). Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons. Redox biology, 11, 73-81 Shental-Bechor, D., Levy, Y. (2008). Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proceedings of the National Academy of Sciences, 105(24), 8256-8261 Simonsen, A., Tooze, S. A. (2009). Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. Journal of Cell Biology, 186(6), 773-782. Spiro, R. G. (1973). Glycoproteins. In Advances in protein chemistry (Vol. 27, pp. 349-467). Academic Press. Spiro, R. G. (2002). Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology, 12(4), 43R-56R. Steen, P. V. D., Rudd, P. M., Dwek, R. A., Opdenakker, G. (1998). Concepts and principles of O-linked glycosylation. Critical reviews in biochemistry and molecular biology, 33(3), 151-208. Taylor, M. E., Drickamer, K. (2011). Introduction to glycobiology. Oxford university press. Ten Hagen, K. G., Fritz, T. A., Tabak, L. A. (2003). All in the family: the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases. Glycobiology, 13(1), 1R-16R. Ten Hagen, K. G., Tetaert, D., Hagen, F. K., Richet, C., Beres, T. M., Gagnon, J., ... Tabak, L. A. (1999). Characterization of a UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity. Journal of Biological Chemistry, 274(39), 27867-27874. Tian, E., Ten Hagen, K. G. (2009). Recent insights into the biological roles of mucin-type O-glycosylation. Glycoconjugate journal, 26(3), 325-334. Tollefsen, S. E., Kornfeld, R. (1983). Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts. Journal of Biological Chemistry, 258(8), 5165-5171. Tsuboi, S., Hatakeyama, S., Ohyama, C., Fukuda, M. (2012). Two opposing roles of O-glycans in tumor metastasis. Trends in molecular medicine, 18(4), 224-232. Varki, A., Cummings, R. D., Esko, J. D., Stanley, P., Hart, G. W., Aebi, M., ... Schnaar, R. L. (2015). Essentials of Glycobiology [internet]. Vliegenthart, J. F., Montreuil, J. (1995). Primary structure of glycoprotein glycans. In New Comprehensive Biochemistry (Vol. 29, pp. 13-28). Elsevier. Wang, X., Wei, S., Zhao, Y., Shi, C., Liu, P., Zhang, C., ... Zhang, H. (2017). Anti-proliferation of breast cancer cells with itraconazole: Hedgehog pathway inhibition induces apoptosis and autophagic cell death. Cancer letters, 385, 128-136. Yang, W. H., Marth, J. D. (2016). Protein glycosylation energizes T cells. Nature Immunology, 17(6), 613-614. Yang, Y., Klionsky, D. J. (2020). Autophagy and disease: Unanswered questions. Cell Death Differentiation, 1-14. Yao, R., Yang, Y., Lian, S., Shi, H., Liu, P., Liu, Y., ... Li, S. (2018). Effects of acute cold stress on liver O-GlcNAcylation and glycometabolism in mice. International journal of molecular sciences, 19(9), 2815. Yin, Z., Pascual, C., Klionsky, D. J. (2016). Autophagy: machinery and regulation. Microbial cell, 3(12), 588. Zhang, C., Deng, X., Qiu, L., Peng, F., Geng, S., Shen, L., Luo, Z. (2018). Knockdown of C1GalT1 inhibits radioresistance of human esophageal cancer cells through modifying β1-integrin glycosylation. Journal of Cancer, 9(15), 2666. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20865 | - |
| dc.description.abstract | 細胞自噬在癌症發生中扮演無可或缺的腳色，而轉譯後修飾亦參與在起始細胞自噬的條件中。前人研究指出，醣基化會藉由調控內質網與高基氏體的平衡進而導致細胞自噬的發生。O-醣基化中，GalNAc 類 O-醣基化為最常見的修飾，其中C1GALT1 為延長醣類鍵結的關鍵酵素。O-醣基化修飾與細胞自噬皆已知有潛力做為癌症治療的對象，但研究尚未指出 O-醣基化與細胞自噬之間的關聯性。此研究分別以基因層面調控 C1GALT1 表現、利用市售藥物 itraconazole 與 benzyl-α-GalNAc 降解 C1GALT1 與抑制 O-醣基化去探討轉錄與轉譯層面對於細胞自噬的影響，再進一步利用流式細胞儀分析報告基因 LC3 去量化自噬小泡的變化。我們發現當抑制C1GALT1 的時候，會使 LC3 的表現量上升並且造成 LC3B 脂質化的累積；且以藥物(itraconazole benzyl-α-GalNAc) 調控 O-醣基化修飾的時候亦有相同的效果。以 pEGFP-LC3 感染細胞觀察到抑制 C1GALT1 會造成自噬小泡的累積，但以mRFP-GFP tandem fluorescent LC3 載體 (ptfLC3) 感染細胞並無觀察到剃除C1GALT1 的細胞之自噬通量上升。我們認為，抑制 C1GALT1，改變細胞中O-醣基化修飾會使 LC3B 脂質化，進而造成自噬小泡的累積。 | zh_TW |
| dc.description.abstract | Autophagy plays an essential role during cancer progression, and post-translational modification is crucial in regulating the initiation of autophagy. Of which, glycosylation has been discovered to impact autophagy via the maintenance of membrane flux between ER and Golgi. GalNAc-type O-glycosylation is the most common type of O-link glycosylation, with core 1 β1,3-galactosyltransferase (C1GALT1) responsible for the elongation of O-glycosylation. As previous studies have shown that C1GALT1 is a potential target for cancer therapeutic, little do we understand the impact of Oglycosylation on autophagy. With multiple approaches, we observed a dynamic induction of LC3 lipidation upon down-regulation of C1GALT1 by genetic manipulation or supplement of O-glycosylation inhibition drugs, such as itraconazole and benzyl-α-GalNAc, suggesting an activation of autophagy. Moreover, the flow cytometry analysis of autophagy reporter system suggested an accumulation of autophagosomes and no increase of autophagic flux. In conclusion, we propose that by modulating C1GALT1, truncated forms of O-glycosylation on surface proteins activate autophagy by triggering lipidated LC3 accumulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T03:07:29Z (GMT). No. of bitstreams: 1 U0001-1908202000202600.pdf: 2991073 bytes, checksum: 77a0f1c92f3a30e02225508a677ea044 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………………………………………………… i 致謝……………………………………………………………………………………………………………………… ii 中文摘要……………………………………………………………………………………………………………… iii Abstract.………………………………………………………………………………………………………… iv Abbreviations………………………………………………………………………………………………… v 目錄………………………………………………………………………………………………………………………… vi Chapter 1. Introduction……………………………………………………………………… 1 1.1 Glycosylation ……………………………………………………………………………………… 1 1.1.1 Types of Glycosylation ………………………………………………………… 2 1.1.2 Mucin type O-Glycosylation ……………………………………………… 4 1.2 Autophagy ………………………………………………………………………………………………… 7 1.2.1 Mechanisms of Autophagy ……………………………………………………… 9 1.2.2 Autophagy and Cancer ……………………………………………………………… 11 1.3 Glycosylation and cell death …………………………………………………13 Chapter 2. Specific Aims and Hypothesis …………………………… 15 Chapter 3. Materials and Methods ……………………………………………… 16 3.1 Materials ………………………………………………………………………………………………… 16 3.1.1 Plasmid ………………………………………………………………………………………………… 16 3.1.2 RNA interference ………………………………………………………………………… 16 3.1.3 Primers ………………………………………………………………………………………………… 17 3.1.4 Antibody ……………………………………………………………………………………………… 18 3.1.5 Chemical Reagents ………………………………………………………………… 18 3.1.6 Others …………………………………………………………………………………………………… 20 3.2 Methods ……………………………………………………………………………………………………… 21 3.2.1 Cell line Cell Culture …………………………………………………… 21 3.2.2 Plasmid/ siRNA Transfection …………………………………………… 21 3.2.3 Virus infection …………………………………………………………………………… 22 3.2.4 Autophagy induction by serum-free starvation…………………… 22 3.2.5 Itraconazole / Benzyl-α-GalNAc supplement ………………………… 22 3.2.6 Plasmid extraction ……………………………………………………………………………………… 23 3.2.7 Miniprep/Midiprep ………………………………………………………………………………………… 23 3.2.8 RNA extraction ………………………………………………………………………………………………… 24 3.2.9 RT-PCR ……………………………………………………………………………………………………………………… 24 3.2.10 Quantitative PCR (qPCR) ……………………………………………………………………… 25 3.2.11 Flow Cytometry ……………………………………………………………………………………………… 25 3.2.12 BCA (Bicinchoninc Acid) Protein Assay ………………………………… 25 3.2.13 Western Blot …………………………………………………………………………………………………… 26 Chapter 4. Results ……………………………………………………………………………………………………… 27 4.1 Changes in O-glycan profile in knock-out C1GALT1 HEK293T cells ………27 4.2 Knock-out C1GALT1 increases lipidated LC3…………………………………………………………………27 4.3 Silencing C1GALT1 increases mRNA expression of autophagy associated genes……………………………………………………………………………………………………………………………………………………………………………28 4.4 Silencing C1GALT1 effect the O-glycan profile and impacts autophagy related protein expression…………………………………………………………………………………………………………………………………………29 4.5 The pEGFP-LC3 reporter system indicates the accumulation of autophagosomes when silencing C1GALT1………………………………………………………………………………………29 4.6 Monitoring autophagy flux by Tandem fluorescent-tagged LC3 in C1GALT1- KO 293T cells ……………………………………………………………………………………………………………………………………………………30 4.7 Pharmacological Degradation of C1GALT1 proteins by Itraconazole changes O-glycan profile and lipidates LC3…………………………………………………………………………………………………31 4.8 Pharmacological effect of O-glycosylation inhibitor Benzyl-α-GalNAc on Oglycan and lipidated LC3 ……………………………………………………………………………………………………………………………………………32 4.9 The effect of Autophagy Inhibitors Chloroquine and Bafilomycin A1 on lipidated LC3 in C1GALT1-KD 293T cells ……………………………………………………………………………………32 Chapter 5. Discussion……………………………………………………………………………………………………………………………………34 5.1 Changes in O-glycosylation lipidates LC3………………………………………………………………………34 5.2 Changes in O-glycan profile by commercial O-glycosylation inhibitors may contribute to anti-cancer effect via mediating autophagy ……………………………………35 5.3 Disrupting C1GALT1 may affect lipidated LC3 by altering ER or Golgi integrity …………………………………………………………………………………………………………………………………………………………………36 Supplement 1. Figures …………………………………………………………………………………………………………………………………39 Supplement 2. References …………………………………………………………………………………………………………………………51 | |
| dc.language.iso | en | |
| dc.subject | LC3 | zh_TW |
| dc.subject | 細胞自噬 | zh_TW |
| dc.subject | O-醣基化 | zh_TW |
| dc.subject | Itraconazole | zh_TW |
| dc.subject | Benzyl-α-GalNAc | zh_TW |
| dc.subject | O-Glycosylation | en |
| dc.subject | LC3 | en |
| dc.subject | Benzyl-α-GalNAc | en |
| dc.subject | Itraconazole | en |
| dc.subject | Autophagy | en |
| dc.title | 抑制C1GALT1調控O-醣基化造成LC3脂質化的累積 | zh_TW |
| dc.title | Changes in O-glycosylation by Silencing C1GALT1 accumulates lipidated LC3 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃偉邦(Wei-Pang Huang),徐嘉琳(Chia-Lin Hsu) | |
| dc.subject.keyword | 細胞自噬,O-醣基化,Itraconazole,Benzyl-α-GalNAc,LC3, | zh_TW |
| dc.subject.keyword | Autophagy,O-Glycosylation,Itraconazole,Benzyl-α-GalNAc,LC3, | en |
| dc.relation.page | 58 | |
| dc.identifier.doi | 10.6342/NTU202004046 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 解剖學暨細胞生物學科所 | |
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