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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周子賓 | |
dc.contributor.author | Wei-Ling Chang | en |
dc.contributor.author | 張瑋玲 | zh_TW |
dc.date.accessioned | 2021-06-16T16:05:02Z | - |
dc.date.available | 2016-07-03 | |
dc.date.copyright | 2013-07-03 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-06-24 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62586 | - |
dc.description.abstract | Rotini(Rti)為人類高基氏體蛋白GOLPH3在果蠅中的同源蛋白,藉由rti 突變造成的性狀,我們研究Rti在發育上扮演的角色。
exostosin (EXT)基因能合成醣基轉移酶,作用於多醣蛋白(HSPGs;Heparan Sulfate Proteoglycans)合成過程中幫助黏多醣(GAG)聚合。我們發現Rti能調控EXT在高基氏體的逆向運輸。當Rti減少時使EXTs的分佈轉移至靠近trans-Golgi的位置,此錯位累積的EXTs 將會被裂解,因而無法再次作用於GAGs長鏈的聚合;相反的,當 Rti大量表現時使EXTs的分佈轉移至靠近ER/cis-Golgi的位置。不管是缺少或大量表現Rti都會造成HSPGs合成出現問題而影響Hh訊息傳遞。 此模式同樣表現於人類的硬骨及軟骨細胞,意即GOLPH3能調控EXT1及EXT2在細胞內高基氏體的逆向運輸並影響EXT1及EXT2蛋白的穩定度。抑癌基因EXT1 或EXT2突變時會使HSPGs的合成出現問題而造成多發性硬軟骨癌(MO; Multiple Osteochondromas),因此我們的研究對多發性硬軟骨癌提供一新的致病機轉,意即GOLPH3調控抑癌基因EXT1及EXT2在高基式體內的分怖情形,錯位的分佈影響蛋白的穩定度,造成EXT1及EXT2蛋白的裂解,進而引發癌症。 rti 突變造成果蠅胚胎背腹軸性異常,為了解rti突變如何造成背腹軸性發育缺失,我們著重於Rti對三個蛋白質水解酶的影響,包括Gastrulation Defective (GD), Snake (Snk), 以及Easter (Ea)。研究發現三個蛋白質水解酶的酶原量在rti突變的胚胎內明顯增加,然而酶原激化後具活性的水解酶量卻減少,此外Snk酶原激化過成提早到卵子期發生。研究顯示Rti影響GD,Snk,以及Ea三個蛋白質水解酶的表現量及酶原激化過程。 簡而言之,Rti/GOLPH3 調控EXT在高基氏體的逆向運輸,此研究對多發性硬軟骨癌提供一新的致病機轉。此外,Rti藉由影響GD,Snk,以及Ea三個蛋白質水解酶來調控果蠅胚胎發育過程中背腹軸性的決定。 | zh_TW |
dc.description.abstract | Rotini, the homologous of human GOLPH3, is a novel Golgi protein found in Drosophila. Based on the phenotypes display by rti GLC embryo, we studied the possible mechanisms of Rti during development.
The exostosin (EXT) genes encode glycosyltransferases required for glycosaminoglycan (GAG) chain polymerization in the biosynthesis of heparan sulfate proteoglycans (HSPGs). As a Golgi protein, Rti mediates the retrograde trafficking of the EXTs glycotransferase enzymes within the Golgi. A reduction in Rti shifts the steady-state distribution of EXTs to the trans-Golgi. These accumulated EXTs tend to be degraded and their re-entrance towards the route for polymerizing GAG chains is disengaged. Conversely, EXTs are mislocalized towards the ER/cis-Golgi when Rti is over-expressed. Both loss of function and overexpression of rti result in incomplete HSPGs and perturb Hedgehog (Hh) signaling. This model is consistent with a role of GOLPH3 in human bone and cartilage cells, whereby GOLPH3 modulates the dynamic retention and protein stability of EXT1/2. Mutation in the tumor suppressor genes EXT1 and EXT2 disturb HSPG biosynthesis and cause multiple osteochondromas (MO). Our data demonstrate that GOLPH3 affects the sub-compartmental distribution of EXT1 and 2 in the Golgi, providing a putative function of GOLPH3 in MO etiology. To study the role of Rti in dorsoventral patterning formation in Drosophila embryo, we focused on three serine proteases, Gastrulation Defective (GD), Snake (Snk), and Easter (Ea). In rti GLC embryos, the protein levels of zymogens form of the three proteases significantly increased, but the processed/cleavage forms are barely detectable. When rti is mutated, the prematurely cleaved Snk has already existed in ovary. These suggest that Rti may affect protein level and cleavage processing of three serine proteases. In summary, Rti/GOLPH3 regulates the retrograde trafficking of EXTs to modulate the dynamic retention of EXTs within the Golgi. An effect of EXTs by GOLPH3 provides a putative role for GOLPH3 in the formation of MO. Besides, Rti affects GD, Snk, and Ea serine proteases to determine the dorsoventral patterning in the Drosophila embryo. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T16:05:02Z (GMT). No. of bitstreams: 1 ntu-102-D96b43003-1.pdf: 23675256 bytes, checksum: 522ecc13ad63342b43a31985d517f482 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 口試委員會審定書 ---------------------------------------- I
致謝 -------------------------------------------------- II 中文摘要 ---------------------------------------------- III ABSTRACT --------------------------------------------- V TABLE OF CONTENTS------------------------------------- VII LIST OF TABLES --------------------------------------- XII LIST OF FIGURES -------------------------------------- XIII ABBREVIATIONS ---------------------------------------- XVII CHAPTER 1: DROSOPHILA GOLPH3, ROTINI, REGULATES GLYCOSAMINOGLYCAN CHAINS POLYMERASES, EXTS, IN THE BIOSYNTHESIS OF HEPARAN SULFATE PROTEOGLYCANS -------- 1 Summary ---------------------------------------------- 2 Introduction ----------------------------------------- 3 I.Heparan sulphate proteoglycans (HSPGs)-------------- 3 II.HSPGs are important for morphogens distributions and signalings-------------------------------------------- 9 III.Rti, the Drosophila GOLPH3, is a Golgi protein that affects the expression of HSPGs ---------------------- 14 IV.Aim------------------------------------------------ 22 Results ---------------------------------------------- 23 I.Rti is not required for the Hh autocleavage process and exerts its function at the plasma membrane------------ 23 II.Rti does not affect the expression pattern of Sdc and Dlp, the core proteins of HSPGs ---------------------- 25 III.Both Rti gain-of-function and loss-of-function reduce EXTs staining in wing imaginal discs------------------ 27 IV.EXT proteins are labile in the absence of rti------ 30 V.The labile Ttv protein in rti mutant clone goes through Hrs-mediated degradation------------------------------ 33 Discussion-------------------------------------------- 37 I.Rti is not required for Hh gene expression and exerts its function after Hh releasing into extracellular matrix- 37 II.Rti regulates HSPGs expression by modulates the protein stability of the EXTs polymerases--------------------- 40 III.The possible role of Rti in EXTs------------------ 43 CHAPTER 2. RTI REGULATES THE RETROGRADE TRAFFICKING OF EXTS IN THE GOLGI COMPLEX---------------------------------- 49 Summary ---------------------------------------------- 50 Introduction------------------------------------------ 51 I.Role of the Golgi complex in glycosylation---------- 51 II.Localization and trafficking of resident Golgi glycosylation enzymes--------------------------------- 52 III.Yeast GOLPH3, Vps74p, mediates the dynamic retention of Glycosyltransferases---------------------------------- 62 IV.Aim------------------------------------------------ 65 Results----------------------------------------------- 67 I.The subcellular distribution of Rti----------------- 67 II.Rti mediates the retrograde transport of EXT enzymes within the Golgi complex------------------------------ 70 III.Rti forms complexes with three EXTs and interacts with COPⅠ--------------------------------------------------- 77 Discussion-------------------------------------------- 83 I.The subcellular localization of Rti and its homology 83 II.Rti mediates Hh signaling by regulating the retrograde trafficking of EXTs in the Golgi---------------------- 84 III.GOLPH3 and its homology may dynamic traffick in all endomembrane system----------------------------------- 87 IV.Rti may be a general factor for intracellular trafficking ------------------------------------------------------- 89 CHAPTER 3. MAMMALIAN GOLPH3 REGULATES THE RETROGRADE TRAFFICKING OF EXTS IN THE GOLGI COMPLEX--------------- 91 Summary------------------------------------------------ 92 Introduction------------------------------------------- 93 I.EXTs are highly evolutionary conserved glycosyltransferases----------------------------------- 93 II.Multiple Osterchondromas are cartilage tumors that genetically linked to EXT------------------------------ 96 III.Golgi phosphoprotein 3 (GOLPH3) represents a new class ofoncoprotein------------------------------------------ 101 IV.Aim------------------------------------------------- 109 Results ----------------------------------------------- 111 I.EXTs are regulated by GOLPH3 in bone, cartilage, and muscle cells------------------------------------------- 111 II.EXTs are not regulated by GOLPH3 inadipose-like cells, stroma cells, and hematopoietic cells------------------ 119 III.EXTs are not regulated by GOLPH3 inepidermal-derived cells-------------------------------------------------- 121 IV.Hh signal transduction is affected in one allele mutation of Drosophila EXT1, ttv ------------------------------- 123 Discussion--------------------------------------------- 125 I.GOLPH3/Rti regulates the retrograde trafficking of EXTs in Drosophila and human cell lines------------------------ 125 II.GOLPH3 may be a new factor for the fomartion of MO-- 128 III.The possible rolesof GOLPH3 in clinical dissease--- 131 CHARPTER 4. A FUNCTION OF RTI IN THE DORSAL/VENTRAL AXIS FORMATION---------------------------------------------- 134 Summary------------------------------------------------ 135 Introduction------------------------------------------- 137 I.Dorsoventral axis formation in the Drosophilaembryonic development-------------------------------------------- 137 II.Serine protease cascade----------------------------- 140 III.The exocyst complex in polarized exocytosis-------- 142 IV.Previous studies of Rti in exocytosis--------------- 145 V.Aim-------------------------------------------------- 148 Results------------------------------------------------ 149 I.Genetic interaction between Rti and GD, Snk, or Ea--- 149 II.The localization of GD-GFP, Snk-GFP, and Ea-GFP in ovary ------------------------------------------------------- 151 III.The protein level of GD-GFP, Snk-GFP, and Ea-GFP--- 154 Discussion--------------------------------------------- 158 I.Rti may be involved in exocytotic trafficking-------- 158 II.Possible mechanisms to increase serine proteases in rti mutation----------------------------------------------- 162 III.The complicated roles of Rti in the dorsoventral axis formation---------------------------------------------- 165 FUTURE ASPECTS----------------------------------------- 167 GOLPH3 may be involved in the formation of human MO disease ------------------------------------------------------- 167 The effects of Rti in Wg signaling in the disc--------- 168 The possible mechanisms of Rti to control dorsoventral formation of Drosophila embryo------------------------- 169 Possible mechanisms of Rti in regulating the transcriptional factor En---------------------------------------------- 170 MATERIALS AND METHODS---------------------------------- 172 Fly stocks and maintenance----------------------------- 172 Cloning constructs------------------------------------- 172 Antibody generation------------------------------------ 174 Heat shock treatment----------------------------------- 175 Fluorescence antibody staining of imaginal discs------- 175 Co-Immunoprecipitation--------------------------------- 177 Immunoelectron Microscopy------------------------------ 177 Immunogold particle quantification--------------------- 178 Cell lines and transfection---------------------------- 179 REFERENCES--------------------------------------------- 180 TABLES------------------------------------------------- 193 FIGURES------------------------------------------------ 199 APPENDIX----------------------------------------------- 264 Ⅰ. THE DROSOPHILA GOLPH3 HOMOLOG REGULATES THE BIOSYNTHESIS OF HEPARAN SULFATE PROTEOGLYCANS BY MODULATING THE RETROGRADE TRAFFICKING OF EXOSTOSINSREFERENCES--------- 264 Ⅱ. DROSOPHILA GOLPH3, ROTINI, PLAYS MULTIPLE ROLES IN EMBRYONIC DORSOVENTRAL PATTERNING---------------------- 274 | |
dc.language.iso | en | |
dc.title | 果蠅高基氏體蛋白質(Rotini)的發育遺傳功能角色探討 | zh_TW |
dc.title | Developmental genetic functions of Drosophila Golgi phosphoprotein 3(GOLPH3), Rotini | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 柯逢春,白麗美,李秀敏,李芳仁 | |
dc.subject.keyword | 高基氏體蛋白質, | zh_TW |
dc.subject.keyword | GOLPH3,Rti, | en |
dc.relation.page | 312 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-06-24 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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