周子賓臺灣大學:分子與細胞生物學研究所張哲維Chang, Che-WeiChe-WeiChang2010-06-022018-07-062010-06-022018-07-062007U0001-0910200715225700http://ntur.lib.ntu.edu.tw//handle/246246/184669多醣蛋白(HSPGs；Heparan Sulfate Proteoglycans)是細胞外間質的一個成員，它的功能之一是調控形態決定素(morphogens)如 Hedgehog (Hh), Wingless (Wg) Decapentaplegic (Dpp)的分佈及訊息傳遞。當多醣蛋白HSPGs的核心蛋白以及多醣鏈聚合所需要的酵素突變時，都會使多醣蛋白HSPGs產生異常，因而導致形態決定素的不正常表現以及缺損的訊息傳遞。先前的研究指出一個果蠅高基氏體蛋白質, 人類GPP34蛋白質的同源物, Rotini (Rti), 突變時會影響形態決定素Hh的行為。本論文再次確認Rti是藉由調控多醣蛋白HSPGs的表現進而影響Hh，而不是直接調控Hh的轉錄、轉譯及Hh的自我裂解程序。 藉由酵母菌雙雜交搜尋系統（yeast-two hybrid screen），本論文首先證實一個與酵母菌的高基氏體回向運輸（Golgi retrograde trafficking）有關的v-SNARE蛋白質, dVti1, 被發現與Rti有交互作用。經GST pull down的分析，確定Rti 與dVti1可以直接的交互作用；在果蠅的S2細胞株以及翅碟細胞裡，Rti與dVti1兩者的分佈在高基氏體有部分的重疊；在遺傳作用上，過量表現的dVti1可以抑制rti突變所造成的多醣蛋白HSPGs表現量的下降。這些資料顯示，Rti與dVti1是高基氏體中多醣蛋白HSPGs生成機制中兩個直接作用的成員。論文更證實Rti對於多醣蛋白HSPGs的核心蛋白，Dally-like protein (Dlp)，的表現並無直接的關係，也未影響Dlp核心蛋白分泌至細胞表面的行為。進一步研究發現，Rti是藉由調控多醣鏈(GAG; glycosaminoglycans)聚合反應中所需要的聚合酵素，如EXT蛋白質家族，進而影響多醣蛋白HSPGs醣鏈的生成；但Rti對於多醣鏈上硫化修飾過程所需的酵素蛋白，如Sulfateless (Sfl)及Slalom（Sll），並無影響。本論文推論：Rti與dVti1參與EXT酵素蛋白在高基氏體中的傳送運輸，以調控多醣鏈GAG的聚合而影響多醣蛋白HSPGs的生成。Heparan sulfate proteoglycans (HSPGs) are components of ECM and critical to distribution and signaling of Hedgehog (Hh), Wingless (Wg) and Decapentaplegic (Dpp) morphogens in Drosophila. Mutations in HSPGs core proteins and in enzymes required for heparan sulfate glycosaminoglycan (HS GAG) chain synthesis will lead to reduction of morphogens expression and aberrant signaling. ere we report that Rotini (Rti), a Drosophila homologue of human GPP34 protein, can regulate expression and signaling of morphogens, such as Hedgehog (Hh). Further studies indicate that Rti affects Hh behaviors by mediating HSPGs expression but not directly regulates Hh transcription, translation and autocleavage process. oreover, a v-SNARE protein, dVti1, involved in Golgi retrograde trafficking in yeast is identified to physically interact with Rti through yeast two-hybrid screen, and this interaction is further confirmed by GST pull down assay. Rti and dVti1 are partially colocalized in Drosophila S2 cells and wing disc cells. Genetically, overexpression of dVti1 can suppress the reduction of HSPGs caused by rti mutation. hese results suggest that Rti and dVti1 are the two directly interacting components involved in mechanism of HSPGs biogenesis. ti is not required for expression of HSPGs core protein, Dally-like protein (Dlp), and, secretion of Dlp to cell surface is either not affected in rti mutant cells. Rti is involved in formation of HS GAG chain by regulating GAG chain polymerases, EXT protein family; however, Rti is not required for expressions of Sulfateless (Sfl) and Slalom (Sll), which are involved in sulfation on GAG chains.ased on these finding, we propose that Rti and dVti1 are involved in the trafficking process of EXT proteins in Golgi complex to regulate the polymerization of GAG chains and biogenesis of HSPGs.Table of contents士論文口試委員審定書謝文摘要bstractist of figuresbbreviationsntroduction 1. Developmental events are governed by positional information established by morphogens 1I. Formation of secreted morphogen gradient…………….3 Posttranslational modification on morphogen molecule 5. Transportation between cells 6. Planar transcytosis 7. Argosomes 8. Cytoneme 8. Receptor/ligand interaction 9. Enhanced degradation 10. HSPGs modulation 12. Core proteins: Glypicans and Syndecan 13. Biogenesis of HSPGs GAG chains 14. HSPGs are required for morphogens distribution and signaling.18II. Hedgehog signaling pathway …21. Autocleavage and lipid modification of Hh 21. Hedgehog signaling pathway 22V. Rotini, a novel segment polarity gene involved in HSPGs expression 24 1. Identification of rotini as a novel segment polarity gene….24 . Rti is a Golgi resided protein, which is implicated to function in Golgi trafficking 253. Hh expression and signaling are impaired in rti mutation...28 4. Rti is required for HSPGs expression………………………29. The aim of this thesis 31aterials and methods 32. Fly stocks and maintenance 32. The autosomal FLP-DFS technique 33. Heat shock treatment 34. GAL4-UAS system 34. Generation of recombinant mutant clones in somatic tissues 35. Fluorescence antibody staining of imaginal discs 36. Yeast Two-hybrid assay 38. GST pull-down assay 38. Western blot 400. Membrane Stripping 421. Cloning constructs 422. Micro-injection and transgenic fly 453. Drosophila S2 cells maintenance 464. dsRNA synthesis 515. dsRNA treatment of S2 cells 54esults 55 . Rti is not required for Hh transcription, expression and autocleavage process 56. Hh processing occurs normally in rtiA4 mutant cells……....57I. Rti interacts and colocalizes with a v-SNARE protein dVti1 to determine the expression of HSPGs 58. The identification of dVti1 (CG3279) through yeast two-hybrid screen 59. Rti physically interacts with dVti1 in GST pull down assay 61. The Golgi protein, Rotini, is partially colocalized with dVti1 in Drosophila S2 cells 62. Overexpression of dVti1 can suppress the reduction of HSPGs mediated by rti mutation 63II. Rotini regulates EXT proteins for the polymerization of HSPGs GAG chain 66. The glycosylation and protein level of Dlp are reduced in rti mutation 67. In wing disc, the expression pattern of Dlp is not affected in rti mutant clones 70 . Rotini regulates EXT proteins for the polymerization of HSPGs GAG chains 72. The subcellular localization of b-GalT7 is not affected by rti loss-of-function 76. rti mutation does not affect the expression of sulfate modification proteins Sfl and Sll 77iscussion 79. Rti is not required for transcription, translation and posttranslational process of Hh and exerts its function after release of Hh into extracellular matrix 79. Rti is involved in GAG chains polymerization by regulating EXT proteins 82. Rti may regulate specific Golgi subunit containing the enzyme complex formed by EXT proteins 87. Through interacting with dVti1, biological function of Rti may be involved in Golgi trafficking of EXT proteins for GAG chain polymerization 90 eference 97iguresapplication/pdf3773972 bytesapplication/pdfen-US型態決定素Hh多醣蛋白HSPGs高基氏體蛋白質GPP34GAG糖鏈聚合酵素EXT 蛋白質細胞內蛋白質運輸HhHSPGsGPP34EXTvesicle transporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/184669/1/ntu-96-R94b43005-1.pdf