Abstract
摘要：先前，我們建立一新穎的果蠅 cFRT2L2R 染色體，可以直接讓我們對由P 跳躍元素所突變的染色體進行基因重組的分析。以此前瞻的技術為平臺，此計劃首先將進行尋找新的具有特殊母源效應的致死基因，以對母源基因在胚胎發育中的功能有更多的瞭解。
硫醣蛋白 HSPGs參與調控Hedgehog (Hh)的分佈及訊息傳遞；當HSPGs的核心蛋白以及多醣鏈(Glycosaminoglycans)在聚合時所需的酵素突變時，HSPGs的生成會產生異常，因而導致Hh的不表現以及訊息傳遞的缺損。
Rotini基因是藉此技術首先找到的人類高基氏體蛋白質的同源物。我們先前初步證實Rti是藉由調控HSPGs的表現而後間接而影響Hh的行為，而不是直接調控Hh的轉錄、轉譯及Hh的自我裂解程序。經Two-hybrids搜尋，果蠅的v-SNARE蛋白質, dVti1, 被發現可能與Rti有交互作用。吾人將以生化、免疫染色與遺傳分析，以確定兩者是否皆分佈在高基氏體中及是否有遺傳上與蛋白質間的交互作用、進而影響HSPGs與Hh的表現量；這將用以確定兩者是否為參與HSPGs生成的兩個直接作用成員。
核心蛋白Dally-like protein (Dlp)或多醣鏈合成的缺損皆影響HSPGs的生成。Rti是否為兩者或其一進入HSPGs生成路徑之所需? Rti 是否影響核心蛋白的表現? Rti 是否影響核心蛋白分泌輸送其至細胞表面的行為? 或者Rti是調控一系列多醣鏈聚合反應中所需要的聚合酵素等進入高基氏體所需要的?
有關HSPGs由細胞質內生成、輸送、分泌到細胞膜表面的機制闕如；本計劃將藉由Rotini 的研究，瞭解此一高基氏體蛋白質調控HSPGs生成的確切作用點，以為細胞質內輸送HSPGs之行為的前瞻性原型研究。
Abstract: Previously, we have developed a novel Drosophila cFRT2L2R chromosome that allows the direct recombination analysis of a P transposon-induced mutation. As a frontier in this field, this project is first aimed to use this efficient protocol as a platform to screen for novel essential gene with a specific maternal effect so as to understand more about maternal contributions in embryogenesis.
Heparan sulfate proteoglycans (HSPGs) regulate the expression level and signal transduction of Hedgehog (Hh) morphogen. Mutations involved in biogenesis of core protein and GAGs glycosaminoglycans can cause the abnormal HSPGs expression and aberrant Hh expression and signaling.
Rotinin (Rti) was isolated based on the cFRT2L2R protocol. It is a Drosophila homologue of human GPP34 Golgi protein. We have preliminarily confirmed that Rti indirectly affects Hh behaviors by mediating HSPGs expression but not directly regulates the transcription, translation and auto-cleavage process of Hh.
A Drosophila v-SNARE protein, dVti1, involved in Golgi retrograde trafficking in yeast is identified to interact with Rti by yeast two-hybrid screen. Biochemical, immunostaining and genetics analyses will be performed to confirm whether Rti and dVti1 have direct physically contact, whether they may co-localize in Golgi compartments and whether they have genetic interaction that can influence the behaviors of HSPGs and Hh. It will be critical to confirm that Rti and dVti1 are two directly interacting components involved in the processes of HSPGs biogenesis.
Defects in core protein, such as Dally-like protein (Dlp), or in the synthesis of GAG side chains can affect the proper formation of HSPGs. We plan to clarify whether Rti is required for the participation of core protein and/or of the enzyme(s) for the synthesis of GAG in the processes of HSPGs biogenesis. That is whether Rti is required for the expression of Dlp protein and for its secretion to cell surface or whether Rti is involved in formation of HS GAG chain by regulating the entrance of polymerases into HSPGs biogenesis..
Up to date, the mechanism for the biogenesis of HSPGs from their formation and transport in cytosol to the secretion toward membrane is not well understood. This project is to explore how a Drosophila Golgi protein, Rotini, can regulate the biogenesis of HSPGs. Its precise action point and to which partner it works with will be determined. Through this study, we will be able to understand how a Golgi protein can regulate the biosynthesis of HSGPs. We believe that this project will be a paradigm to study how the cell can control the behavior of HSPGs transportation.
Keyword(s)
多醣蛋白
多醣鏈聚合酵素
高基氏體蛋白質
果蠅
Heparan sulfate proteoglycans
glycosaminoglycans
Golgi protein
Drosophila
