脊髓小腦運動失調症第十七型：微生物模型之藥物篩選
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2009
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目前已知道有十種遺傳性神經退化疾病為多麩醯胺 (polygluta-
mine, poly-Q)相關的疾病，其中第十七型脊髓小腦萎縮症 (SCA 17)，起因於染色體6q27位置上的TBP基因之轉譯區中，發生CAG三核苷酸重複擴增所導致。因此SCA17的可能分子致病機制為：突變的TBP蛋白質上具有延長的poly-Q片段，導致蛋白質結構錯誤摺疊及聚集，最後形成不溶性的包涵體，因而造成細胞死亡。已知在活體 (in vivo)和試管內 (in vitro)實驗中也可以發現，抑制錯誤延長性poly-Q片段的蛋白聚集，可以抑制poly-Q相關疾病的產生。本研究建立TBP-Lac Zα 融合蛋白於大腸桿菌中的表達系統，藉由β半乳糖苷酶之α互補作用，建構出微生物快速藥物篩選系統，以期加速相關藥物的開發。質體建構方面，是以pGEX 質體為基礎建構數種蛋白表達質體，包括帶有不同延長poly-Q的pGEX-TBP-lac Zα以及pGEX-Lac Zα重組質體。蛋白表達方面，將相關質體轉型至數種大腸桿菌 (E. coli)，以藍白篩選的方式，找出合適的菌種與表達條件，用以表達可溶性的短延長TBP蛋白 (培養液呈藍色)、及不可溶性長延長TBP蛋白 (培養液呈白色)。利用表現型分析、基因型分析、蛋白質電泳、西方轉漬分析以及β半乳糖苷酶活性分析，確立藥物篩選系統的可行性並驗證之。結果發現JM109大腸桿菌表達系統符合篩藥條件 (即pGEX-Lac Zα呈藍色、pGEX-fTBP40Q-Lac Zα呈白色)，故pGEX-fTBP40Q-Lac Zα/JM109可以作為篩藥模式。藥物篩選方面，目前以具有潛力之藥物 (例如海藻糖與剛果紅)做測試，結果發現這兩種藥物似乎無法抑制不可溶性長延長TBP蛋白凝集。本篇論文提供一種可以大量快速進行SCA17藥物篩檢的微生物模式，新穎的合成藥物仍在持續篩檢中，以找出有效抑制不可溶性長延長TBP蛋白凝集之藥物以供細胞及動物模式做進一步的藥物測試。
Abstract Many neurodegenerative diseases are linked to abnormally expanded CAG repeats in the coding regions of responsible genes. One of them, spinocerebellar ataxia type 17 (SCA 17) was identified with CAG trinucleotide repeat expansion in the TATA-box binding protein (TBP) gene on chromosome 6q27. The possible molecular pathogenic mechanisms of SCA17 could be aggregation caused by mutant TBP with expanded polyglutamine strentches and dysfuction of transcriptional regulation by loss of function. Although the detail pathogenic mechanism is unknown in poly-Q disease, inhibition of aggregation is effective to protect cell in vitro and in vivo. In this study, an E. coli. expression system was established to express the TBP-Lac Zα fusion proteins. By structural α complementation of β Galactosidase (β Gal), we anticipate this system could be a bacterium-based model for rapid drug screening to inhibit protein aggregation. In plasmid construction, we used pGEX plasmid to construct several protein expression vectors including those harboring TBP-Lac Zα fusion genes with various length of polyQ tract and pGEX-Lac Zα. In protein expression, several E. coli. strains were tested to express these recombinant proteins by blue-white screening, and expression conditions were obtained to express soluble short poly-Q TBP as blue color culture, and aggregated long poly-Q TBP as white color culture. We validated this drug screening system by phenotypic screening, genotyping, SDS-PAGE, Western blotting and β gal activity assay. The result showed that JM109 E. coli. expression system can express pGEX-Lac Zα as blue color culture and pGEX-fTBP40Q-Lac Zα as white color culture. We used pGEX-fTBP40Q-Lac Zα/JM109 as drug screening model. In drug screening, several candidate drugs capable of inhibiting poly-Q protein aggregation (such as trehalose and congo red) were tested to prevent TBP aggregation. The results show that trehalose and congo red can not inhibit long poly-Q TBP aggregation by blue-white screening. This study could provide a high-throughput microbial drug screening system.
Abstract Many neurodegenerative diseases are linked to abnormally expanded CAG repeats in the coding regions of responsible genes. One of them, spinocerebellar ataxia type 17 (SCA 17) was identified with CAG trinucleotide repeat expansion in the TATA-box binding protein (TBP) gene on chromosome 6q27. The possible molecular pathogenic mechanisms of SCA17 could be aggregation caused by mutant TBP with expanded polyglutamine strentches and dysfuction of transcriptional regulation by loss of function. Although the detail pathogenic mechanism is unknown in poly-Q disease, inhibition of aggregation is effective to protect cell in vitro and in vivo. In this study, an E. coli. expression system was established to express the TBP-Lac Zα fusion proteins. By structural α complementation of β Galactosidase (β Gal), we anticipate this system could be a bacterium-based model for rapid drug screening to inhibit protein aggregation. In plasmid construction, we used pGEX plasmid to construct several protein expression vectors including those harboring TBP-Lac Zα fusion genes with various length of polyQ tract and pGEX-Lac Zα. In protein expression, several E. coli. strains were tested to express these recombinant proteins by blue-white screening, and expression conditions were obtained to express soluble short poly-Q TBP as blue color culture, and aggregated long poly-Q TBP as white color culture. We validated this drug screening system by phenotypic screening, genotyping, SDS-PAGE, Western blotting and β gal activity assay. The result showed that JM109 E. coli. expression system can express pGEX-Lac Zα as blue color culture and pGEX-fTBP40Q-Lac Zα as white color culture. We used pGEX-fTBP40Q-Lac Zα/JM109 as drug screening model. In drug screening, several candidate drugs capable of inhibiting poly-Q protein aggregation (such as trehalose and congo red) were tested to prevent TBP aggregation. The results show that trehalose and congo red can not inhibit long poly-Q TBP aggregation by blue-white screening. This study could provide a high-throughput microbial drug screening system.
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第十七型脊髓小腦萎縮症, β半乳糖苷酶, α互補作用, 藥物篩選, SCA17, β gal, α complementation, drug screening