脊髓小腦運動失調症第十七型之微生物藥物篩檢模式：以微生物抗氯黴素能力作為篩檢標記

High-Throughput Microorganism-Based Screening of Novel Molecules for SCA 17 Treatment: Monitoring TBP solubility by chloramphenicol resistance

永資陵(Zi-Ling Yong)

指導教授 ： 李冠群

國立臺灣師範大學/理學院/生命科學研究所/碩士(2010年)

摘要

目前已知有十種經遺傳的神經退化性疾病是和多麩醯胺(polyglutamine, polyQ)相關的疾病，其中第十七型脊髓小腦萎縮症(spinocerebellar ataxia type 17, SCA 17)是由於染色體上6q27位置的TATA binding protein (TBP)基因之轉譯區中，CAG三核苷酸發生重複擴增所導致。TBP基因所轉譯出來的蛋白質為細胞內重要的轉錄調節因子，而擴增的polyQ可能會影響到TBP蛋白的功能，因此推測SCA 17可能分子的致病機制為：突變的TBP蛋白質其擴增的polyQ片段，會導致蛋白質錯誤摺疊，最後形成不溶性的包涵體 (inclusion body)，進而造成神經細胞的功能異常、病變甚至死亡。而在活體(in vivo)和離體(in vitro)實驗中也可以發現，抑制錯誤延長性polyQ片段的蛋白聚集，可以抑制polyQ相關疾病的產生。本研究在大腸桿菌中建立TBP-chloramphenicol acetyltransferase (CAT，氯黴素乙醯轉移酶) 融合蛋白的表達系統，此細菌藥物篩檢模式是將TBP蛋白凝集現象與細菌細胞失去氯黴素抗藥性現象結合，亦即以抗藥性蛋白做為genetic marker protein，任何有助於蛋白溶解度提升之藥物，皆可藉由細菌本身抗藥性提升而被篩選出來。經表現型分析、基因型分析、西方轉漬分析及CAT 活性分析後，發現以Tuner、JM109、JM109(DE3)及BL21(DE3)大腸桿菌分別做為pGEX及pET system的表達宿主，此二表達系統符合篩藥條件(即長擴增的TBP片段抗藥性低於短擴增的TBP片段)，故Tuner/pGEX-tTBP60Q-CAT、JM109/pGEX-tTBP60Q-CAT、JM109(DE3)/pGEX-tTBP60Q-CAT及BL21(DE3)/pET-tTBP60Q-CAT可以作為篩藥模式。在建立篩檢模式之正控制組方面，選定Tuner/pGEX-tTBP60Q-CAT、JM109/pGEX-tTBP60Q-CAT及BL21(DE3)/pET-tTBP60Q-CAT微生物模式，以具有潛力之藥物(例如海藻糖及剛果紅)做測試，結果發現海藻糖及剛果紅似乎無法抑制不可溶性長擴增TBP蛋白凝集，可能是因海藻糖及剛果紅無法抑制此系統表達之長擴增TBP蛋白之凝集。當使用低溫誘導處理時，結果發現長擴增TBP蛋白溶解度提升，且其抗藥性有增加之現象，證明此系統可做為其他藥物之篩檢模式。本研究提供一種可以大量快速進行SCA17藥物篩檢的微生物模式，經由表現型分析、基因型分析、西方轉漬及CAT活性分析，找尋具有抑制長擴增TBP蛋白凝集之潛力藥物。

關鍵字

SCA 17 ； 氯黴素 ； 藥物篩檢

並列摘要

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 trinucleo- tide repeat expansion in the TATA-box binding protein (TBP) gene on chromosome 6q27. The possible molecular pathogenic mechanisms in 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, we establish an E. coli. expression system to express the TBP-chloramphenicol acetyltransferase (CAT) fusion proteins. We constructed a simple in vivo system for assessing protein solubility that involves expressing a fusion of an expanded polyQ TBP with CAT, the enzyme responsible for conferring bacterial resistance to chloramphenicol. In plasmid construction, we used pGEX and pET plasmid to construct several protein expression vectors including those harboring TBP-CAT fusion genes with various length of polyQ tract、pGEX-CAT and pET-CAT. In protein expression, several E. coli. strains were tested to express these recombinant proteins by Cm resistance, and expression conditions were obtained to express CAT fuses with insoluble TBP-Qn, they confer significantly lower or even no Cm resistance, and vice versa. We validated this drug screening system by phenotypic screening, genotyping, Western blotting and CAT assay. The result showed that Tuner and BL21(DE3) E. coli. expression system can express CAT fuses (Tuner/pGEX system、JM109/pGEX system、JM109(DE3)/pGEX system and BL21(DE3)/pET system) with insoluble TBP-Qn, they confer significantly lower Cm resistance, and vice versa. We used Tuner/pGEX-tTBP60Q-CAT and BL21(DE3)/pET-tTBP60Q-CAT as drug screening model. In model test of positive control, 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 Cm resistance. Maybe trehalose and congo red could not suppress the expression of this system to extend the length of TBP protein aggregation. To prove that this system can be used as the screening model of other drugs, thus reducing the culture temperature, was found poly-Q protein reduced aggregation, and the resistance was increasing. This study could provide a high-throughput microbial drug screening system, and this system can be used as other drug screening models and analysis by phenotype, genotype analysis, Western blot and CAT activity analysis to find inhibit the aggregation of long extension of TBP protein potential drug.

參考文獻

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