- 學位論文
脊髓小腦運動失調症第十七型之微生物藥物篩檢模式：以 β-半乳糖苷酶結構互補特性偵測蛋白溶解度
Screening of Novel Molecules for SCA 17 Treatment by Using a Microbial Model: Monitoring Protein Solubility by β-Galactosidase Structural Complementation
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摘要
多種遺傳性神經退化疾病為多麩醯胺 (polyglutamine, polyQ) 相關疾病，其中第十七型脊髓小腦萎縮症 (spinocerebellar ataxias type 17, SCA17)，於染色體 6q27 位置上的 TATA-box binding protein (TBP) 基因之轉譯區，發生 CAG 三核苷酸重複擴增導致，可能的分子致病機制為突變的 TBP 蛋白上具有擴增的多麩醯胺片段，造成蛋白結構錯誤摺疊，導致轉錄調控功能喪失。因此，建立一個可篩選出有助於抑制 TBP 蛋白聚集之藥物的篩檢模式，對於治療第十七型脊髓小腦萎縮症是極重要之事。根據 β-半乳糖苷酶 (β-galactosidase, β-gal) 蛋白結構中的 α 和 ω 二片段可互補形成具活性之酵素的特性。本研究建立表達 TBP/α 融合蛋白之大腸桿菌表達系統，此系統藉由含多麩醯胺擴增的 TBP 蛋白不正常聚集，連帶影響 α 片段構形，而無法形成有活性的 β-半乳糖苷酶，導致無法代謝 X-gal，菌落無法呈現藍色的表現型。經表現型分析、基因型分析、西方轉漬分析及 β-半乳糖苷酶比活性分析，發現以 JM109(DE3) 大腸桿菌作為pGEX-5x-3-fTBP(Q40)-lacZα 質體之表達宿主，表達出不溶性 fTBP(Q40)/α 融合蛋白，菌體不具有 β-半乳糖苷酶活性，呈現出符合微生物藥物篩檢系統所必須具備的非藍色表現型。故選定 JM109(DE3)/pGEX-5x-3-fTBP(Q40)-lacZα 作為微生物藥物篩檢的模式菌株。並以海藻糖處理模式菌株，發現海藻糖可抑制 fTBP(Q40)/α 融合蛋白聚集，可作為此微生物藥物篩檢系統之正控制組。利用盤式培養方式培養此菌株，可以應用於高通量的藥物篩檢，預期藉此系統可以快速篩選出可抑制多麩醯胺擴增之蛋白聚集的藥物，加速第十七型脊髓小腦萎縮症的藥物開發。
關鍵字
第十七型脊髓小腦萎縮症並列摘要
Many neurodegenerative diseases are linked to abnormally expanded CAG repeats in the coding regions of responsible genes. Spinocerebellar ataxia type 17 (SCA 17) was identified in CAG tri-nucleotide 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 (polyQ) strentches and dysfuction of transcriptional regulation by loss of function. An efficient cell-based screening for TBP aggregation inhibitors is important in drug discovery for SCA17 treatment. By structural complementation between the α- and ω-peptides of β-galactosidase (β-gal), the solubility of polyQ-expanded TBP can be monitored simply by the blue color of the culture and correlated with β-gal activity.In this study, we established an Escherichia coli. expression system to express the TBP/α fusion proteins. The results of phenotyping, genotyping, western blotting and β-gal activity assay showed that when α peptide was fused with insoluble polyQ-expanded TBP(Q40), it becomes misfolding and confer no β-gal activity. For this reason, the E. coli JM109(DE3) harboring pGEX-5x-3-fTBP(Q40)-lacZα can serve as a microbial model to screen for compounds that inhibit the polyQ-expanded TBP aggregation. Using this model, we found that trehalose showed potency to inhibit polyQ-expanded TBP aggregation and can be used as a positive control. Therefore, in a plate assay format, this microbial model is suitable for rapidly screening novel compounds that are capable of inhibiting the aggregation of polyQ proteins.
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