第十七型脊髓小腦共濟失調症致病機轉：伴隨蛋白的保護功能與TATA結合蛋白CAG三核苷重複擴增造成不正常蛋白質摺疊之研究

Abstract

摘要 遺傳性第十七型脊髓小腦萎縮症(SCA17)與染色體6q27位置的TATA binding protein (TBP)基因的CAG三核苷重複擴增相關。TBP廣泛表現在中樞神經系統及周邊組織。臨床上SCA17病患症狀很廣泛，致病機轉亦未完全清楚。為探討SCA17疾病致病機轉，我們建立短暫大量表現及穩定誘導正常TBP-Q36及多麩醯胺擴增TBP-Q61的人類胚胎腎293細胞，並利用差異性螢光標記二維電泳、質譜、免疫轉漬等方法，分析蛋白質的表現。以doxycycline誘導表現後，擴增的TBP-Q61形成聚集，且活化的caspase-3皆顯著增加。蛋白質體分析顯示23個蛋白質的差異表現在1.35倍以上。進一步以二維電泳及西方免疫轉漬確認HSPA5、HSPA8、PARK7的差異表現。淋巴細胞的蛋白質分析顯示，與正常人的淋巴細胞相較，帶有多麩醯胺擴增TBP的病患淋巴細胞，其HSPA5、HSPA8、HSPB1的表現顯著下降。進一步利用lenti病毒轉染，檢視geldanamycin對HSPA5表現及SCA17性狀的調節。在多麩醯胺擴增TBP等位基因的檢測方面，分析了臺灣地區帕金森氏症、阿茲海默氏症、非典型帕金森氏症候群患者的TBP基因CAG 三核苷酸重複，共發現6個擴增的等位基因(44 ~ 46Q)。此類非典型小腦萎縮症病患的報導，有助於疾病性質的瞭解。綜合上述，實驗結果顯示利用蛋白質體分析來找出和SCA17疾病致病機轉相關的差異表現蛋白，有助於致病機轉的瞭解，並可能依據來發展治療策略。

Abstract Expansion of the CAG repeat of the TATA-box binding protein (TBP) gene has been identified as the causative mutations in spinocerebellar ataxia 17 (SCA17). TBP is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17 clinical presentation is broad. The underlying molecular changes of SCA17 are rarely explored. To study the molecular mechanisms underlying SCA17, transient overexpressed and stably induced isogenic 293 cells expressing normal TBP-Q36 and expanded TBP-Q61 were generated and analyzed the expressed proteins using two-dimensional difference in gel electrophoresis (2D-DIGE), followed by mass spectrometry and immunoblotting. Upon induction with doxycycline, the expanded TBP-Q61 formed aggregates with significant increase in cleaved caspase-3. Proteomics study identified a total of 23 proteins with expression changes greater than 1.35 fold. The altered expression of HSPA5, HSPA8 and PARK7 were further validated by 2D and Western immunoblot analyses. In lymphoblastoid cells, significant lower HSPA5, HSPA8 and HSPB1 expression levels were observed in cells with expanded TBP than that of the control cells. Using lentiviral transduced human neuroblastoma cell models, the effects of geldanamycin on HSPA5 expression and SCA17 phenotype were assessed. Genetic screening of triplet expansion in the TBP gene in Taiwanese Parkinson's disease, Alzheimer's disease and atypical parkinsonism revealed a total of 6 expanded alleles (44 ~ 46) in patients group. Reports of additional patients are crucial for better understanding the nature of the disease. Together, this study illustrates the utility of proteomics to identify alterations of proteins which may shed insights into the pathogenesis and lead to therapeutic interventions for this disease.