| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 95 === Autosomal dominant spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia of gait and limbs variably associated with dementia, dysarthria, etc. More than 28 SCA types have been described. Among them, SCA type 17 (SCA17) is caused by an expanded polyglutamine (polyQ) in the TATA-box binding protein (TBP), a general transcription initiation factor. In addition to SCA17, neurodegenerative diseases including HD, SBMA, DRPLA and SCA types 1, 2, 3, 6, 7 are also caused by expansion of a CAG repeat tracts encoding expanded polyQ tracts. The expanded polyQ tract causes a conformational change in the polypeptide to promote misfolding and aggregation of the disease protein, leading to the death of neurons. Unrelated expanded alleles characterized in familial and sporadic ataxia patients are found ranging from 43~66 repeats as opposed to 25~42 repeats in the general population. We screened the SCA17 TBP gene in 197 normal controls and in patients with various neurodegenerative diseases: 13 ataxia, 103 Parkinson's disease, 122 dementia, 101 essential tremor, and 29 chorea and dystonia. The most common TBP allele contains 36 repeats and no expanded allele was found. Then biochemical and yeast assays were developed to screen effective chemical compounds which may inhibit polyglutamine protein aggregation. GST fused Qn, tTBP-Qn (truncated N-terminal TBP), nTBPQn (N-terminal TBP) and fTBPQn (full-length TBP) (n = 3 ~ 61) were overexpressed in E. coli BL21 cells and purified by affinity chromatography. After factor Xa cleavage to remove GST, the TBPQn proteins were used in drug screening by filter-trap assay and western blotting. However, neither controls (Congo red and trehalose) nor tested chemical compounds inhibit polyQ protein aggregation efficiently. This might be due to the low solubility of TBP and the suitability of 1C2 antibody. In addition, the expression of tTBPQ20/Q54-EGFP、nTBPQ20/Q48-EGFP under the control of the GAL1 promoter was induced by galactose in erg6 and W303 yeast strains. No growth inhibition was observed in yeast expressing full-length or N-terminal TBPQ54-EGFP proteins. This might be due to that low expression level was induced or the length of polyQ is not long enough to exhibit toxicity in yeast. Nevertheless, the study may provide information for future research design to develop assay to screen novel chemical inhibitors of aggregation.
|
|---|
