| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 89 === Abstract (English)
Hereditary progressive dystonia/ Dopa-responsive dystonia (HPD/DRD) with marked diurnal fluctuation is an autosomal dominantly inherited dystonia caused by abnormalities of the gene of GTP cyclohydrolase I (GCH) located on the 14q22.1-q22.2. GCH is the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin (BH4), an essential factor for phenylalanine hydroxylase, tyrosine hydroxylase (TH), tryptophan hydroxylase and nitric oxide synthase.
In HPD/DRD patients, the GCH enzyme activity is reduced to 20% of its normal value. The modern mechanism is unclear but a dominant-negative effect has thought to be a factor for the reduced activity. Because GCH is a homodecameric protein, the mutant GCH protein may inhibit or destabilize the wild-type. In many reports, mutant GCH proteins, e.g. D134V, G201E and G203R, were unstable and greatly reduced in patients or cell culture expression. To enhance the stability of mutant GCH-G201E (G201E) protein, we coexpressed G201E, Hsp70 and Hsp40 in HEK 293 cells. Western blot analysis showed an increase on the stability of G201E protein for 4- to 5-fold but a little effect on the solubility of G201E protein.
We next showed that there was no increase on the activity of G201E while coexpressed with Hsp70 and Hsp40. With cross-linking assay, we found the aberration of GCH activity of G201E was due to the inability of homodecamer formation for the enhanced G201E protein. On the other hand, we found the enhanced G201E protein did form heterocomplex with wild-type one by cotransfection and co-immunoprecipitation experiments. These results demonstrated that Hsp70 and Hsp40 not only help to stabilize G201E protein in the cell but also facilitate G201E protein to form complex with wild-type GCH protein.
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