GTP Cyclohydrolase I 突變及Splicing之分子結構機轉(2/3)

Abstract

GTP cyclohydrolase I regulates the level of tetrahydrobiopterin and in turn the activities of nitric oxide synthase and aromatic amino acid hydroxylases. Mutations of the GCH gene induce human diseases including malignant hyperphenylalaninemia (HPA) and dopa-responsive dystonia (DRD). DRD is often caused by dominant GCH mutations with residual GCH activities up to 15% of normal. GCH protein is a multimeric protein composed of 10 identical subunits, and we have demonstrated that both some dominant GCH mutations and the type II cDNA exerted a dominant-negative effect toward the wild-type GCH protein. In this study, we demonstrated the expression of wild type GCH protein (by transfecting BHK cells) increased after heat shock. The mRNA levels were not changed, but we are not able to detect change in protein stability by pulse-chase and immunoprecipitation study. Cotransfection of hsp40 or hsp70 could not mimic the effect of heat shock. On the contrary, cotransfection of hsp27 greatly decreased the expression of GCH proteins. This study suggests regulation of GCH protein expression by a complex cellular reaction bringing up by heat shock and the heat shock proteins.

GTP 環狀水解酵素(GCH)突變造成二種疾病：肌肉張力不全(dopa-responsive dystonia, DRD)與苯酮尿症(malignant phenylketonuria)，後者伴隨嚴重的神經傷 害。GCH 基因的突變，有些會以一種dominant-negative 的方式，影響到細胞中 正常的GCH 蛋白。我們最近也發現GCH 基因的第二型splicing 也有 dominant-negative 的效果。當我們將細胞進行熱休克後，轉染到BHK 細胞中之 GCH 基因會表現出比較大量的蛋白。GCH 基因的轉錄沒有改變， 在 pulse-chase/immunoprecipitation 實驗中也看不到蛋白質穩定性的變化。轉染時如 果同時加入hsp40 及hsp70 時，GCH 蛋白的表現並不會有變化。反而是轉染時 如果同時加入hsp27 時，GCH 蛋白的表現量會有明顯的減低。這個研究顯示GCH 蛋白的表現會受到細胞內複雜，和熱休克有關之機制的控制。