去泛素化酵素 Ubiquitin C-terminal Hydrolase之基質專一性研究

Abstract

真核細胞的數種類泛素蛋白質 (ubiquitin-like proteins) 中，以 NEDD8 (neuronal precursor cell expressed developementally downregulated protein 8) 和泛素的相似度最高，然而兩者卻各自擁有專屬之酵素系統且參與不同的生理途徑。目前已知多數的去泛素化酵素 (deubiquitinating enzyme, DUB) 只對泛素作用，然而 Ubiquitin C-terminal Hydrolase (UCH) 家族則是被報導對泛素及 NEDD8 皆具有活性，而且對泛素仍具有較高之活性。本研究的目的即是探討 UCH 家族對泛素和 NEDD8 的辨識與作用機制。 研究顯示 UCH 具有高度保守性之酵素活性區，然而不同 UCH 之活性區外的 crossover loop 長度彼此相異且影響到對泛素鏈 (ubiquitin chains) 的活性，因此我們假設 UCH不同長度之 crossover loop 亦會影響其作用於泛素和 NEDD8 的活性。本研究改變 UCH 成員的 crossover loop 長度，觀察其對泛素及 NEDD8 的活性是否會跟著改變。結果顯示 UCH 活性區附近的 crossover loop不是主要決定基質專一性和活性的位置。因此，本研究推想會造成酵素對基質的親和力有所差異的原因，應該來自兩個基質上不同的保守性胺基酸。為進一步觀察 UCH 對突變特定胺基酸後之基質的活性是否改變，本研究針對泛素及 NEDD8 之4號、12號、14號、63號、64號及72號的保守性胺基酸進行突變，並且製備其重組蛋白以進行UCH的活性分析。實驗結果顯示，泛素與NEDD8上的保守性胺基酸並非決定其與UCH之間的結合和專一性辨識的分子決定基 (molecular determinant)。

In eukaryotes, NEDD8 (neural precursor cell expressed developmentally downregulated protein 8) is the most close relative to ubiquitin (Ub) among several kinds of ubiquitin-like proteins. However, Ub and NEDD8 have their unique enzyme systems involved in different cellular pathways. Most of ubiquitinating enzymes (DUB) are specific for Ub, whereas the UCH family is reported to have NEDD8 cross-reactivity. This work is aimed to study the substrate recognition and catalytic mechanism of UCH for processing Ub and NEDD8. It is reported that the length of the active-site crossover loop defines the substrate specificity of UCH for ubiquitin chains. Thus, this study examined whether the length of the active-site crossover loop was also involved in the substrate specificity of UCH. The result showed that mutation of the active-site crossover loop did not impede UCH’s catalytic activity. To investigate whether the replacement of the conserved amino acid residues of Ub with the corresponding residues of NEDD8 may affect the substrate specificity, mutations of the residues at position 4, 12, 14, 63, 64 and 72 were applied to test the possibility. The experimental results demonstrated that the conserved amino acid residues at residues 4, 12, 14, 63, 64 and 72 were not the molecular determinants for substrate binding and specific recognition.