USP2與Ube1專一性辨認泛素之分子決定基研究

Abstract

泛素 (uibiquitin, Ub) 與類泛素蛋白質 (ubiquitin-like protein) 的修飾作用參與許多細胞中的重要生理路徑。眾多的類泛素蛋白質中，又以NEDD8 (Neuronal precursor cell Expressed, Developmentally Down-regulated 8) 與Ub的序列相似度最高 (80%的胺基酸序列相同)，但是兩者所參與的生理路徑卻截然不同，因此研究各自的專屬酵素系統如何專一性地針對這兩者進行辨識與作用就顯得相當重要。已知泛素蛋白酶USP2與NEDD8蛋白酶SENP8選擇基質的辨認位置並不相同，但是USP2如何達到專一性辨認Ub的分子機轉仍不清楚。本研究藉由分析USP2與Ub之共結晶的立體結構，推測第4、12與14號胺基酸是兩者交互作用的主要區域，因此推測靠近Ub分子N端的區域，可能為USP2辨識基質的關鍵位置。實驗結果顯示若將Ub之4、12及14號的胺基酸分別更換為NEDD8之序列後，USP2水解這些突變基質的能力明顯降低；其中，以突變了第4號胺基酸的抑制效果最佳，突變12與14號的胺基酸亦有明顯的抑制效果，而僅突變51或72號胺基酸的Ub則完全無法抑制USP2的酵素活性。重要的是，只有當4、12、14與72號胺基酸皆同時突變才能完全抑制USP2的活性，顯示72號胺基酸仍可能扮演著輔助辨識的角色。本研究亦分析了4、12、14及72號胺基酸，是否也是ubiquitin E1 activating enzyme Ube1辨認基質的重要分子決定基 (molecular determinant)。實驗結果顯示Ube1對同時突變這四個胺基酸的突變株的作用能力下降了，可見此四個分子決定基在泛素修飾系統中扮演著提供高度專一性的重要角色。

Ub and ubiquitin-like protein modification involve in lots of important physiological pathways. Of all known ubiquitin-like proteins, NEDD8 (Neuronal precursor cell Expressed, Developmentally Down-regulated 8) is the closest relative to ub in amino acid sequence identity. In spite of their highly identity, ub and NEDD8 participate in distinct pathways, indicating that they should be distinguished and activated by individual specific enzyme systems. Previous studies showed that ub-specific peptidase USP2 and NEDD8-specific peptidase SENP8 perform different substrate specificity for recognizing ub and NEDD8. By investigating the crystal structure of ub-USP2 binding complex and comparing the amino acid sequence of ub and NEDD8, residues 4, 12 and 14 of ub were proposed as important molecular determinants. Mutations at F4K, T12E and T14E of ub partially inhibited its hydrolysis by USP2. In this study, the experimental results further revealed that residues 4, 12, 14 and 72 of ub can also be the molecular determinants for specific recognition by ubiquitin E1 activating enzyme Ube1. The present data showed that Ube1 cannot catalyze the ubiquitin mutant with the simultaneous mutations at residues 4, 12, 14 and 72, suggesting that these four molecular determinants are conserved among different enzymes in the ubiquitin processing and activation pathways.