Study on the molecular mechanism for substrate specific recognition by ubiquitin-like protein peptidases

• Main Authors: Yung-Cheng Shin, 辛永誠
• Other Authors: Shih-Chung Chang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/v6j75c

博士 === 國立臺灣大學 === 生化科技學系 === 105 === There are more than ten kinds of ubiquitin-like proteins (Ubls) have been found. Ubls have high similarity of amino acid sequences and three-dimensional structures with ubiquitin, but perform different physiological functions in cells. NEDD8 (Neural Precursor Cell Expressed, Developmentally Down-Regulated 8) and ubiquitin (Ub) have the highest similarity (up to 80%) among different Ubls, whereas the respective proteases SENP8 and USP2 still can react to NEDD8 or Ub correctly. Studies have shown that when proteins with wrong NEDD8 or Ub modification will possibly lead to disease, so it’s important to explore the specific identification mechanism between NEDD8 and Ub modification systems. In the present study, the fusion protein of Ub and NEDD8, CrRUB1 (Chlamydomonas reinhardtii related to ubiquitin 1), was utilized as the substrate for investigating the catalytic specificity of SENP8. By using the site-directed mutagenesis method for replacing the amino acid residues 51 and 72 of CrNEDD8 with their CrUb equivalents, and vice versa, the experimental results showed that CrNEDD8 with N51E/A72R mutations was not cleaved by SENP8, but CrUb with E51N/R72A mutations was efficiently cleaved by SENP8, implying that residues 51 and 72 are the major determinants for specific recognition by SENP8. However, the mutation of residues 51 and 72 of CrNEDD8 and CrUb didn’t largely influence the activity of USP2, suggesting that USP2 and SENP8 have different substrate identification mechanism. The three-dimensional structure of human USP2 and Ub reveals that residues 4, 12 and 14 of Ub could interact with USP2, and these three residues were not conserved in NEDD8. The experimental results showed that Ub4K/12E/14E/72A mutant was not cleaved by USP2, but N84F/12T/14T/72A mutant was efficiently cleaved by USP2, indicating that these four residues were the important determinants for distinguishing between ubiquitin and NEDD8 by USP2. In addition, USP21 also displays the same substrate recognition mechanism. In contrast, Ub E1-activating enzyme Ube1 can still react to NEDD8 and the Ub4K/12E/14E/72A mutant, suggesting that not all the enzymes involved in ubiquitination pathway have the ability to distinguish Ub and NEDD8.