The Molecular Determinants for Ubiquitin and NEDD8 Discrimination by Human Deubiquitinases
Date Issued
2012
Date
2012
Author(s)
Chen, Jou-Han
Abstract
Ubiquitination is a posttranslational modification resulted from the attachment of ubiquitin’s C-terminal glycine to lysine residues on target proteins. There are several ubiquitin-like proteins sharing similar protein structure and modification pathway with ubiquitin. NEDD8 (neuronal precursor cell expressed developmentally downregulated protein 8) is its closest relative. Before conjugation, ubiquitin needs to be processed by deubibiquitinases to expose the C-terminal di-glycine motif. Most of deubiquitinases are specific for ubiquitin, whereas some are reported with NEDD8 cross-reactivity. The purpose of this work is to study the substrate recognition mechanism of USP and UCH by which they can discriminate between ubiquitin and NEDD8.
By examining the crystal structure of ubiquitin-USP2 binding complex and the amino acid sequence of ubiquitin and NEDD8, residues 4, 12, 14, 51 and 72 of ubiquitin were mutated to corresponding residues in NEDD8 and vice versa. By fusing ubiquitin and NEDD8 with C-terminal His tag, hydrolysis of the substrate by USP2 was analyzed by western blotting using the anti-His antibody. The data showed that mutations at F4K, T12E and T14E of ubiquitin partially inhibited its hydrolysis by USP2, due to the steric hindrance and electric repulsion between the mutated residues and USP2. Residue 72, on the other hand, may act as a helper since it interacts with USP2 and further inhibited hydrolysis in T12E/T14E/R72A ubiquitin mutant than T12E/T14E mutant. However, the E51N mutation of ubiquitin showed minor effects on catalytic inhibition, therefore it may not serve as a recognition site for USP2. Moreover, amino acids interacting with residues 4, 12, 14 and 72 of ubiquitin are conserved among USP members, suggesting a general recognition mechanism in the USP protein family. In this work, the experimental result also showed that UCHL1 contains dual specificity for processing ubiquitin and NEDD8.
Subjects
ubiquitin
deubiquitinas
Type
thesis
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