Human antizyme-mediated binding inhibition and degradation of ornithine decarboxylase

• Main Authors: Chu-Ju Lee, 李竺如
• Other Authors: Hui-Chih Hung
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/84539204626555863876

碩士 === 國立中興大學 === 生命科學系所 === 104 === ODC is a pyridoxal-5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of ornithine to putrescine. This reaction is the first and rate-limiting step in polyamine biosynthesis. Polyamines such as putrescine, spermidine, and spermine are small organic cations which can bind to DNA, RNA, and protein to control cellar proliferation, differentiation, apoptosis, and gene expression. Antizyme (AZ) is a regulatory protein of ODC, which can bind to ODC and cause the dimer dissociation to lose ODC enzymatic activity. However, the AZ binding induces a conformational change to expose C-terminal tail of ODC to be recognized by 26S proteasome. In the AZ-1 inhibition experiments, we aligned the sequences of AZ1, AZ2 and AZ3 and found two charged residues, R199 and E219, these two charged residues were mutated to alanine. The results revealed that AZ1 E219A played a critical role in inhibiting ODC enzyme activity. According to the experiments of ODC enzyme activity analysis, AZ isoforms have differential ODC binding affinities and inhibition abilities. AZ1 is stronger than AZ3. We compared the sequences between AZ1 and AZ3 to find the different residues and substituted the AZ3 residues for corresponding AZ1 residues. Enzyme activity inhibition assay showed that N152S and N178K are critical residues for AZ3 to increase its inhibiting ability. For in vitro degradation, sixteen residues on AZ1 were mutated to alanine according to the crystal structure of AZ-ODC complex and sequence alignment of AZ isoforms. The residues S120、N129 and G137 may be the major residues for ODC degradation. Based on the above results, we could find the important AZ amino acid residues that have significant effect on ODC inhibition and degradation.