Crystallization and preliminary X-ray crystallographic analysis of the human antizyme inhibitor in complex with antizyme

• Main Authors: Pei-Ying Lee, 李佩螢
• Other Authors: 詹迺立
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/00733463085145140166

碩士 === 國立中興大學 === 生物化學研究所 === 97 === Polyamines are aliphatic cations which play critical roles during cell proliferation, cell death and differentiation. Intracellular polyamine levels are controlled by regulating the protein level of ornithine decoboxylase (ODC), the first enzyme in the polyamine biosynthesis pathway. Antizyme (AZ) is a negative regulator of cellular ODC level and polyamine transport. The elevation of cellular polyamine concentration stimulates a programmed +1 frameshift during translation of the AZ messenger RNA (mRNA), and allows the expression of functional full-length AZ. AZ binds and inactivates ODC by forming AZ-ODC heterodimer, and promotes an ubiquitin-independent degradation by the 26S proteasome, which consequently reduces polyamine synthesis. Mammalian cells contain another relevant regulatory protein termed antizyme inhibitor (AZI), which is homolous to ODC but lacks decarboxylating activity. AZI has a higher affinity for AZ than does ODC, therefore ODC may released from AZ in the presence of AZI and restores the ODC activity. While AZ promotes protolytic destruction of ODC, the degradation of AZI is ubiquitin-dependent and does not require interaction with AZ. Moreover, AZ binding actually stabilizes AZI by inhibiting its ubiquitination. Besides controlling cellular polyamines homeostasis, it has been shown that AZI may regulate the replication of centriole and participate in cell division. To understand how AZ interacts with AZI and to explain how AZ inhibits the poly-ubiqitination of AZI, this study is aimed to determine the structure of AZ-AZI complex. A number of crystallization conditions have been indentified from which crystals of AZ-AZI complex can be obtained. However, these crystals only diffracted X-ray to ~ 7Å resolution. Preliminary analysis indicated that AZ-AZI crystals belongs to space group P3, with unit-cell parameters a = b = 163.11 Å, c = 149.69 Å, α= β= 90°, γ =120°. Additive screen、crystal annealing、optimal buffer searching and in situ proteolysis had been performed to improve crystal quality. However, a useful crystallization condition for AZ-AZI complex remains to be identified.