Structural studies of human antizyme isoforms in complex with its inhibitor

• Main Authors: Sheng Ou Yang, 歐陽陞
• Other Authors: Nei-Li Chan
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/88989425896937799978

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 104 === Abstract Polyamines are multivalent organic polycations ubiquitously present in eukaryotic cells. With their polycationic characteristics, polyamines can bind to proteins and nucleic acids via electrostatic interactions to modulate their structures and functions, in turn affecting cell growth and differentiation. However, abberant accumulation of polyamines is linked to tumorigenesis, thus the intracellular concentrations of polyamines are tightly regulated. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthesis pathway, can regulate the polyamines abundance in cells. Expression of full-length Az (Antizyme) is increased in response to high cellular polyamines levels through the polyamine-induced translational +1 frameshifting mechanism. Az can block the formation of catalytically active ODC homodimer by forming an Az-ODC heterodimer, which not only inhibits ODC enzymatic activity but also trigger ODC degradation via the 26s proteasome in an ubiquitin-independent manner. Furthermore, Az also inhibits the uptake of extracellular polyamine to reduce the cellular polyamide level. Therefore, Az is a negative regulator of cellular polyamines. In mammals, the Az family is consisted of three members (isoforms 1~3). Among them, isoform 1(Az1) and 2 (Az2) exhibit a similar tissue distribution and high sequence similarity. However, previous studies have shown that only Az1 can efficiently promote ODC degradation in vitro and Az2 most likely functions as a reservoir for transient and reversible suppression of ODC activity. In contrast, AzIN can effectively replenish ODC activity by competing with ODC for Az1 via the formation Az-AzIN heterodimer, which also prolongs the half-life of iv AzIN by preventing its ubiquitination. A recent study show that an mRNA-editing event mediated by the ADAR1 (Adenosine Deaminase Acting on RNA-1) on AzIN mRNA can promote HCC (Human Hepatocellular Carcinoma) by causing a serine-to-glycine substitution at residue 367 of AzIN. The mutated AzIN (AzINS367G) exhibits a higher affinity toward Az, producing a Az1-AzINS367G protein complex with enhanced stability. To understand the structural details regarding the formation of Az1-AzIN complex, our laboratory has determined the crystal structure of a truncated Az1 in complex with ODC and obtained a lower resolution 5.8 A crystal structure of Az1110-228-AzIN. The main objective of this work is to obtain crystal structures of Az-AzIN at higher resolution and elucidate the interactions between Az1 and AzIN in atomic detail. Toward this goal, two strategies were proposed: (1) obtaining a higher resolution crystal structure of Az2-AzIN by exploting the high similarity between Az1 and Az2 and (2) using the more stable of Az1-AzINS367G complex for structural analysis. We have constructed a series of recombinant expression vectors for producing Az2-AzIN and Az1-AzINS367G. The reconstituted complexes were purified by using immobilized metal affinity, ion exchange and gel filtration chromatography for crystallization trials. We have successfully identified a condition by which Az1119-228-AzINS367G can be crystallized. However, like the previous Az1110-228-AzIN crsytals produced previously, these crystals diffract only to low resolution. The attemps for crystallzing Az2-AzIN is not yet successful at this time. We will examine whether the diffraction quality of the Az1119-228-AzINS367G crystals can be further improved and continue to search for new crystallization conditions for Az2-AzIN in the future.