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Anti-Enzyme Inhibitor 1 is a protein encoded by the AZIN1 gene in humans. Antizyme inhibition factor-1 (AZIN1) is an important protein factor involved in the regulation of polyamine metabolism in cells. It can regulate cell proliferation through various pathways. High polyamine content is necessary for the rapid proliferation of normal cells and tumor cells, thus inhibiting the synthesis of cellular polyamines as a new strategy for anti-tumor therapy.
Figure 1. RNA-edited antizyme inhibitor 1 (AZIN1) interferes with the formation of the antizyme–ornithine decarboxylase (AZ–ODC) complex, contributing to cancer development. (Qiu S, et al. 2016)
Ornithine decarboxylase (ODC) is a key enzyme in the synthesis of polyamines, and ornithine decarboxylase antizyme (AZ) is a naturally occurring ODC inhibitor in cells, which can form AZ-ODC heterodimer with ODC, then targeted delivery of ODC to 26S proteasome degradation. Studies have found that there is also a protein inhibitor (AZI) that inhibits AZ in cells. It is homologous to ODC but lacks decarboxylase activity. AZIN1 is a member of the AZI family and widely distributed in tissues of cells throughout the body. It can compete with ODC to bind AZ and form a more stable AZIN1-AZ dimer, saving ODC from AZ-ODC dimer and recovering its catalytic activity. At the same time, the degradation rate of ODC slows down, increasing its intracellular levels to promote the synthesis of polyamines.
AZIN1 upregulates intracellular polyamine levels, and high polyamine levels inhibit AZIN1 expression at both the transcriptional and posttranscriptional levels, forming a feedback regulatory loop. Murakami et al. found that polyamines negatively regulate the transcription of AZIN1 gene. ODC inhibitor DFMO can also increase AZIN1 gene transcription while reducing intracellular polyamine levels. Moreover, treatment with putrescine or spermidine can inhibit AZIN1 transcription. In addition, polyamines can also control AZIN1 activity by affecting alternative splicing of AZIN1 precursor mRNA.
AZIN1 Regulates Cellular Polyamine Metabolism through RNA Editing
RNA editing is an important step in the post-transcriptional modification of some genes. The A→I editing of RNA is a process in which adenine (A) is converted to inosine nucleoside (I) by oxidative deamination under the catalysis of adenosine deaminase acting on RNA (ADAR). It has been found that the ADAR gene family includes at least three members, ADAR1, ADAR2, and ADAR3. Qin et al. found that ADAR1 is highly expressed in human esophageal squamous cell carcinoma and is closely related to poor prognosis. To investigate the effects of A→I editing on AZIN1 function, this team transfected wild-type AZIN1 (wt/AZIN1) and A→I edited AZIN1 (edt/AZIN1) into KYSE180 or EC109 esophageal cancer cells, respectively. AZIN1's function has a gain-of-function that allows cells to proliferate faster and migrate and erode more. Therefore, the A→I editing of AZIN1 may be an important factor in the occurrence of esophageal squamous cell carcinoma.
In a study of hepatocellular carcinoma, Chen et al.obtained similar conclusions. They found that AZIN1 in hepatoma cells has a high frequency A→I editing phenomenon, which is also catalyzed by ADAR1 and highly expressed in cancer cells, causing the 367th serine residue in the AZIN1 polypeptide chain to be converted into a glycine residue. The research team transfected AZIN1 (GFP-wt/AZIN1) or A→I -edited AZIN1 (GFPedt/AZIN1) into PLC8024 and QGY7703 hepatoma cells, respectively, and found that the ability of proliferation and clone formation in soft agar medium of GFP-edt/AZIN1 transfected cells was significantly increased. The functional gain analysis found that AZIN1 edited by A→I have a stronger binding ability to AZ, which can more effectively release ODC in AZ-ODC complex, increasing the content of polyamines in cells and promoting cell proliferation. Therefore, A→I editing can make AZIN1 have a stronger ability to promote the development of liver cancers.
AZIN1 Mediates Cellular Polyamine Metabolism via miRNA
The main function of AZIN1 is to relieve the inhibition of ODC by AZ, promoting polyamine synthesis and increasing the level of polyamines in cells. This function of AZIN1 is closely related to TGF-β-mediated renal fibrosis. Elevated levels of intracellular polyamines downregulate TGF-beta receptor expression and decrease TGF-beta/Smad3 signaling pathway activity, thereby inhibiting fibrosis. When TEC was treated with TGF-β, miR-433 expression was up-regulated but AZIN1 expression was decreased. At the same time, intracellular ODC activity and polyamine content were also significantly decreased, and TGF-β/Smad3 signaling pathway activity was increased to promote fibrosis. High expression of AZIN1 in cells reverses the above functions of TGF-β. These studies suggest that down-regulation of AZIN1 expression is an important basis for the development of renal fibrosis, while high expression of AZIN1 can prevent renal fibrosis by inhibiting the activity of TGF-β/Smad3 signaling pathway.
AZIN1 Interacts with Cyclin D1 to Regulate Cell Proliferation
AZIN1 also directly interacts with cyclins to regulate cell proliferation. Cyclin D1 is an important cyclin that promotes cell proliferation and is highly expressed in many tumors. It regulates the expression of genes involved in DNA replication and DNA damage checkpoints. The activation of these genes is required for the cell cycle to enter the S phase from the G1 phase, thereby exerting its function of promoting cell proliferation.
In vitro protein degradation experiments showed that under the conditions of wild-type AZIN1 protein or mutant AZIN1 protein, the stability of cyclin D1 increased and the degradation rate slowed down. The AZ, deletion mutant AZIN1 and cyclin D1 genes were transfected into HEK293 cells in different ways, and then co-immunoprecipitation analysis showed that the mutant AZINΔ117-140 could directly interact with cyclin D1 and form a complex. However, when AZ was highly expressed, the AZIN1-cyclin D1 complex disappeared, indicating that AZIN1 can form a protective complex with cyclin D1, but its affinity is lower than that of AZ-cyclin D1 complex. These studies suggest that high expression of AZ and/or inhibition of AZIN1 expression may inhibit tumor cell proliferation by down-regulating cyclin D.
AZIN1 Affects Cell Proliferation by Interfering with Centrosome Replication
The centrosome replication of the centrosome plays a key role in maintaining normal mitosis and genetic stability of the cell. Abnormal expansion of the centrosome or the appearance of aneuploidy is an important factor in tumorigenesis and development. Recent studies have found that both AZ and AZIN1 can be enriched in the centrosome and the changes in the content and ratio of the two in the centrosome have a significant impact on the structure of the centrosome. When AZ is highly expressed in cells, cells with abnormal number of centrosomes are significantly reduced. However, cells with high expression of AZIN1 significantly increase the number of cells with abnormal number of centrosomes. Down-regulation of AZIN1 expression alone can inhibit cell proliferation, and AZIN1 silencing inhibits centrosome replication as a result of cell cycle arrest. This study found that AZIN1-siRNA can reduce aberrant replication of centrosomes. Therefore, the effect of AZIN1 on centrosome replication is independent of the progression of the cell cycle. A large number of progeny central granules appeared in cells with high expression of AZIN1 but the parental central granules did not increase significantly, indicating that the increase in centrosome caused by AZIN1 was due to excessive replication of the progeny centrosome.