Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions

Abstract

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2–polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone—2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2–polyamine complex formation. Thus, the (OAZ1)2–polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds—2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells

“…The activation of polyamine catabolism decreased levels of OAZ1 and increased levels of AZIN1, presumably to allow the induction of the biosynthetic pathway. One should note that regulation by OAZ1 is likely to be regulated in post-transcriptional step, as functional antizyme is produced upon a polyamine-dependent frameshift during the translation of its mRNA [ 50 ] and, probably, during its polyamine-dependent dimerization [ 51 ]. In Huh7.5 cells, the mRNA of the agmatinase (AGMAT) was also detected, which was not observed in the non-tumor HepaRG line, as was revealed through both RNAseq and RT-qPCR analysis ( Figure 4 a,d).…”

C-Methylated Spermidine Derivatives: Convenient Syntheses and Antizyme-Related Effects

“…The association constant (K a ) for the complex of Spm with mOAZ1-6xHis was about three times lower than Spd. At the same time, the thermodynamic parameters of complex formation (ΔH and TΔS ratio) for these two polyamines were similar; this suggests that their interaction with mOAZ1 was of the same nature [ 31 ]. Surprisingly, among 1-MeSpd, 2-MeSpd, and 3-MeSpd, only 1-MeSpd and 3-MeSpd induced mOAZ1-6xHis dimerization like Spd, whereas 2-MeSpd did not.…”

“…Polyamines may stabilize the stem-loop structure of mRNA starting after the stop codon, and these interactions may slow down the elongation speed and allow ribosomes to pass through the stop codon [ 52 ]. Recently, using RNA melting temperature analysis, we compared the ability of 1-MeSpd, 2-MeSpd, and 3-MeSpd to stabilize the structure of a model 72-mer 2′-O-Me-oligoribonucleotide containing the +1 frameshifting site, hairpin, and pseudoknot of the mOAZ1 mRNA and demonstrated that 2-MeSpd is a slightly worse stabilizer than Spd, 1-MeSpd, and 3-MeSpd [ 31 ]. This correlates with a poor induction of OAZ1 synthesis (Western blot analysis) in DU145 cells grown in the presence of 2-MeSpd [ 31 ].…”

“…Recently, using RNA melting temperature analysis, we compared the ability of 1-MeSpd, 2-MeSpd, and 3-MeSpd to stabilize the structure of a model 72-mer 2′-O-Me-oligoribonucleotide containing the +1 frameshifting site, hairpin, and pseudoknot of the mOAZ1 mRNA and demonstrated that 2-MeSpd is a slightly worse stabilizer than Spd, 1-MeSpd, and 3-MeSpd [ 31 ]. This correlates with a poor induction of OAZ1 synthesis (Western blot analysis) in DU145 cells grown in the presence of 2-MeSpd [ 31 ]. It may be assumed that the second position of the Spd molecule is essential for the stabilization of the hairpin structure of OAZ1 mRNA; indeed, among the studied C -methylated Spd analogs, 2,2-Me 2 Spd turned out to be the worst stabilizer, similar to 2-MeSpd ( Table 2 ).…”

“…Recently, we discovered that a full-length mouse OAZ1 can dimerize in the presence of polyamines and/or Spd C -methylated analogs; in the latter case, dimerization efficiency depends on the methyl group position. Moreover, it was shown that the dimerization potential demonstrated by 2-MeSpd was almost negligible [ 31 ]. Changing the stereo configuration of the chiral center(s) of C -methylated polyamine analogs gives an opportunity to precisely regulate the productivity of their interaction with enzymes and cells.…”