Minimal Antizyme Peptide Fully Functioning in the Binding and Inhibition of Ornithine Decarboxylase and Antizyme Inhibitor

Ju-Yi Hsieh,Hui-Chih Hung,Guang-Yaw Liu,Jung-Yen Yang,Chih-Li Lin

doi:10.1371/journal.pone.0024366

Ju-Yi Hsieh, Hui-Chih Hung + Show 3 more

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https://doi.org/10.1371/journal.pone.0024366

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Abstract

Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with K d,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.

Highlights

Polyamines such as putrescine, spermidine, and spermine can bind to DNA, RNA, and proteins to control a variety of physiological functions including DNA replication, gene regulation, cell cycling, apoptosis, post-translational modification and protein synthesis [1,2,3]
We first examined the inhibitory effect of these AZ peptides toward ornithine decarboxylase (ODC) enzyme activity (Figure 2) and calculated the IC50 values for these AZ peptides (Table 1)
We are the first group using biochemical and biophysical methods to demonstrate a minimal fully functional human AZ peptide, AZ_95-176, which is sufficient for both the inhibition of ODC enzyme activity and the binding of Antizyme inhibitor (AZI)

Summary

Introduction

Polyamines such as putrescine, spermidine, and spermine can bind to DNA, RNA, and proteins to control a variety of physiological functions including DNA replication, gene regulation, cell cycling, apoptosis, post-translational modification and protein synthesis [1,2,3]. Elevated polyamine levels cause cell growth and differentiation in eukaryotes and may be highly associated with the development of cancer [2,4,5,6]. Due to the fact that elevated ODC activity increases polyamine levels in cells and that overexpression of ODC is associated with neoplastic transformation of cells [9,10,11,12], ODC is considered an oncogenic enzyme [7]. Inhibitors of ODC and the polyamine synthesis pathway have excellent therapeutic potential for many cancers [2,5,12,13]

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