Minimal antizyme peptide fully functioning in the binding and inhibition of ornithine decarboxylase and antizyme inhibitor

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 (IC(50): 0.20 µM) similar to that of AZ-95-228 (IC(50): 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 IC(50) values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC(50) 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.

Figure 1. Homology model of human antizyme.

(A) A homology model of human AZ using the NMR structure of rat AZ as the template . (B) The secondary structure elements of human AZ from 95–228 consisting of two α-helices and eight β-strands. (C) A diagram of all truncated AZ peptides.

Figure 2. Relative ODC enzyme activity in the presence of AZ peptides.

The activity of ODC was inhibited by various concentrations of AZ peptides. ODC concentrations were fixed at 20 µg/mL (0.19 µM). The IC₅₀ values of AZ peptides shown in Table 1 were derived from fitting these inhibition curves. The molar ratio refers to AZ peptide versus ODC monomer.

Figure 3. Continuous sedimentation coefficient distribution of human ODC in the presence of AZ peptide.

The concentration of ODC was fixed at 0.3 mg/mL with concentrations of AZ ranging from 0.015 to 0.2 mg/mL (the molar ratio of AZ/ODC ranged from 0.12 to 2) in a buffer of 30 mM Tris-HCl (pH 7.4) and 25 mM NaCl at 20°C. The sedimentation velocity data were globally fitted with the SEDTHAT program to obtain K _d values for the AZ peptide-ODC complex (Table 2).

Figure 4. Relative ODC enzyme activities in the presence of AZ and AZI.

ODC enzymes equilibrated with equimolar AZI were inhibited by various concentrations of AZ peptides. The concentrations of AZI and ODC were fixed at 18.9 µg/mL and 20 µg/mL, respectively (the ratio of AZI monomer versus ODC monomer was fixed at 1). Panels A–H show the inhibition curves of ODC for each AZ peptide in the absence (closed circles) and in the presence (open circles) of AZI. Panel I displays the inhibition curves in the presence of AZI for different AZ peptides.

Figure 5. Docked structure of the mouse AZ-ODC complex.

The molecular docking structure of the mouse AZ-ODC complex demonstrates a heterodimer consisting of an ODC monomer and an AZ monomer. The putative AZ-binding site in ODC is colored yellow, and segment 95–176 of AZ is purple. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA, USA).