Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2

Abstract

Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect on ODC. Whereas ODC and AZIN1 are well-characterized proteins, little is known on the structure and stability of AZIN2, the lastly discovered member of this regulatory circuit. In this work we first analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified conserved residues in the antizyme-binding element, whose substitution drastically affected the capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the proteasome inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that of proteasome. These findings provide new relevant information on this unique regulatory mechanism of polyamine metabolism.

Keywords: AZ, antizyme; AZBE, antizyme-binding element; AZIN, antizyme inhibitor; Antizyme; Antizyme-binding element; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; GDT_TS, global distance test total score; HA, hemagglutinin; HEK, human embryonic kidney; Homology modeling; ODC, ornithine decarboxylase; PAGE, polyacrylamide gel electrophoresis; Polyamines; Proteasome inhibitors; Protein degradation; RMSD, root-mean-square deviation; TGN, trans-Golgi network.

Fig. 1

Biochemical studies of the AZIN2 quaternary structure in transfected cells. (A) Left panel: Cross-linking analysis of transfected cell lysates of AZIN2 and ODC. HEK 293T cells were transiently transfected with AZIN2-FLAG, ODC-FLAG, or co-transfected with AZIN2-FLAG and AZ1, and the cell lysates were incubated with 1 mM bissulfosuccinimidylsuberate (BS3) for 1 h. The proteins were then separated by SDS-PAGE, transferred onto PVDF membrane, which was then incubated with an anti-FLAG antibody. Note that the band corresponding to AZIN2 monomers disappears after cross-linking. Right panel (marked with an asterisk): AZIN2 blot similar to that shown in the left panel, but using a longer exposure time to the detection reagent. Note that the tag is now found in high molecular weight bands. (B) Migration pattern of AZIN2-FLAG and ODC-FLAG under native conditions. Samples were analyzed by non-denaturing PAGE, blotted to PVDF and probed with an anti-FLAG antibody. (C) Size-exclusion chromatography of AZIN2-FLAG and ODC-FLAG. AZIN2-FLAG or ODC-FLAG cell lysates were resolved by a gel filtration column (Zorbax GF-250) and fraction aliquots were analyzed by Western blot or assayed for ODC activity. The arrowhead marks the elution fraction in which bovine serum albumin (BSA) was eluted.

Fig. 2

(A) AZIN2 is unable to form heterodimers with ODC. Cells were transfected with ODC-HA or co-transfected with ODC-HA and AZIN2-FLAG or ODC-FLAG. Samples from cell lysates were analyzed by Western blot using an anti-HA antibody. In addition, samples were inmunoprecipitated (IP) with anti-FLAG affinity gel beads for 3 h. After washing, the eluted proteins were subjected to Western blot analysis and incubation with an anti-HA antibody (lower row). ODC-FLAG but not AZIN2-FLAG interacted with ODC-HA. The blots shown are representative of three experiments. (B) ODC activity in cells transfected with ODC alone or in presence of AZIN2.

Fig. 3

Structural model of mouse AZIN2. The upper row presents the predicted model in the ribbon (A) and the surface (B) representation, colored according to the predicted local deviation from the real structure (i.e., the predicted error of the model), as calculated by MetaMQAPII: blue indicates low predicted deviation of Cα atoms down to 0 Å, red indicates unreliable regions with deviation >5 Å, green indicates intermediate values. (C) Predicted tertiary structure of AZIN2 in the ribbon representation with the AZBE region in yellow and the N terminus region formed by the 70–110 residues in red. The side chains of the amino acids included in the AZBE region (111–145) were also shown and colored according to the type of atom (red, oxygen; blue, nitrogen).

Fig. 4

Conserved residues of the putative AZBE region of mouse AZIN2. (A) Region encompassed by residues 117–143 of mouse AZIN2, showing the invariant residues (shown in blue) found by multiple alignment of the AZBE amino acid sequences from different AZIN2 orthologues and paralogues (ODC and AZIN1). See Ref. . (B) Scheme of the different mutations in AZIN2 affecting the conserved residues of the AZBE region, and the electric charge of this region in the different AZIN2 mutants.

Fig. 5

Mutations of certain conserved residues of the AZBE region negatively affect the interaction of AZIN2 with AZ1. (A) HEK 293T cells were transfected with wild type AZIN2-FLAG (AZIN2 Wt), or with variants with single substitutions (K116A and K142A), double substitutions (KK/AA), triple substitutions (ELK/AAA) or with the AZBE-deleted protein (DelAZBE), together with AZ1-HA. (B) Cells were transfected with AZIN2 Wt or variants with single substitutions (A124S, E139A and L140A), double substitutions (KK/AA and EL/AA), and triple substitutions (ELK/AAA), together with AZ1-HA. The cell lysates were immunoprecipitated with anti-FLAG affinity gel beads for 3 h. After washing, the eluted proteins (IP) were separated by electrophoresis and Western blot analysis was performed. AZ1-HA was detected using an anti-HA antibody.

Fig. 6

AZIN2 variants were less effective than unaltered AZIN2 in regulating the polyamine homeostasis. (A) Influence of AZIN2 and its variants on the endogenous ODC activity of HEK 293T cells. The cells were transiently transfected with AZIN2 (WT) or with some AZIN2 variants. Twenty-four hours after transfection ODC activity was assayed in the cell extracts as indicated in Section 4. Control cells were transfected with the empty vector. (B) Influence of AZIN2 and its variants on ODC activity in cells co-transfected with ODC and AZ1. ODC and AZIN2 protein levels were analyzed by Western blot. The molar ratio of the different constructs (ODC-FLAG/AZIN2-FLAG/AZ) was 10:10:1. (C) Putrescine uptake by COS7 cells transfected with AZ3 or co-transfected with AZ3 and AZIN2 variants. Control cells were transfected with the empty vector. Data are expressed as mean ± S.E. of triplicate determinations.

Fig. 7

Half-lives of AZIN2 and its paralogues ODC and AZIN1 in HEK 293T cells. (A) Cells were transfected with AZIN2, AZIN1 or ODC tagged with FLAG. Twenty-four hours after transfection, cycloheximide (100 μM) was added, the cells harvested at the indicated times and ODC levels were detected by measuring the enzymatic activity, whereas AZIN1 and AZIN2 protein levels were determined by Western blot analysis using an anti-FLAG antibody. (B) Influence of the AZs in the half-life of AZIN2. Cells were transfected with AZIN2-FLAG alone or co-transfected with AZIN2-FLAG and each of the three AZ isoforms. Transfected cells were treated as indicated above and protein levels were determined by Western blot analysis and incubation with anti-FLAG antibody as shown in (C). The indicated values of % degradation represent a mean value of three repetitions.

Fig. 8

AZIN2 expression increases the stability of the three antizymes in HEK293 cells. (A) Cells were transiently transfected with each AZ-HA alone, or together with wild type AZIN2 or AZIN2 with deletion of the AZBE region (AZIN2ΔAZBE). Twenty-four h after transfection, cells were lysed and AZ-HA levels were determined by Western blot analysis, using an anti-HA antibody. (B) ODC stabilizes AZ2 and AZ3 but promotes AZ1 degradation. HEK293 cells were transfected with each AZ-HA alone, or together with ODC or AZIN2. AZ expression was analyzed as in A. (C) Differential effect of ODC and C-terminal truncated ODC (ΔCtODC) on AZ1 stability in 293 cells. Cells were transiently transfected with each AZ-HA alone, or together with ODC, ΔCtODC or AZIN2. Twenty hours after transfection, cells were incubated with cycloheximide (100 μM) for 90 min, and after cell lysis AZ1-HA levels were analyzed by Western blotting, using an anti-HA antibody. Results are representative of three separate experiments.

Fig. 9

Effect of the administration of the proteasome inhibitor MG132 on the protein levels of AZIN2 and its paralogues. (A) HEK 293T cells transiently transfected with ODC-FLAG, AZIN1-FLAG or AZIN2-FLAG for 20 h were treated with 100 μM cycloheximide alone or in combination with 50 μM MG132 for additional 4 h. Protein levels were determined by Western blotting and incubation with anti-FLAG antibody. Loading controls were performed using anti-Actin antibody. (B) Twenty hours after transfection of the 293 cells with AZIN2-FLAG, cells were incubated with different doses of MG132 dissolved in DMSO or DMSO alone for additional 4 h, and the expression of AZIN2 was analyzed as described in B. (C) HEK 293T cells transfected with AZIN2-FLAG were incubated for 5 h with no inhibitor or with the proteasomal inhibitor MG132 (50 μM) alone or in combination with inhibitors of lysosomal degradation (ammonium chloride 50 mM, chloroquine 200 μM). (D) HEK 293T cells transfected with AZIN2-FLAG were incubated for 5 h with ammonium chloride (50 mM) or MG132 (50 μM) alone, and with a combination of both compounds. AZIN2 protein levels were assayed as in B. Actin was determined as a loading control. Results shown are representative of at least three separate experiments.

Fig. 10

Visualization of key residues of the AZBE site in the 3D structure of mouse ODC and AZIN2. The figure shows the residues found within a distance of 4 Å from residues L139 and L140 in ODC (blue) and AZIN2 (orange), respectively. This image was produced using UCSF Chimera from the data available in Protein Data Bank for mouse ODC (PDB: 7ODC) and the final model of AZIN2 generated by comparative modeling. Residues were colored according to the type of atom (red, oxygen; blue, nitrogen; yellow, sulfur).