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Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2.

Ramos-Molina B, Lambertos A, Lopez-Contreras AJ, Kasprzak JM, Czerwoniec A, Bujnicki JM, Cremades A, Peñafiel R - FEBS Open Bio (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.

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.

No MeSH data available.


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.
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f0035: 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.

Mentions: To determine the half-life of AZIN2 and to compare this with those of ODC and AZIN1, we transiently transfected HEK 293T cells with constructs encoding the FLAG-tagged tested proteins, and after transfection the cells were incubated with cycloheximide at different times. Fig. 7A shows that under overexpression conditions the half-life of AZIN2 (∼90 min) was much lower than that of ODC (>8 h), but significantly higher than that of AZIN1 (∼40 min). Note that transfected ODC was quite stable in contrast to the short half-life (lower than 30 min) found for the endogenous ODC of HEK 293T cells (data not shown), probably due to the marked differences in the ODC/AZ ratio between transfected and non-transfected cells. In addition, we analyzed the effect of AZs on AZIN2 stability by means of the co-transfection of HEK 293T cells with AZIN2-FLAG and each of the three different AZs. Fig. 7B shows that the degradation of AZIN2 was reduced by the presence of any of the three AZ isoforms, but the protective effect was higher in the case of AZ1 and AZ2 (t1/2=543 ± 93, 492 ± 99 and 155 ± 10 min in presence of AZ1, AZ2 and AZ3, respectively). Accordingly, the AZIN2 steady state levels were always higher in presence of the AZs (Fig. 7C), which is in agreement with previous works [37,38]. Although it is known that AZs are able to bind and regulate ODC [18,24], and as shown here AZs stabilize AZIN2, little is known on the effect of AZ-binding proteins on AZs stability. Fig. 8A shows that the co-expression of AZIN2 with each of the three AZs, markedly increased the steady state levels of AZs in the transfected cells, and that such stabilization of AZs was not observed when the cells were transfected with AZBE-deleted AZIN2, a mutated form of AZIN2, that, as shown in Fig 5A, is unable to bind to AZs. These results suggest that AZs and AZIN2 mutually stabilize each other by the formation of an AZ-AZIN2 complex. Similarly to AZIN2, ODC stabilized AZ2 and AZ3 but, however, the effect on AZ1 was different since ODC elicited the decrease of AZ1 protein. This discrepancy appears to be related with the degradation of ODC by the proteasome, since the co-transfection of AZ1 with the truncated form of ODC, lacking the 21 residues of the C-terminal region (ΔCt-ODC), a stable form of ODC [9], protected AZ1 from degradation as efficiently as AZIN2 (Fig. 8C).


Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2.

Ramos-Molina B, Lambertos A, Lopez-Contreras AJ, Kasprzak JM, Czerwoniec A, Bujnicki JM, Cremades A, Peñafiel R - FEBS Open Bio (2014)

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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0035: 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.
Mentions: To determine the half-life of AZIN2 and to compare this with those of ODC and AZIN1, we transiently transfected HEK 293T cells with constructs encoding the FLAG-tagged tested proteins, and after transfection the cells were incubated with cycloheximide at different times. Fig. 7A shows that under overexpression conditions the half-life of AZIN2 (∼90 min) was much lower than that of ODC (>8 h), but significantly higher than that of AZIN1 (∼40 min). Note that transfected ODC was quite stable in contrast to the short half-life (lower than 30 min) found for the endogenous ODC of HEK 293T cells (data not shown), probably due to the marked differences in the ODC/AZ ratio between transfected and non-transfected cells. In addition, we analyzed the effect of AZs on AZIN2 stability by means of the co-transfection of HEK 293T cells with AZIN2-FLAG and each of the three different AZs. Fig. 7B shows that the degradation of AZIN2 was reduced by the presence of any of the three AZ isoforms, but the protective effect was higher in the case of AZ1 and AZ2 (t1/2=543 ± 93, 492 ± 99 and 155 ± 10 min in presence of AZ1, AZ2 and AZ3, respectively). Accordingly, the AZIN2 steady state levels were always higher in presence of the AZs (Fig. 7C), which is in agreement with previous works [37,38]. Although it is known that AZs are able to bind and regulate ODC [18,24], and as shown here AZs stabilize AZIN2, little is known on the effect of AZ-binding proteins on AZs stability. Fig. 8A shows that the co-expression of AZIN2 with each of the three AZs, markedly increased the steady state levels of AZs in the transfected cells, and that such stabilization of AZs was not observed when the cells were transfected with AZBE-deleted AZIN2, a mutated form of AZIN2, that, as shown in Fig 5A, is unable to bind to AZs. These results suggest that AZs and AZIN2 mutually stabilize each other by the formation of an AZ-AZIN2 complex. Similarly to AZIN2, ODC stabilized AZ2 and AZ3 but, however, the effect on AZ1 was different since ODC elicited the decrease of AZ1 protein. This discrepancy appears to be related with the degradation of ODC by the proteasome, since the co-transfection of AZ1 with the truncated form of ODC, lacking the 21 residues of the C-terminal region (ΔCt-ODC), a stable form of ODC [9], protected AZ1 from degradation as efficiently as AZIN2 (Fig. 8C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.

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.

No MeSH data available.