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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.


(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.
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f0010: (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.

Mentions: Although all these results pointed out for the inability of AZIN2 to form homodimers, in contrast to ODC, we wondered whether AZIN2 may form heterodimers with ODC. To answer this question we carried out immunoprecipitation assays using cells co-transfected with constructs of AZIN2 tagged with the FLAG epitope (AZIN2-FLAG) and ODC tagged either with the hemagglutinin epitope (ODC-HA) or the FLAG epitope (ODC-FLAG). Fig. 2A shows that in cells co-transfected with both ODC constructs, dimers formed by ODC-HA and ODC-FLAG monomers could be detected by Western blotting after co-immunoprecipitation. However, no evidence of heterodimer formation between AZIN2 and ODC was found in the cells co-transfected with AZIN2-FLAG and ODC-HA. In agreement with these results, ODC activity was not decreased by co-transfection with AZIN2 (Fig. 2B). Note that the formation of AZIN2-ODC heterodimers would have decreased the activity of ODC due to the lack of decarboxylating activity of AZIN2 [32,52].


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)

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

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Show All Figures
getmorefigures.php?uid=PMC4066113&req=5

f0010: (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.
Mentions: Although all these results pointed out for the inability of AZIN2 to form homodimers, in contrast to ODC, we wondered whether AZIN2 may form heterodimers with ODC. To answer this question we carried out immunoprecipitation assays using cells co-transfected with constructs of AZIN2 tagged with the FLAG epitope (AZIN2-FLAG) and ODC tagged either with the hemagglutinin epitope (ODC-HA) or the FLAG epitope (ODC-FLAG). Fig. 2A shows that in cells co-transfected with both ODC constructs, dimers formed by ODC-HA and ODC-FLAG monomers could be detected by Western blotting after co-immunoprecipitation. However, no evidence of heterodimer formation between AZIN2 and ODC was found in the cells co-transfected with AZIN2-FLAG and ODC-HA. In agreement with these results, ODC activity was not decreased by co-transfection with AZIN2 (Fig. 2B). Note that the formation of AZIN2-ODC heterodimers would have decreased the activity of ODC due to the lack of decarboxylating activity of AZIN2 [32,52].

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.