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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.


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.
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f0045: 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.

Mentions: It is known that AZINs are degraded in a ubiquitin-dependent manner by the proteasome [37,48,52], in contrast to ODC that is degraded by the proteasome without ubiquitination [11]. To obtain further information about possible differences in the degradative processes of these proteins, we incubated HEK 293T cells, transfected with the different paralogue constructs, with cycloheximide in absence and presence of the proteasome inhibitor MG132. Fig. 9A shows that the protein levels of AZIN1 and AZIN2 decreased after treatment with cycloheximide, as expected, due to the high turnover of these two proteins. However, the co-administration of cycloheximide with the proteasomal inhibitor MG132 caused differential effects on AZIN protein levels. Thus, whereas AZIN1 was not decreased under this condition, due to the inhibitory effect of MG132 on degradation, AZIN2 was still degraded. This unexpected effect of MG132 on AZIN2 was corroborated by incubating AZIN2-transfected cells with MG132 alone (Fig. 9B). To determine whether the down-regulation of AZIN2 by the proteasome inhibitor could be mediated by channeling AZIN2 to an alternative degradative route to that of the proteasome 26S, we performed a set of experiments in which the AZIN2-transfected cells were incubated simultaneously with MG132 and different inhibitors of lysosomal function, such as ammonium chloride and chloroquine. As shown in Fig. 9C, the inhibition of the lysosomal degradation pathway by ammonium chloride or chloroquine partially prevented the degradation of AZIN2.


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)

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.
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Related In: Results  -  Collection

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f0045: 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.
Mentions: It is known that AZINs are degraded in a ubiquitin-dependent manner by the proteasome [37,48,52], in contrast to ODC that is degraded by the proteasome without ubiquitination [11]. To obtain further information about possible differences in the degradative processes of these proteins, we incubated HEK 293T cells, transfected with the different paralogue constructs, with cycloheximide in absence and presence of the proteasome inhibitor MG132. Fig. 9A shows that the protein levels of AZIN1 and AZIN2 decreased after treatment with cycloheximide, as expected, due to the high turnover of these two proteins. However, the co-administration of cycloheximide with the proteasomal inhibitor MG132 caused differential effects on AZIN protein levels. Thus, whereas AZIN1 was not decreased under this condition, due to the inhibitory effect of MG132 on degradation, AZIN2 was still degraded. This unexpected effect of MG132 on AZIN2 was corroborated by incubating AZIN2-transfected cells with MG132 alone (Fig. 9B). To determine whether the down-regulation of AZIN2 by the proteasome inhibitor could be mediated by channeling AZIN2 to an alternative degradative route to that of the proteasome 26S, we performed a set of experiments in which the AZIN2-transfected cells were incubated simultaneously with MG132 and different inhibitors of lysosomal function, such as ammonium chloride and chloroquine. As shown in Fig. 9C, the inhibition of the lysosomal degradation pathway by ammonium chloride or chloroquine partially prevented the degradation of AZIN2.

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.