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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.


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. [59]. (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.
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f0020: 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. [59]. (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.

Mentions: Although we initially reported that AZIN2 interacts with the three antizymes [32], little is known on the molecular aspects of this interaction. In ODC, an important structural element for the binding to antizymes is the AZBE domain. This region was early identified by comparing trypanosomal ODC, an enzyme isoform that is not regulated by AZ, with mouse ODC [44]. The AZBE site spans from residues 117 to 140 of mouse ODC. In a previous work, we found that the deletion of this sequence abolished the capacity of AZIN2 to stimulate polyamine uptake, presumably because this variant does not interact with AZs [58]. In order to determine the residues of the AZBE region of AZIN2 implicated in the interaction with AZs, we considered the five conserved amino acid residues (K116, A124, E139, L140 and K142) of this region in mouse AZIN2 (Fig. 4A), previously deduced [59] by using a multi-alignment sequence analysis of the AZBE region of AZIN2 orthologues and those of corresponding paralogues provided by the ENSEMBL genome database. Given that several conserved acidic glutamate residues are present in the region of AZ1 necessary for the interaction with ODC [60] and that the putative AZBE site of ODC contains various basic residues, one might expect an electrostatic binding between AZ1 and ODC. In agreement with this hypothesis, a recent study has associated the electric charge of the AZBE site to the interaction ODC-AZ1 [61]. In the case of mouse AZIN2 the total charge of the putative AZBE site is +2, due to the presence of five basic (K116, K122, K126, R130 and K142) and three acidic residues (D135, E137 and E139) (see Fig. 4A). Note that two of the conserved residues are positively charged lysines (K116 and K142) that could interact electrostatically with the conserved glutamic residues of AZ1. To determine the influence of the electric charge on the interaction between AZIN2 and AZ, we first specifically generated several AZIN2 variants by substituting conserved lysine or glutamic residues by neutral uncharged alanines. Thus, we obtained several single, double and triple substitutions in which the electric charge was maintained (A124S; L140A; E139A/L140A/K142A), reduced (K116A; K142A; K116A/K142A) or increased (E139A; E139A/L140A) (see Fig. 4B).


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)

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. [59]. (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.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: 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. [59]. (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.
Mentions: Although we initially reported that AZIN2 interacts with the three antizymes [32], little is known on the molecular aspects of this interaction. In ODC, an important structural element for the binding to antizymes is the AZBE domain. This region was early identified by comparing trypanosomal ODC, an enzyme isoform that is not regulated by AZ, with mouse ODC [44]. The AZBE site spans from residues 117 to 140 of mouse ODC. In a previous work, we found that the deletion of this sequence abolished the capacity of AZIN2 to stimulate polyamine uptake, presumably because this variant does not interact with AZs [58]. In order to determine the residues of the AZBE region of AZIN2 implicated in the interaction with AZs, we considered the five conserved amino acid residues (K116, A124, E139, L140 and K142) of this region in mouse AZIN2 (Fig. 4A), previously deduced [59] by using a multi-alignment sequence analysis of the AZBE region of AZIN2 orthologues and those of corresponding paralogues provided by the ENSEMBL genome database. Given that several conserved acidic glutamate residues are present in the region of AZ1 necessary for the interaction with ODC [60] and that the putative AZBE site of ODC contains various basic residues, one might expect an electrostatic binding between AZ1 and ODC. In agreement with this hypothesis, a recent study has associated the electric charge of the AZBE site to the interaction ODC-AZ1 [61]. In the case of mouse AZIN2 the total charge of the putative AZBE site is +2, due to the presence of five basic (K116, K122, K126, R130 and K142) and three acidic residues (D135, E137 and E139) (see Fig. 4A). Note that two of the conserved residues are positively charged lysines (K116 and K142) that could interact electrostatically with the conserved glutamic residues of AZ1. To determine the influence of the electric charge on the interaction between AZIN2 and AZ, we first specifically generated several AZIN2 variants by substituting conserved lysine or glutamic residues by neutral uncharged alanines. Thus, we obtained several single, double and triple substitutions in which the electric charge was maintained (A124S; L140A; E139A/L140A/K142A), reduced (K116A; K142A; K116A/K142A) or increased (E139A; E139A/L140A) (see Fig. 4B).

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.