title: 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.
authors: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.
authors: Bruno
authors: B
authors: Ramos-Molina
authors: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.
authors: Ana
authors: A
authors: Lambertos
authors: Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.
authors: Andrés J
authors: AJ
authors: Lopez-Contreras
authors: Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland.
authors: Joanna M
authors: JM
authors: Kasprzak
authors: Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland.
authors: Anna
authors: A
authors: Czerwoniec
authors: International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors: Janusz M
authors: JM
authors: Bujnicki
authors: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.
authors: Asunción
authors: A
authors: Cremades
authors: Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain ; Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain.
authors: Rafael
authors: R
authors: Peñafiel
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