Structural and Degradative Aspects of Ornithine Decarboxylase Antizyme Inhibitor 2
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FOB 199 No. of Pages 12, Model 5G 6 June 2014 FEBS Open Bio xxx (2014) xxx–xxx 1 journal homepage: www.elsevier.com/locate/febsopenbio 5 6 3 Structural and degradative aspects of ornithine decarboxylase 4 antizyme inhibitor 2 a a b c 7 Q1 Bruno Ramos-Molina , Ana Lambertos , Andrés J. Lopez-Contreras , Joanna M. Kasprzak , c d a a,⇑ 8 Anna Czerwoniec , Janusz M. Bujnicki , Asunción Cremades , Rafael Peñafiel 9 a Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain 10 b Genomic Instability Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 11 c Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland 12 Q2 d International Institute of Molecular and Cell Biology, Warsaw, Poland 13 14 article info abstract 3116 17 Article history: Ornithine decarboxylase (ODC) is the key enzyme in the polyamine biosynthetic pathway. ODC levels 32 18 Received 13 May 2014 are controlled by polyamines through the induction of antizymes (AZs), small proteins that inhibit 33 19 Revised 23 May 2014 ODC and target it to proteasomal degradation without ubiquitination. Antizyme inhibitors (AZIN1 34 20 Accepted 27 May 2014 and AZIN2) are proteins homologous to ODC that bind to AZs and counteract their negative effect 35 21 Available online xxxx 36 22 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 37 analyzed structural aspects of AZIN2 by combining biochemical and computational approaches. We 38 23 Keywords: demonstrated that AZIN2, in contrast to ODC, does not form homodimers, although the predicted 39 24 Antizyme 25 Antizyme-binding element tertiary structure of the AZIN2 monomer was similar to that of ODC. Furthermore, we identified 40 26 Homology modeling conserved residues in the antizyme-binding element, whose substitution drastically affected the 41 27 Polyamines capacity of AZIN2 to bind AZ1. On the other hand, we also found that AZIN2 is much more labile than 42 28 Protein degradation ODC, but it is highly stabilized by its binding to AZs. Interestingly, the administration of the protea- 43 29 Proteasome inhibitors some inhibitor MG132 caused differential effects on the three AZ-binding proteins, having no effect 44 30 on ODC, preventing the degradation of AZIN1, but unexpectedly increasing the degradation of 45 AZIN2. Inhibitors of the lysosomal function partially prevented the effect of MG132 on AZIN2. These 46 results suggest that the degradation of AZIN2 could be also mediated by an alternative route to that 47 of proteasome. These findings provide new relevant information on this unique regulatory mecha- 48 nism of polyamine metabolism. 49 Ó 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This 50 is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 51 52 53 54 55 1. Introduction Ornithine decarboxylase (ODC) is the key biosynthetic enzyme that 61 converts ornithine into putrescine, which is the precursor for the 62 56 Polyamines are small cationic molecules essential for cell physiological polyamines, spermidine and spermine. Different 63 57 growth, proliferation, differentiation and apoptosis [1–4].In studies have revealed that ODC and polyamines play an important 64 58 mammals, the intracellular polyamine levels are tightly controlled role in the development of cancer [6–8]. ODC activity is highly 65 59 by the regulation of different processes including their biosynthe- dependent on the intracellular polyamine concentrations, being 66 60 sis, degradation and transport across the plasma membrane [5]. rapidly down regulated under high polyamine amounts, acting at 67 transcriptional, translational and post-translational levels [9]. 68 Post-translational regulation of ODC is mainly mediated by the 69 Abbreviations: AZ, antizyme; AZBE, antizyme-binding element; AZIN, antizyme 70 inhibitor; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; ODC, action of a family of small proteins named antizymes (AZs), whose ornithine decarboxylase; GDT_TS, global distance test total score; HA, hemagglu- synthesis is stimulated by increased polyamine levels [10]. AZs 71 tinin; HEK, human embryonic kidney; PAGE, polyacrylamide gel electrophoresis; bind and inhibit ODC and target it to proteasomal degradation 72 RMSD, root-mean-square deviation; TGN, trans-Golgi network without ubiquitination [11,12]. Besides, AZs inhibit extracellular 73 ⇑ Corresponding author. Address: Department of Biochemistry and Molecular polyamine uptake presumably by interacting with the polyamine 74 Biology B and Immunology, Faculty of Medicine, University of Murcia, 30100 Murcia, Spain. Tel.: +34 868 88 7174; fax: +34 868 88 4150. transport system [13–15]. In mammals, the family of AZs is com- 75 E-mail address: [email protected] (R. Peñafiel). posed by three isoforms (AZ1-3), AZ1 being the most predominant 76 http://dx.doi.org/10.1016/j.fob.2014.05.004 2211-5463/Ó 2014 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Please cite this article in press as: Ramos-Molina, B., et al. Structural and degradative aspects of ornithine decarboxylase antizyme inhibitor 2. FEBS Open Bio (2014), http://dx.doi.org/10.1016/j.fob.2014.05.004 FOB 199 No. of Pages 12, Model 5G 6 June 2014 2 B. Ramos-Molina et al. / FEBS Open Bio xxx (2014) xxx–xxx 77 form in most cells and tissues [16,17]. Despite the fact that all AZ ODC-FLAG under the same experimental conditions. Cross-linking 139 78 isoforms are able to inhibit ODC and the polyamine uptake, only analysis clearly showed, as expected, the presence of ODC dimers 140 79 AZ1 clearly induces ODC proteasomal degradation [11,18]. In fact, (Fig. 1A). In the case of AZIN2, the putative dimer band was not 141 80 there are controversial data on the capacity of AZ2 for targeting detected. However, after the cross-linking reaction the monomer 142 81 ODC to degradation in vivo and in vitro [19–21], and recently it band almost disappeared, detecting faint staining of bands 143 82 has been reported that AZ3, a testis-specific isoform [22,23],is corresponding to higher molecular weight than the putative dimer 144 83 unable to target ODC to degradation [21]. (Fig. 1A). Since AZIN2 is located in the endoplasmic reticulum- 145 84 In addition to AZs, the ODC activity is also indirectly regulated Golgi intermediate compartment (ERGIC) and in the trans-Golgi 146 85 by a family of proteins named antizyme inhibitors (AZINs). These network (TGN) [39,51], these higher molecular weight bands 147 86 proteins, closely related to ODC but without enzymatic activity, may correspond to cross-linked species of the AZIN2 monomer 148 87 also interact with antizymes, even more efficiently than ODC, with membrane proteins of these compartments, to which AZIN2 149 88 counteracting the effects of antizymes on ODC [24,25]. In mam- is likely to be associated. Interestingly, when AZIN2 was co-trans- 150 89 mals, the AZIN family is formed by two members: AZIN1 and fected with AZ1, after cross-linking experiments the main band of 151 90 AZIN2. AZIN1 is a ubiquitous protein that regulates intracellular AZIN2 detected was mainly the monomer, probably due to the fact 152 91 polyamine levels and cell growth [26–29]. Mice with disruption that AZ1 prevents the binding of AZIN2 to proteins of the outer sur- 153 92 of the AZIN1 gene die soon after birth [30]. On the other hand, face of the ERGIC or the Golgi apparatus, in agreement with previ- 154 93 AZIN2 was first found to be expressed in testis and brain [31,32], ous data [39,51], and therefore, the formation of the cross-linked 155 94 but more recent analyses have indicated that it is also expressed species of higher molecular weight. To corroborate the monomeric 156 95 in specific type of cells and secretory tissues [33–36]. Although state of AZIN2 under physiological conditions, we next studied cell 157 96 the physiological role of AZIN2 is mostly unknown, there are data extracts of transfected cells with AZIN2 (or ODC), using polyacryl- 158 97 suggesting that it is involved in cell growth, vesicular trafficking, amide gel electrophoresis (PAGE) under non-denaturing condi- 159 98 secretion and spermiogenesis [36–39]. Interestingly, overexpres- tions. Fig. 1B shows that whereas in the case of ODC two bands 160 99 sion of AZIN2 has been observed in certain pathologies [40]. were found, corresponding to monomeric and dimeric forms of 161 100 Numerous studies have demonstrated that ODC is enzymati- ODC, only the lower molecular weight band corresponding to the 162 101 cally active as homodimer [9,41,42], and that in solution there is monomer was detected for AZIN2. Finally, size exclusion chroma- 163 102 an equilibrium between the monomeric inactive form and the tography was used to analyze the size of transfected AZIN2 and 164 103 dimeric one [41,43]. It is also well known that antizyme exerts 104 its negative effect on ODC activity by binding to the ODC monomer, 105 preventing the formation of the active ODC dimer, and by targeting (A) 106 the inactive ODC to the 26S proteasome, where ODC is degraded 107 without ubiquitination [11]. Initial studies based on the differential 108 affinity for AZ between murine and trypanosomal ODC, led to the 109 identification of the antizyme-binding element (AZBE), as a 110 sequence spanning the residues from 117 to 140 in mouse ODC 111 [44]. Subsequently, the analysis of the tertiary structure of ODC 112 revealed that this region is present in two a-helices included in a 113 TIM-like a/b-barrel domain, in which several basic residues are 114 exposed toward the surface [45,46].