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Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

ABSTRACT: Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ95-228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the ?1 helix, ?5 and ?6 strands, and connecting loop between ?6 and ?2 (residues 142-178), which is the posterior part of AZ95-228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ95-228 (residues 120-145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

SUBMITTER: Hsieh JY

PROVIDER: S-EPMC6995573 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

Hsieh Ju-Yi JY Liu Yen-Chin YC Cheng I-Ting IT Lee Chu-Ju CJ Wang Yu-Hsuan YH Fang Yi-Shiuan YS Liu Yi-Liang YL Liu Guang-Yaw GY Hung Hui-Chih HC

Biomolecules 20191212 12

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ<sub>95-228</sub>) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified th ...[more]

PMID: 31842334

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Project description:Endotoxemia impairs hypoxic pulmonary vasoconstriction which leads to systemic hypoxemia. This derogation is attributable to increased activity of nitric oxide synthase 2 and arginase metabolism. Gene expression analysis has shown increased expression of ornithine decarboxylase in lungs of endotoxemic mice, a downstream enzyme of arginase metabolism. The aim of this study was to investigate whether inhibition of ornithine decarboxylase increases hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice. Mice received lipopolysaccharides or saline intraperitoneal, and hypoxic pulmonary vasoconstriction was measured using an isolated perfused mouse lung model. Additional mice with and without endotoxemia were pretreated with the ornithine decarboxylase-inhibitor difluoromethylornithine before examination of hypoxic pulmonary vasoconstriction. Hypoxic pulmonary vasoconstriction was defined as the difference of pulmonary arterial pressure between normoxic and hypoxic ventilation. In addition, lung tissue was analyzed using real-time quantitative polymerase chain reaction, Western blot and immunohistochemistry. Lipopolysaccharides caused an up-regulation of ornithine decarboxylase mRNA level (2.73 ± 0.19-fold increase, p < 0.05) as well as ornithine decarboxylase protein level (4.05 ± 0.37-fold increase, p < 0.05). Endotoxemia attenuated hypoxic pulmonary vasoconstriction when compared with untreated control mice (26.3 ± 9.7% vs. 67.0 ± 17.5%). Difluoromethylornithine (20, 100, 500 mg kg-1 body weight intraperitoneal) restored hypoxic pulmonary vasoconstriction in lungs of endotoxemic mice in a dose-dependent way (25.8 ± 9.9%, 57.3 ± 17.2%, 62.3 ± 12.4%) and decreased hypoxic pulmonary vasoconstriction in control mice (53.6 ± 13.6%, 40.0 ± 14.9%, 35.9 ± 12.4%). These results show that endotoxemia induces ornithine decarboxylase expression and suggest that ornithine decarboxylase contributes to the endotoxemia-induced impairment of hypoxic pulmonary vasoconstriction. Inhibition of ornithine decarboxylase might be a target in the therapy of diseases with inflammation impaired hypoxic pulmonary vasoconstriction, like the sepsis-associated acute respiratory distress syndrome (ARDS).

Project description:Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.

Project description:Repeated injections of 1,3-diaminopropane, a potent inhibitor of mammalian ornithine decarboxylase, induced protein-synthesis-dependent formation of macromolecular inhibitors or ;antienzymes' [Heller, Fong & Canellakis (1976) Proc. Natl. Acad. Sci. U.S.A.73, 1858-1862] to ornithine decarboxylase in normal rat liver. Addition of the macromolecular inhibitors, produced in response to repeated injections of diaminopropane, to active ornithine decarboxylase in vitro resulted in a profound loss of the enzyme activity, which, however, could be partly recovered after passage of the enzyme-inhibitor mixture through a Sephadex G-75 columin in the presence of 0.4m-NaCl. This treatment also resulted in the appearance of free inhibitor. In contrast with the separation of the enzyme and inhibitory activity after combination in vitro, it was not possible to re-activate, by using identical conditions of molecular sieving, any inhibited ornithine decarboxylase from cytosol fractions obtained from animals injected with diaminopropane. However, the idea that injection of various diamines, also in vivo, induces acute formation of macromolecular inhibitors, which reversibly combine with the enzyme, was supported by the finding that the ornithine decarboxylase activity remaining after diaminopropane injection appeared to be more stable to increased ionic strength than the enzyme activity obtained from somatotropin-treated rats. Incubation of the inhibitory cytosol fractions with antiserum to ornithine decarboxylase did not completely abolish the inhibitory action of either the cytosolic inhibitor or the antibody. A single injection of diaminopropane produced an extremely rapid decay of liver ornithine decarboxylase activity (half-life about 12min), which was comparable with, or swifter than, that induced by cycloheximide. However, although after cycloheximide treatment the amount of immunotitrable ornithine decarboxylase decreased only slightly more slowly than the enzyme activity, diaminopropane injection did not decrease the amount of the immunoreactive protein, but, on the contrary, invariably caused a marked increase in the apparent amount of antigen, after some lag period. The diamine-induced increase in the amount of the immunoreactive enzyme protein could be totally prevented by a simultaneous injection of cycloheximide. These results are in accord with the hypothesis that various diamines may result in rapid formation of macromolecular inhibitors to ornithine decarboxylase in vivo, which, after combination with the enzyme, abolish the catalytic activity but at the same time prevent the intracellular degradation of the enzyme protein.

Dataset Information

Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

Publications

Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase.

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