Enzyme Ornithine Decarboxylase Research Articles

Exogenous proline and proline-enriched Lolium perenne leaf extract protects against phytotoxic effects of nickel and salinity in Pisum sativum by altering polyamine metabolism in leaves

Pea plants (Pisum sativum L.) were exposed to NaCl and/or NiCl2 stress to investigate whether pure proline and Lolium perenne L. (LP) leaf extract could efficaciously protect against the phytotoxicity generated by nickel and/or salinity stress in pea. Both stress factors (salinity and nickel) significantly inhibited growth, total chlorophyll content, photosynthetic activity, stomatal conductance, intercellular CO2 level, relative water content (RWC), and membrane stability index (MSI). However, superoxide, hydrogen peroxide, proline, glycine betaine, total free amino acids, total soluble sugars, total phenolic compounds, tocopherol contents, lipid peroxidation, and electrolyte leakage were significantly increased. Similarly, free, soluble-conjugated, and insoluble-bound polyamine contents [spermine (Spm), spermidine (Spd), and putrescine (Put)] and activities of polyamine-biosynthetic enzymes (arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase) in leaves were also increased in response to nickel and/or salinity stress. Exogenously applied proline and LP leaf extracts significantly overcame the nickel- and/or salinity-induced toxic effects on growth, RWC, and various photosynthetic attributes. The follow-up treatment with proline and LP leaf extract detoxified the stress caused by NiCl2 and/or NaCl by suppressing the lipid peroxidation and electrolyte leakage, accelerating the activities of polyamine-biosynthetic enzymes, and improving the MSI, leaf polyamines (Spm, Spd, and Put), and organic osmolytes (free proline, glycine betaine, total free amino acids, total soluble sugars, total phenols, and tocopherol contents). Natural proline (LP leaf extract) proved better than pure proline in improving growth, photosynthetic activity, RWC, organic osmolytes, and polyamine metabolism. Since LP leaf extract is enriched with substantial amounts of proline along with many other essential nutrients, it was as efficacious as pure proline in improving growth, some major physiological attributes, and polyamine metabolism in pea under nickel and/or salinity stress. Thus, it can be used as an alternative inexpensive source of proline for its utilization as a mitigating agent for protecting plants against the phytotoxic effects of nickel and/or salinity stress.

Novel Interaction of Ornithine Decarboxylase with Sepiapterin Reductase Regulates Neuroblastoma Cell Proliferation

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein–protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC–SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan–Meier analysis (log-rank test, P=5×10−4), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.

Hairless and the polyamine putrescine form a negative regulatory loop in the epidermis

Hairless (HR) is a nuclear protein with corepressor activity that is highly expressed in the skin and hair follicle. Mutations in Hairless lead to hair loss accompanied by the appearance of papules (atrichia with papular lesions), and similar phenotypes appear when the key polyamine enzymes ornithine decarboxylase (ODC) and spermidine/spermine N(1) -acetyltransferase (SSAT) are overexpressed. Both ODC and SSAT transgenic mice have elevated epidermal levels of putrescine, leading us to investigate the mechanistic link between putrescine and HR. We show here that HR and putrescine form a negative regulatory network, as epidermal ODC expression is elevated when HR is decreased and vice versa. We also show that the regulation of ODC by HR is dependent on the MYC superfamily of proteins, in particular MYC, MXI1 and MXD3. Furthermore, we found that elevated levels of putrescine lead to decreased HR expression, but that the SSAT-TG phenotype is distinct from that found when HR is mutated. Transcriptional microarray analysis of putrescine-treated primary human keratinocytes demonstrated differential regulation of genes involved in protein-protein interactions, nucleotide binding and transcription factor activity, suggesting that the putrescine-HR negative regulatory loop may have a large impact on epidermal homeostasis and hair follicle cycling.

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

A Modified Spectrophotometric and Methodical Approach to Find Novel Inhibitors of Ornithine Decarboxylase Enzyme: A Path through the Maze

A Modified Spectrophotometric and Methodical Approach to Find Novel Inhibitors of Ornithine Decarboxylase Enzyme: A Path through the Maze

Abstract LB-179: Phase I trial of relapsed neuroblastoma with DFMO alone and in combination with etoposide.

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. There is no curative treatment for children with relapsed NB, and the 5-year survival rate is <10%. These more aggressive forms of NB respond poorly to chemotherapeutic approaches, and there is a need for agents with novel mechanisms of action. Ornithine decarboxylase (ODC), a gene encoding for the ODC enzyme that is pivotal in polyamine biosynthesis, has been shown to be upregulated in NB. High polyamine content and elevated ODC activities have been associated with poor prognosis in NB. Suppression of polyamines in NB has been shown in preclinical studies to be effective to reduce tumor. Methods: The primary aim of this phase I study was to determine the safety and maximum tolerated dose (MTD) of DFMO as a single agent and in combination with etoposide in pediatric patients with refractory or recurrent NB. The secondary aims were to evaluate the pharmacokinetics of DFMO, the treatment response and biological correlates in urine. A dose escalation trial of daily oral DFMO for one 21-day cycle, followed by four 21 day cycles of DFMO and oral etoposide was completed. Response was evaluated by CT or MRI and MIBG imaging. Measurement of urinary catecholamines and bone marrow disease were used to determine a clinical response. Urinary polyamines were measured at day 1 of each cycle. Results: Twenty-one patients were enrolled between Mar 2010 and Oct 2012. The median age was 10 years old, with a range of 1-17 years old. Patients were enrolled at one of four escalating doses (500mg/m2-1500mg/m2 BID). No dose-limiting toxicities (DLTs) or serious adverse events (SAEs) were seen. The biological effect of urinary polyamine normalization was seen at these dose levels, therefore this study did not escalate to a final MTD. While a phase I study is not powered to evaluate anti-tumor efficacy, seventeen patients with NB were evaluable. Of these; 15 had stable disease and 2 had progressive disease. Of note, 1 subject obtained a complete response in their bone marrow while four patients have been on study progression free for over a year. Spot urine samples from patients had measurable levels of polyamines monoacetylspermidine (MASpd) and diacetylspermine (DASpm). Treatment was associated with decreased urinary MASpd over the first cycle for all patients. Patients with disease progression during the first 3-months of treatment had average increases in urinary DASpm and histamine over this time, while this parameter decreased over time in those patients exhibiting 3-month stabilization of disease. Conclusion: The biologically active dose of DFMO is 500-1500 mg/m2 BID and is well tolerated by pediatric patients. Prolonged stabilization of disease was seen with clinical benefit in greater than 50% of patients. Urine polyamines appear to be a marker for response to DFMO therapy. This biological correlate will be further explored in a Phase II study currently underway. Citation Format: Giselle L. Sholler, Eugene W. Gerner, Genevieve Bergendahl, Bonnie J. LaFleur, Alyssa VanderWerff, Takamaru Ashikaga, William Ferguson, William Roberts, Don Eslin, Joel Kaplan, Jacqueline M. Kraveka, Deanna Mitchell, Kathleen Neville, Randal Wada, Nehal Parikh, Leonard Sender, Timothy Higgins, Andre Bachmann. Phase I trial of relapsed neuroblastoma with DFMO alone and in combination with etoposide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-179. doi:10.1158/1538-7445.AM2013-LB-179

Environmental pesticide exposure modulates cytokines, arginase and ornithine decarboxylase expression in human placenta

To evaluate the cytokine balance and enzymatic alterations induced by environmental pesticide exposure during pregnancy, this transversal study explored placentas derived from non-exposed women (control group-CG), and from women living in a rural area (rural group-RG), collected during intensive organophosphate (OP) pesticide spraying season (RG-SS) and during non-spraying season (RG-NSS). The exposure biomarkers blood cholinesterase and placental carboxylesterase (CaE) were significantly decreased in RG-SS. Among the cytokines studied IL-8, IL-6, TNFα, IL-10, TGFβ and IL-13, the expression frequency of IL-13 increased in RG-SS. Arginase and ornithine decarboxylase (ODC) enzymes were induced in syncytiotrophoblast and endothelial cells. Interestingly, the decrease in CaE activity was associated with arginase and ODC activity induction. These findings suggest that environmental pesticide exposure impacts the placenta by increasing the expression frequency of the anti-inflammatory cytokine IL-13, which may be related to the up-regulation of enzymes implicated in tissue repair.

Structural Insight into DFMO Resistant Ornithine Decarboxylase from Entamoeba histolytica: An Inkling to Adaptive Evolution

BackgroundPolyamine biosynthetic pathway is a validated therapeutic target for large number of infectious diseases including cancer, giardiasis and African sleeping sickness, etc. α-Difluoromethylornithine (DFMO), a potent drug used for the treatment of African sleeping sickness is an irreversible inhibitor of ornithine decarboxylase (ODC), the first rate limiting enzyme of polyamine biosynthesis. The enzyme ODC of E. histolytica (EhODC) has been reported to exhibit resistance towards DFMO.Methodology/Principal FindingThe basis for insensitivity towards DFMO was investigated by structural analysis of EhODC and conformational modifications at the active site. Here, we report cloning, purification and crystal structure determination of C-terminal truncated Entamoeba histolytica ornithine decarboxylase (EhODCΔ15). Structure was determined by molecular replacement method and refined to 2.8 Å resolution. The orthorhombic crystal exhibits P212121 symmetry with unit cell parameters a = 76.66, b = 119.28, c = 179.28 Å. Functional as well as evolutionary relations of EhODC with other ODC homologs were predicted on the basis of sequence analysis, phylogeny and structure.Conclusions/SignificanceWe determined the tetrameric crystal structure of EhODCΔ15, which exists as a dimer in solution. Insensitivity towards DFMO is due to substitution of key substrate binding residues in active site pocket. Additionally, a few more substitutions similar to antizyme inhibitor (AZI), a non-functional homologue of ODCs, were identified in the active site. Here, we establish the fact that EhODC sequence has conserved PLP binding residues; in contrast few substrate binding residues are mutated similar to AZI. Further sequence analysis and structural studies revealed that EhODC may represent as an evolutionary bridge between active decarboxylase and inactive AZI.

Peri-ovulatory putrescine to reduce aneuploid conceptions

Women experience diminished fertility, increased risk of miscarriages and congenital birth defects in their late 30s and early 40s, 10-15 years before reaching menopause. Egg aneuploidy (having an incorrect number of chromosomes) is the most important etiology for these reproductive problems in older women. Science correspondent Jon Cohen wrote a News piece in 2002 [1] on the quest to understand the mechanisms of maternal aging-related aneuploidies in humans, which is still applicable today. Although we have made impressive progress in understanding how aneuploidies arise, we know little about providing medical interventions to reduce them. “My Holy Grail is to deliver some sort of aneuploidy-reduction pill, …”, Dr. Terry Hassold, a leading geneticist in the field, is quoted as saying. Our recent study [2] may be a step towards developing such a pill. Aneuploid eggs result in aneuploid conceptions, most of which are lost either prior to clinical recognition of pregnancies or by miscarriages. The small proportion that survive inevitably carry major birth defects. Accurate egg aneuploidy rates in the general population are not known, but numerous analyses of IVF samples have revealed staggeringly high rates. Notably, a recent study of more than 20,000 IVF eggs revealed significant age-related aneuploidies, reaching >40% in the 40 year old age group [3]. Furthermore, this study [3] employs fluorescence in situ hybridization of polar bodies and is therefore limited in the number of chromosomes (five) that can be analyzed simultaneously. The true aneuploidy rates in older women, considering all 23 chromosomes, would be expected to be much higher. This unusually high egg aneuploidy rate in humans is likely due to a combination of our longevity and the peculiar oogenesis (the process of generating eggs) in all vertebrates including humans. Unlike vertebrate males that continuously generate mature sperm from germ line stem cells through adulthood, in vertebrates oogenesis begins during the embryonic stage when germ line stem cells initiate meiosis and develop into primary oocytes. By birth, the females have developed a finite number of primary oocytes arrested in meiotic prophase that comprises their lifetime egg supply. The prophase oocytes contain chromosome bivalents each having four chromatids linked together by a combination of sister chromotid cohesion and non-sister cross-over (Figure ​(Figure1),1), with this linkage persisting for decades in women. During ovulation the two sister chromatids are segregated to the same cell (egg or 1st polar body) in a process called oocyte maturation; in the subsequent fertilization, the sisters are segregated between the 2nd polar body and the haploid egg. As females age, chromosome cohesion becomes progressively weakened [4], predisposing the oocytes to greater risk of premature separation of sister chromatids (PSSC) during oocyte maturation [2-5] (Figure ​(Figure11). Figure 1 Premature separation of sister chromatids (PSSC) during oocyte maturation Given the great time lapse, it seems daunting that any measure can be found to keep these primary oocytes “fresh” while women age. Our study however suggests that a metabolic deficiency during ovulation in older females, which can be easily corrected, is an important compounding factor in age-related egg aneuploidies. It has been known for more than four decades [6] that during mammalian ovulation, the ovaries exhibit a transitory (i.e. a few hours) rise in the enzyme ornithine decarboxylase (ODC) and its product putrescine and yet its physiological function remains enigmatic. We found that inhibition of ODC during ovulation results in significant increase of incidence of PSSC in eggs of young mice. Interestingly, older mice exhibit a diminished ODC rise in the ovaries, and a corresponding increase of PSSC in eggs. Most remarkably, a combination of putrescine supplementation in mouse drinking water leading up to oocyte retrieval and in oocyte maturation medium reduced the incidence of PSSC in eggs by more than 50% [2]. Putrescine is a naturally occurring, small metabolite easily absorbed and distributed throughout the organism. A high dose of putrescine supplementation similar to that used in our study has been given safely to neonatal calves and pigs as an ingredient of soybean-based milk replacement [7]. However, post-conception putrescine supplementation in mouse drinking water compromises fetal development, likely due to excess reactive oxygen species produced by placental diamine oxidase oxidizing the exogenous putrescine [8]. In contrast, periovulatory putrescine supplementation is safe for gestation, because exogenous putrescine administered around ovulation is cleared long before placental development (Tao and Liu, in preparation). Nonetheless, further studies, including that employing primates, are required to formulate effective and safe putrescine supplementation protocols to reduce egg aneuploidies, both in IVF clinics and in natural conception.

Amelioration of chilling stress by arginine in tomato fruit: Changes in endogenous arginine catabolism

Freshly harvested tomato fruit were pretreated with 0.2mM arginine at −35kPa for 0.5min and then stored at 2°C for 28d to investigate the effect of exogenous arginine treatment on endogenous arginine catabolism in relation to chilling injury (CI). Arginine treatment reduced the CI index of fruit and enhanced accumulation of polyamines, especially putrescine, and proline, which resulted from the increased activities of the catabolic enzymes arginase, arginine decarboxylase, ornithine decarboxylase and ornithine δ-aminotransferase at most sampling times. Nitric oxide synthase activity was also increased by arginine treatment, which at least partly contributed to the increased nitric oxide concentration. These results revealed that the reduction in CI by exogenous arginine may be due to the accumulation of putrescine, proline and nitric oxide induced by activating the different pathways of endogenous arginine catabolism.

Docking and molecular dynamics studies of peptide inhibitors of ornithine decarboxylase: a rate-limiting enzyme for the metabolism of Fusarium solani

Fusarium solani causes a wide variety of diseases in plants. Polyamine biosynthesis is responsible for the growth and pathogenicity of the fungus. The initial step of this pathway involves the decarboxylation of ornithine to putrescine, and is catalyzed by the enzyme ornithine decarboxylase (ODC). Inhibiting this process may be a promising approach for the management of fungal disease in various crops. Therefore, there is a need to develop inhibitors of ODC that have higher binding capacity than ornithine. Fifteen peptides were designed and modeled based on physicochemical properties of residues in the active site of ODC. The peptide GLIWGNGPF showed the highest dock score. It is assumed that the de novo design of peptides could be a potential approach to inhibit polyamine biosynthesis. Molecular dynamics studies make an important contribution to understanding the effect of the binding of peptides and the stability of an ODC-peptide complex system. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:8.

Mechanistic insights into the dual inhibition strategy for checking Leishmaniasis

Leishmaniasis 1 1These authors contributed equally. is an endemic disease mainly caused by the protozoan Leishmania donovani (Ld). Polyamines have been identified as essential organic compounds for the growth and survival of Ld. These are synthesized in Ld by polyamine synthesis pathway comprising of many enzymes such as ornithine decarboxylase (ODC), spermidine synthase (SS), and S-adenosylmethionine decarboxylase. Inhibition of these enzymes in Ld offers a viable prospect to check its growth and development. In the present work, we used computational approaches to search natural inhibitors against ODC and SS enzymes. We predicted three-dimensional structures of ODC and SS using comparative modeling and molecular dynamics (MD) simulations. Thousands of natural compounds were virtually screened against target proteins using high throughput approach. MD simulations were then performed to examine molecular interactions between the screened compounds and functional residues of the active sites of the enzymes. Herein, we report two natural compounds of dual inhibitory nature active against the two crucial enzymes of polyamine pathway of Ld. These dual inhibitors have the potential to evolve as lead molecules in the development of antileishmanial drugs.

Biochemical, Mutational and In Silico Structural Evidence for a Functional Dimeric Form of the Ornithine Decarboxylase from Entamoeba histolytica

Background Entamoeba histolytica is responsible for causing amoebiasis. Polyamine biosynthesis pathway enzymes are potential drug targets in parasitic protozoan diseases. The first and rate-limiting step of this pathway is catalyzed by ornithine decarboxylase (ODC). ODC enzyme functions as an obligate dimer. However, partially purified ODC from E. histolytica (EhODC) is reported to exist in a pentameric state.Methodology and ResultsIn present study, the oligomeric state of EhODC was re-investigated. The enzyme was over-expressed in Escherichia coli and purified. Pure protein was used for determination of secondary structure content using circular dichroism spectroscopy. The percentages of α-helix, β-sheets and random coils in EhODC were estimated to be 39%, 25% and 36% respectively. Size-exclusion chromatography and mass spectrophotometry analysis revealed that EhODC enzyme exists in dimeric form. Further, computational model of EhODC dimer was generated. The homodimer contains two separate active sites at the dimer interface with Lys57 and Cys334 residues of opposite monomers contributing to each active site. Molecular dynamic simulations were performed and the dimeric structure was found to be very stable with RMSD value ∼0.327 nm. To gain insight into the functional role, the interface residues critical for dimerization and active site formation were identified and mutated. Mutation of Lys57Ala or Cys334Ala completely abolished enzyme activity. Interestingly, partial restoration of the enzyme activity was observed when inactive Lys57Ala and Cys334Ala mutants were mixed confirming that the dimer is the active form. Furthermore, Gly361Tyr and Lys157Ala mutations at the dimer interface were found to abolish the enzyme activity and destabilize the dimer.ConclusionTo our knowledge, this is the first report which demonstrates that EhODC is functional in the dimeric form. These findings and availability of 3D structure model of EhODC dimer opens up possibilities for alternate enzyme inhibition strategies by targeting the dimer disruption.

Evidence of a Role for Antizyme and Antizyme Inhibitor as Regulators of Human Cancer

Antizyme and its endogenous antizyme inhibitor have recently emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis. Antizyme was originally isolated as a negative modulator of the enzyme ornithine decarboxylase (ODC), an essential component of the polyamine biosynthetic pathway. Antizyme binds ODC and facilitates proteasomal ODC degradation. Antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate. In this review, we summarize the current literature on the role of antizyme and antizyme inhibitor in cancer, discuss how the ratio of antizyme to antizyme inhibitor can influence tumor growth, and suggest strategies to target this axis for tumor prevention and treatment.

Mechanism of Formation of the Internal Aldimine in Pyridoxal 5′-Phosphate-Dependent Enzymes

In this paper we studied the mechanism of formation of the internal aldimine, a common intermediate to most pyridoxal 5'-phosphate (PLP)-dependent enzymes. A large model based on the crystal structure from the human ornithine decarboxylase (ODC) enzyme was constructed and in total accounts for 504 atoms. The reaction mechanism was investigated using the ONIOM methodology (B3LYP/6-31G(d)//AM1), and the final energies were calculated with the M06/6-311++G(2d,2p)//B3LYP/6-31G(d) level of theory. It was demonstrated that the reaction is accomplished in three sequential steps: (i) the nucleophilic attack of Lysine69 to PLP, (ii) the carbinolamine formation, and (iii) a final dehydration step. For the carbinolamine formation, several mechanistic hypotheses were explored, and the preferred pathway assigns a key role for the conserved active site Cys360. The overall reaction is exergonic in -9.1 kcal/mol, and the rate-limiting step is the dehydration step (E(a) = 13.5 kcal/mol). For the first time, we provide an atomistic portrait of this mechanism in an enzymatic environment. Moreover, we were able to assign a novel role to Cys360 in the ODC reaction mechanism that was never proposed.

Minimal Antizyme Peptide Fully Functioning in the Binding and Inhibition of Ornithine Decarboxylase and Antizyme Inhibitor

Antizyme (AZ) is a protein with 228 amino acid residues that regulates ornithine decarboxylase (ODC) by binding to ODC and dissociating its homodimer, thus inhibiting its enzyme activity. Antizyme inhibitor (AZI) is homologous to ODC, but has a higher affinity than ODC for AZ. In this study, we quantified the biomolecular interactions between AZ and ODC as well as AZ and AZI to identify functional AZ peptides that could bind to ODC and AZI and inhibit their function as efficiently as the full-length AZ protein. For these AZ peptides, the inhibitory ability of AZ_95-228 was similar to that of AZ_WT. Furthermore, AZ_95-176 displayed an inhibition (IC50: 0.20 µM) similar to that of AZ-95-228 (IC50: 0.16 µM), even though a large segment spanning residues 177–228 was deleted. However, further deletion of AZ_95-176 from either the N-terminus or the C-terminus decreased its ability to inhibit ODC. The AZ_100-176 and AZ_95-169 peptides displayed a noteworthy decrease in ability to inhibit ODC, with IC50 values of 0.43 and 0.37 µM, respectively. The AZ_95-228, AZ_100-228 and AZ_95-176 peptides had IC50 values comparable to that of AZ_WT and formed AZ-ODC complexes with K d,AZ-ODC values of 1.5, 5.3 and 5.6 µM, respectively. Importantly, our data also indicate that AZI can rescue AZ peptide-inhibited ODC enzyme activity and that it can bind to AZ peptides with a higher affinity than ODC. Together, these data suggest that these truncated AZ proteins retain their AZI-binding ability. Thus, we suggest that AZ_95-176 is the minimal AZ peptide that is fully functioning in the binding of ODC and AZI and inhibition of their function.

Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N 1-acetyltransferase (SSAT) overexpressing mice

Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N(1)-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1β and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α(1)-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response.

Insulin-like growth factor (IGF) signalling and genome-wide transcriptional regulation in fast muscle of zebrafish following a single-satiating meal

Male zebrafish (Danio rerio) were fasted for 7 days and fed to satiation over 3 h to investigate the transcriptional responses to a single meal. The intestinal content at satiety (6.3% body mass) decreased by 50% at 3 h and 95% at 9 h following food withdrawal. Phosphorylation of the insulin-like growth factor (IGF) signalling protein Akt peaked within 3 h of feeding and was highly correlated with gut fullness. Retained paralogues of IGF hormones genes were regulated with feeding, with igf1a showing a pronounced peak in expression after 3 h and igf2b after 6 h. Igf-I receptor transcripts were markedly elevated with fasting, and decreased to their lowest levels 45 min after feeding. igf1rb transcripts increased more quickly than igf1ra transcripts as the gut emptied. Paralogues of the insulin-like growth factor binding proteins (IGFBPs) were constitutively expressed, except for igfbp1a and igfbp1b transcripts, which were significantly elevated with fasting. Genome-wide transcriptional responses were analysed using the Agilent 44K oligonucleotide microarray and selected genes validated by qPCR. Fasting was associated with the upregulation of genes for the ubiquitin-proteasome degradation pathway, anti-proliferative and pro-apoptotic genes. Protein chaperones (unc45b, hspd1, hspa5, hsp90a.1, hsp90a.2) and chaperone interacting proteins (ahsa1 and stip1) were upregulated 3 h after feeding along with genes for the initiation of protein synthesis and mRNA processing. Transcripts for the enzyme ornithine decarboxylase 1 showed the largest increase with feeding (11.5-fold) and were positively correlated with gut fullness. This study demonstrates the fast nature of the transcriptional responses to a meal and provides evidence for differential regulation of retained paralogues of IGF signalling pathway genes.

Critical Factors Governing the Difference in Antizyme-Binding Affinities between Human Ornithine Decarboxylase and Antizyme Inhibitor

Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC50 value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K d) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K d value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K d value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K d value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.

Abstract 248: Control of prostate tumor growth and centriole amplification by antizyme and antizyme inhibitor

Abstract The endogenous antizyme inhibitor (AZI) is best characterized for its role in regulating polyamine homeostasis. Polyamines are organic cations synthesized by the enzyme ornithine decarboxylase (ODC), a protein which is negatively regulated and targeted for proteasomal degradation by antizyme (AZ). AZI is a potential oncogene which promotes cell growth not only by inhibiting antizyme (AZ) activity, but also by releasing ODC from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing AZI shRNA. AZI knockdown was confirmed by western blot and RT-PCR. To examine the ability of these cells to form tumors in vivo, 1×106 cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. Cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo, and had significantly smaller end tumor weights. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a novel and valid therapeutic target for cancer treatment. In addition to their effects on cell proliferation, previous studies in our laboratory have shown that both AZI and AZ localize to centrosomes and are important for controlling centrosome duplication. Centrosome amplification is a frequent and early event in tumorigenesis. We found that overexpression of AZI and knockdown of AZ both result in cells with daughter centriole amplification due to abnormal centriole synthesis. To gain insight into the mechanism by which AZ and AZI modulate centriole synthesis, we are currently analyzing whether proteins that regulate centrosome duplication interact with either AZI or AZ. We are also using Tet-inducible overexpression vectors to monitor the effect of increased AZ or AZI levels on centriole amplification, and exploring how localization of these proteins to the centrosome contributes to the centrosome amplification phenotype. These studies provide a much greater understanding of AZI function, and suggest that high AZI levels may contribute to tumorigenesis both by promoting cell growth and inducing centrosome amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 248. doi:10.1158/1538-7445.AM2011-248