Potential urinary and plasma biomarkers of peroxisome proliferation in the rat: identification of N-methylnicotinamide and N-methyl-4-pyridone-3-carboxamide by 1H nuclear magnetic resonance and high performance liquid chromatography

This study identified two potential novel biomarkers of peroxisome proliferation in the rat. Three peroxisome proliferator-activated receptor (PPAR) ligands, chosen for their high selectivity towards the PPARα, -δ and -γ subtypes, were given to rats twice daily for 7 days at doses known to cause a pharmacological effect or peroxisome proliferation. Fenofibrate was used as a positive control. Daily treatment with the PPARα and -δ agonists produced peroxisome proliferation and liver hypertrophy. ¹H nuclear magnetic resonance spectroscopy and multivariate statistical data analysis of urinary spectra from animals given the PPARα and -δ agonists identified two new potential biomarkers of peroxisome proliferation - N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY) - both endproducts of the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway. After 7 days, excretion of NMN and 4PY increased 24- and three-fold, respectively, following high doses of fenofibrate. The correlation between total NMN excretion over 7 days and the peroxisome count was r=0.87 (r²=0.76). Plasma NMN, measured using a sensitive high performance liquid chromatography method, was increased up to 61-fold after 7 days' treatment with high doses of fenofibrate. Hepatic gene expression of aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) was downregulated following treatment with the PPARα and -δ agonists. The decrease was up to 11-fold compared with controls in the groups treated with high doses of fenofibrate. This supports the link between increased NMN and 4PY excretion and regulation of the tryptophan-NAD⁺ pathway in the liver. In conclusion, NMN, and possibly other metabolites in the pathway, are potential non-invasive surrogate biomarkers of peroxisome proliferation in the rat.     

Accelerated development of uroporphyria in mice heterozygous for a deletion at the uroporphyrinogen decarboxylase locus

Three weeks after a single dose of iron-dextran and Aroclor 1254, mice maintained continuously on delta-aminolevulinic acid supplemented drinking water showed significantly elevated levels of hepatic uroporphyrin and depressed (25% of normal) uroporphyrinogen decarboxylase (URO-D) activity. Depressed URO-D activity was paralleled by the ability of heat denatured cytosol to inhibit rhURO-D activity. Mice heterozygous for a targeted disruption at the URO-D locus (URO-D+/-) exhibited half the URO-D activity of homozygous controls prior to treatment. After treatment, these animals showed URO-D activity and rhURO-D inhibitory activity comparable to similarly treated wild type (URO-D +/+) mice but with significantly greater uroporphyrin accumulation. With only 10 days of treatment, URO-D +/- but not URO-D +/+ mice showed changes similar in magnitude to those seen after 21 days. Prior to treatment, URO-D genotype did not influence overall hepatic P450 concentration in either sex and there was no significant difference between sexes. The treatment regimen significantly elevated P450 in animals of either URO-D genotype and in both sexes, although the induction response at the 10-day point was attenuated in URO-D +/- mice. From differences in the CO absorbance maximum, and by P450 activity analysis, this attenuated induction response resulted from an attenuation of the CYP2B not the CYP1A induction.     

OMP decarboxylase--An enigma persists

In 1995, Radzicka and Wolfenden reported that the rate enhancement produced by orotidine 5'-phosphate decarboxylase (ODCase) approaches 10(17), making this enzyme the most effective pure protein catalyst known in Nature [A. Radzicka, R. Wolfenden, Science 267 (1995) 90-93]. Over the last 12 years, there have been many hypotheses put forward to explain that impressive effect. In this perspective, we provide a summary of the reaction pathways under consideration for ODCase, highlight the supporting and refuting data, and suggest experiments designed to further test each of the candidate pathways.     

Protein kinase CK2 links polyamine metabolism to MAPK signalling in Drosophila

MAPK signalling is a complex process not only requiring the core components Raf, MEK and Erk, but also many proteins like the scaffold protein KSR and several kinases to specifically localize, modulate and fine-tune the outcome of the pathway in a cell context specific manner. In mammals, protein kinase CK2 was shown to bind to the scaffold protein KSR and to phosphorylate Raf proteins at a conserved serine residue in the negative-charge regulatory (N−) region, thereby facilitating maximal activity of the MAPK signalling pathway. In this work we show that in Drosophila CK2 is also bound to KSR. However, despite the presence of a corresponding serine residue in the N-region of DRaf, CK2-mediated phosphorylation of DRaf takes place on a serine residue at the N-terminus and is required for Erk activation. Previous work identified polyamines as regulators of CK2 kinase activity. The main cellular source of polyamines is the catabolism of amino acids. Evidence is provided that phosphorylation of DRaf by CK2 is modulated by polyamines, with spermine being the most potent inhibitor of the reaction. We suggest that CK2 is able to monitor intracellular polyamine levels and translates this information to modulate MAPK signalling.     

Evolution and Multiplicity of Arginine Decarboxylases in Polyamine Biosynthesis and Essential Role in Bacillus subtilis Biofilm Formation

Arginine decarboxylases (ADCs; EC 4.1.1.19) from four different protein fold families are important for polyamine biosynthesis in bacteria, archaea, and plants. Biosynthetic alanine racemase fold (AR-fold) ADC is widespread in bacteria and plants. We report the discovery and characterization of an ancestral form of the AR-fold ADC in the bacterial Chloroflexi and Bacteroidetes phyla. The ancestral AR-fold ADC lacks a large insertion found in Escherichia coli and plant AR-fold ADC and is more similar to the lysine biosynthetic enzyme meso-diaminopimelate decarboxylase, from which it has evolved. An E. coli acid-inducible ADC belonging to the aspartate aminotransferase fold (AAT-fold) is involved in acid resistance but not polyamine biosynthesis. We report here that the acid-inducible AAT-fold ADC has evolved from a shorter, ancestral biosynthetic AAT-fold ADC by fusion of a response regulator receiver domain protein to the N terminus. Ancestral biosynthetic AAT-fold ADC appears to be limited to firmicute bacteria. The phylogenetic distribution of different forms of ADC distinguishes bacteria from archaea, euryarchaeota from crenarchaeota, double-membraned from single-membraned bacteria, and firmicutes from actinobacteria. Our findings extend to eight the different enzyme forms carrying out the activity described by EC 4.1.1.19. ADC gene clustering reveals that polyamine biosynthesis employs diverse and exchangeable synthetic modules. We show that in Bacillus subtilis, ADC and polyamines are essential for biofilm formation, and this appears to be an ancient, evolutionarily conserved function of polyamines in bacteria. Also of relevance to human health, we found that arginine decarboxylation is the dominant pathway for polyamine biosynthesis in human gut microbiota.     

A novel bifunctional molybdo-enzyme catalyzing both decarboxylation of indolepyruvate and oxidation of indoleacetaldehyde from a thermoacidophilic archaeon, Sulfolobus sp. strain 7

An enzyme, which catalyzes both decarboxylation of indolepyruvate and subsequent oxidation of indoleacetaldehyde into indoleacetate, was purified from a thermoacidophilic archaeon, Sulfolobus sp. strain 7. The enzyme showed a M_r of 280 kDa on gel filtration and was composed of three subunits (a, 89; b, 30; and c, 19 kDa), possibly in a stoichiometry of 2:2:2. Mo and Fe were detected. Thiamine pyrophosphate was absent. Biotin was suggested to bind to the b-subunit. The first step, the decarboxylation reaction, was specific for 2-oxoacids with an aromatic group, while in the second reaction, various aldehydes including glyceraldehyde, which is a glycolytic intermediate in the organism, were oxidized.     

Quaternary structure of the oxaloacetate decarboxylase membrane complex and mechanistic relationships to pyruvate carboxylases

The oxaloacetate decarboxylase primary Na(+) pump (OAD) is an essential membrane protein complex that functions in the citrate fermentation pathway of some pathogenic bacteria under anaerobic conditions. OAD contains three different subunits: Oad-α, a biotinylated extrinsic protein that catalyzes the α-ketodecarboxylation of oxaloacetate; Oad-γ, a structural bitopic membrane protein whose cytosolic tail (named as Oad-γ') binds tightly to Oad-α; and Oad-β, a multispan transmembrane α-helical protein that constitutes the Na(+) channel. How OAD is organized structurally at the membrane and what the molecular determinants are that lead to an efficient energy coupling mechanism remain elusive. In the present work, we elucidate the stoichiometry of the native complex as well as the low resolution structure of the peripheral components of OAD (Oad-α and Oad-γ') by small angle x-ray scattering. Our results point to a quaternary assembly similar to the pyruvate carboxylase complex organization. Herein, we propose a model in which the association in pairs of Oad-α dimers, mediated by Oad-γ, results in the acquisition of a functional oligomeric state at the bacterial membrane. New structural insights for the conformational rearrangements associated with the carboxylbiotin transfer reaction within OAD are provided.     

Antizyme affects cell proliferation and viability solely through regulating cellular polyamines

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.     

FXYD proteins stabilize Na,K-ATPase: amplification of specific phosphatidylserine-protein interactions

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C₁₂E₈), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E₁ ↔ E₂ conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.     

Discovery of Potent and Selective Inhibitors of Trypanosoma brucei Ornithine Decarboxylase

Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of human African trypanosomiasis is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Here, we describe the use of an enzymatic high throughput screen of 316,114 unique molecules to identify potent and selective inhibitors of ODC. This screen identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.