Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction

Purine catabolic pathway in Bacillus subtilis is consisted of more than 14 genes. Among these genes, pucL and pucM are required for uricase activity. While PucL is known to encode the uricase itself, the function of PucM is still unclear although this protein is also indispensable for uric acid decomposition. Here, we provide evidence that PucM, a transthyretin-related protein, functions to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. Based on these results, we propose that transthyretin-related proteins present in diverse organisms are not functionally related to transthyretin but actually function as a hydroxyisourate hydrolase.     

Structure-based characterization of canine-human chimeric uricases and its evolutionary implications

Uricase was lost in hominoids during primate evolution, but the inactivation mechanism remains controversial. To investigate the inactivation process of hominoid uricase, chimeric constructions between canine and human uricase were employed to screen the target regions that may contain labile or inactivated mutations in deduced human uricase. Four chimeric uricases were constructed and showed different enzymatic characteristics. Homology modeling, rational site-directed mutagenesis and DNA alignment were used to analyze the changes. Arg119 is conserved in functional mammalian uricases and its side-chains are crucial in maintaining the stability of the β-barrel core. A single CGT (Arg) to CAT (His) mutation at codon 119 that is shared by the human and great ape clade greatly reduces this stability and could cause the loss of uricase activity. We speculate that this missense mutation occurred first and inactivated the uricase protein in humans and great apes and that later the known nonsense mutation at codon 33 occurred and silenced the uricase gene. A single GTC (Val) to GCC (Ala) mutation at codon 296 in canine uricase is regarded as deleterious structural mutation, but such kinds of deleterious mutations have been widely accumulated in extant mammalian uricases. We speculate that a reduction in uricase activity has been an evolutionary tendency in mammals. Moreover, from structure-activity analysis of helix 2 in ancestral primate uricase, we suggest that before the inactivation of hominoid uricase, deleterious structural evolutionary changes had occurred in ancestral primates. The loss of hominoid uricase should be caused by progressive multistep mutations rather than a single mutation event.     

Uricase protein sequences: conserved during vertebrate evolution but absent in humans

Uricase is a peroxisomal liver enzyme that catalyzes the oxidation of uric acid to allantoin during purine catabolism. It is present in vertebrates in most species of fish, amphibians, and mammals but its enzymatic activity is absent in hominoids. We have used Western blot analysis in a comparative study to establish a homology among uricases from different species of vertebrates. Using antibodies against denatured rat liver uricase, we have been able to detect for the first time cross-reactivity with the uricase of species ranging in the evolutionary scale from fish to primates (macaque). Our results suggest that these uricases have a common evolutionary origin. Our conclusion is also supported by the fact that uricase from different species exhibits identical tissue, subcellular localization, and similarity of molecular weights. This study was extended to include human liver samples. Using the same approach but with a more sensitive detection system (alkaline phosphatase instead of peroxidase), we did not detect polypeptide species related to rat uricase in human fetal or adult liver samples, which indicates that during hominoid evolution, the mutational event responsible for the loss of uricase activity in humans precluded formation of a translatable uricase mRNA.     

Fructose,uricase, and the Back‐to‐Africa hypothesis

In the Middle Miocene (approximately 17 to 12 Ma) at least two radiations of fossil apes from East Africa into Eurasia occurred, and, while controversial, some paleoanthropological studies suggest that one of the Eurasian lineages may have returned to Africa to evolve into humans and the African apes. Here, we present a novel argument supporting this hypothesis. Specifically, the global cooling that occurred in the middle Miocene rendered hominoids living in Europe at risk for starvation as seasonal climate change resulted in less availability of fruits during the winter months. During this time, a mutation in uricase occurred in early hominids that resulted in a rise in serum uric acid. Uric acid has been found to potentiate the effect of fructose to increase fat stores, suggesting that the mutation provided a survival advantage. Such a survival advantage would have been less likely to occur in Africa, where the continued presence of tropical rainforests would have been more likely to provide food throughout the year. Furthermore, Miocene apes in Europe were in protected sites where geographic isolation could have allowed the uricase mutation to be rapidly expressed in the entire population. While speculative, we suggest that the uricase mutation supports an extra‐Africa origin of humans.     

Protection of dopaminergic cells by urate requires its accumulation in astrocytes

Urate is the end product of purine metabolism and a major antioxidant circulating in humans. Recent data link higher levels of urate with a reduced risk of developing Parkinson's disease and with a slower rate of its progression. In this study, we investigated the role of astrocytes in urate-induced protection of dopaminergic cells in a cellular model of Parkinson's disease. In mixed cultures of dopaminergic cells and astrocytes oxidative stress-induced cell death and protein damage were reduced by urate. By contrast, urate was not protective in pure dopaminergic cell cultures. Physical contact between dopaminergic cells and astrocytes was not required for astrocyte-dependent rescue as shown by conditioned medium experiments. Urate accumulation in dopaminergic cells and astrocytes was blocked by pharmacological inhibitors of urate transporters expressed differentially in these cells. The ability of a urate transport blocker to prevent urate accumulation into astroglial (but not dopaminergic) cells predicted its ability to prevent dopaminergic cell death. Transgenic expression of uricase reduced urate accumulation in astrocytes and attenuated the protective influence of urate on dopaminergic cells. These data indicate that urate might act within astrocytes to trigger release of molecule(s) that are protective for dopaminergic cells.     

Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Apical Voltage-Driven Urate Efflux Transporter NPT4 in Renal Proximal Tubule

Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).     

Phylogenetic Articulation of Uric Acid Evolution in Mammals and How It Informs a Therapeutic Uricase

The role of uric acid during primate evolution has remained elusive ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule in our serum. It has been difficult to generate a neutral or adaptive explanation in part because the uricase enzyme evolved to become a pseudogene in apes thus masking typical signals of sequence evolution. Adding to the difficulty is a lack of clarity on the functional role of uric acid in apes. One popular hypothesis proposes that uric acid is a potent antioxidant that increased in concentration to compensate for the lack of vitamin C synthesis in primate species ∼65 Ma. Here, we have expanded on our previous work with resurrected ancient uricase proteins to better resolve the reshaping of uricase enzymatic activity prior to ape evolution. Our results suggest that the pivotal death-knell to uricase activity occurred between 20 and 30 Ma despite small sequential modifications to its catalytic efficiency for the tens of millions of years since primates lost their ability to synthesize vitamin C, and thus the two appear uncorrelated. We also use this opportunity to demonstrate how molecular evolution can contribute to biomedicine by presenting ancient uricases to human immune cells that assay for innate reactivity against foreign antigens. A highly stable and highly catalytic ancient uricase is shown to elicit a lower immune response in more human haplotypes than other uricases currently in therapeutic development.     

Cloning, sequence analysis and expression in Escherichia coli of the gene encoding a uricase from the yeast-like symbiont of the brown planthopper, Nilaparvata lugens

A urate oxidase (uricase; EC 1.7.3.3) gene of the yeast-like fungal endosymbiont of the brown planthopper, Nilaparvata lugens, was cloned, and sequenced together with its flanking regions. The gene comprised a open reading frame of 987 bp, that was split into two parts by a single 96 bp intron. The encoded uricase was 296 amino acids with 62% sequence identity with that of Aspergillus flavus. The molecular weight deduced was 32,882, and the predicted isoelectric point was 6.06. The symbiont's uricase conserved all the known consensus motifs, except the C-terminal PTS-1, Ser-basic-Leu. The leucine at the third position of PTS-1 was replaced by serine in the C-terminus of the symbiont's uricase. The symbiont's uricase gene was successfully expressed in Escherichia coli, and the product, tagged with histidine residues, was purified. The symbiont's uricase, thus produced, was as active as those from plants and animals, but less active than those from other fungi.     

The moose,purine degradation,and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.     

A steady-state kinetic investigation of the uricase reaction mechanism

The steady-state kinetics of porcine liver uricase has been investigated over an extensive range of both urate and oxygen concentration. Spectrophotometric assay of the accumulation of reaction intermediate which absorbs strongly in the ultraviolet region as well as the oxygen electrode were used to minimize inaccuracies in initial velocity measurements. The Superoxide radical could not be detected as a product of the uricase reaction. Reciprocal plots with both urate and oxygen showed extensive nonlinearity. Mechanisms involving second binding sites on uricase appear unlikely in view of the structural properties of the enzyme. A random substrate binding scheme has been shown to be consistent with all of the nonlinear reciprocal plots and is thus suggested as the most probable mechanism of action for uricase.     

Biogenesis of peroxisomes in regenerating rat liver. I. Sequential changes of catalase and urate oxidase detected by ultrastructural cytochemistry

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".     

The transthyretin-related protein family.

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.     

The transthyretin-related protein: structural investigation of a novel protein family

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.     

Regulation of the purine catabolic enzymes in Neurospora crassa.

Neurospora crassa can utilize purines and their metabolic products as a nitrogen source. Regulation of the five enzymes required for uric acid metabolism was studied. The first three enzymes of this catabolic pathway are controlled in a complex manner that involves both induction and repression. Both uricase and allantoicase were induced by uric acid while allantoinase was induced by either uric acid or allantoin. Synthesis of all three of these enzymes was repressed by the end product, ammonia. The ure-2 mutant, which is urease deficient and cannot derive ammonia from purines, shows a hyperinducibility of these same three enzymes. The last two enzymes of the pathway, ureidoglycollase and urease, were found to be constitutive.     

A human genomic sequence highly homologous to the 3'-untranslated region of rat uricase mRNA

A human genomic sequence was isolated from a library using a rat uricase cDNA probe. Sequence analysis has shown that it is highly homologous to the 3'-untranslated region of rat uricase mRNA. Total loss of uricase activity in human is, therefore, not due to total loss of the gene. Discovery of high degree of conservation of the non-coding region of the gene would be of great interest as we attempt to learn the process of gene evolution.     

Supplementation of Poultry Feeds with Dietary Zinc and Other Minerals and Compounds to Mitigate Nitrogen Emissions—A Review

One of the environmental challenges that the poultry industry has been faced with is ammonia emission from manure. One way to reduce nitrogen excretion and emissions is supplementing dietary trace minerals to inhibit the activity of microbial uricase, a key enzyme converting nitrogen compounds in the manure into ammonia. Several dietary minerals are commercially available as economic alternatives for reducing ammonia emissions in poultry. In this review, we discuss different mineral elements including zinc as feed amendment minerals that could be used to reduce ammonia emission. Issues discussed include potential for inhibiting microbial uricase, dietary supplementation levels, growth performance, toxicity, their influence on manure nitrogen emission, and potential mineral accumulation in soil. In addition, we discuss other minerals and compounds that have the potential to reduce ammonia volatilization by inhibiting microbial uricase and growth of uric acid-utilizing microorganisms.     

Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

Induction and de novo synthesis of uricase, a nitrogen-regulated enzyme in Neurospora crassa.

Two efficient procedures are presented for the purification of the purine catabolic enzyme uricase from Neurospora crassa. A specific antiserum for uricase was prepared and used to examine the regulation of uricase expression. Even when wild-type cells are growing under full nitrogen repression conditions, they possess a considerable basal level of uricase. Induction results in a severalfold increase in the level of this enzyme and reflects de novo enzyme synthesis. Identical forms of uricase were translated in vitro from RNA isolated from control and induced cells, but, unexpectedly, induced cells contained less translatable uricase mRNA than did control cells. Although uricase is localized in peroxisomes, the enzyme subunit appears to be synthesized in mature form without any requirement for processing.     

Characterization of the bacteriophage PhiKMV DNA ligase

Gene 17 product (gp17) of the Pseudomonas aeruginosa-infecting bacteriophage phiKMV shows in silico similarity to T7 DNA ligase. In a semi-quantitative activity assay, it is shown that gp17 is a functional, ATP-dependent DNA ligase, in spite of some structural differences related to DNA-binding properties). Enzymatic activity of His6-based purified expression product was optimised (4 degrees C at 24h for sticky end double-stranded DNA fragments) and estimated at 0.5 Weiss U/microg.