Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h-1 , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).

Cysteine Enrichment Mediates Co-Option of Uricase in Reptilian Skin and Transition to Uricotelism.

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with ß-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin.

Cloning, Heterologous Expression, and Characterization of a Neutral Uricase from Arthrobacter sp. CSAJ-16 in Cangshan Mountain.

Uricase (or Urate oxidase), a key enzyme involved in purine metabolism, is commonly used in treating conditions such as gout, hyperuricemia, and tumor lysis syndrome. In this study, a uricase-producing strain (named CSAJ-16) was isolated from the soil sample of Cangshan Mountain, Yunnan Province, China. This strain was identified as Arthrobacter sp. CSAJ-16. Based on the gene sequence alignment, the uricase gene (named aruox) of Arthrobacter sp. CSAJ-16 was amplified and heterologously expressed. The recombinant uricase (ArUOX) was about 32 kDa. The optimal pH and temperature of ArUOX were pH 7 and 20°C, respectively. The ArUOX remained above 50% relative activity after incubation at 37°C for 100 min or at pH 6.0-8.6 for 24 h. Moreover, metal ions such as K+, Mg2+, Ca2+, Ba2+ and Pb2+ can significantly enhance the activity of ArUOX (> 200%). These enzymatic properties indicate that ArUOX has potential applications in pharmaceutical enzymes and uric acid detection kits.

The effects of free Cys residues on the structure, activity, and tetrameric stability of mammalian uricase.

Mammalian uricases contain four conserved cysteine (Cys) residues, but little is known about their structures and functions. In this study, we first confirmed that all four Cys residues are free and not involved in disulfide bond formation, using canine uricase as a model protein. Cys residues had a greater effect on stability than on activity based on single Cys-to-Ser (serine) substitutions. Circular dichroism (CD) and homology modeling indicated that C188S reduces ß-sheet contents and inter- and intra-subunit hydrophobic interaction, potentially impairing the core tetrameric ß-barrel structure of the tunneling-fold protein, and ultimately decreased the tetrameric stability. Additionally, the inactivation of C188S during the stability tests may be a complex process involving depolymerization followed by irregular aggregation. Double mutations or thiol blockage of Cys188 and Cys195 significantly disrupted the formation and stability of tetrameric uricase, which may be mediated by the free thiols in Cys residues. The present results demonstrated that the free Cys residues are essential for tetrameric formation and stability in mammalian uricase. This implies that free cysteine residues, although not involved in disulfide bonding, may play important structural roles in certain proteins, underscoring the significance of the hydrophobic characteristics of the free thiols in Cys residues. KEY POINTS: • Four Cys residues are not involved in disulfide bonding in mammalian uricase. • The hydrophobicity of free thiols is critical for tetrameric stability in uricase. • Free Cys residues can serve structural roles without involving in disulfide bonds.

Uricase-Deficient Larval Zebrafish with Elevated Urate Levels Demonstrate Suppressed Acute Inflammatory Response to Monosodium Urate Crystals and Prolonged Crystal Persistence.

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox-/- larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.

Comparison of 3 hyperuricemia mouse models and evaluation of food-derived anti-hyperuricemia compound with spontaneous hyperuricemia mouse model.

Hyperuricemia animal models have long been used for evaluating food-derived anti-hyperuricemia compounds. Fructose and potassium oxonate are commonly used for developing hyperuricemia mouse model. Recent research also developed spontaneous hyperuricemia model by uricase knockout (Uox-/-). In this work, we evaluated 3 kinds of models with the same gene background to illustrate the differences between the treatments. Unlike the uric acid levels in potassium oxonate (224.79 ± 33.62 µmol/L) and Uox-/- groups (458.39 ± 38.29 µmol/L), fructose treatment did not lead to higher serum uric acid level (174.93 ± 30.46 µmol/L) comparing to the control group (153.53 ± 40.96 µmol/L). However, abnormal glycometabolism only developed in the fructose and the Uox-/- group. In addition, anemia, inflammasome and severe renal injury occurred in the Uox-/- group. The Uox-/- mice were then treated with puerarin and allopurinol, and found that puerarin could reduce serum uric acid and alleviated the serious renal damage associated with high uric acid. Thus, the Uox-/- mice could be a suitable model for screening and evaluating anti-hyperuricemia compounds.

Engineered Escherichia coli Nissle 1917 with urate oxidase and an oxygen-recycling system for hyperuricemia treatment.

Hyperuricemia is the second most prevalent metabolic disease to human health after diabetes. Only a few clinical drugs are available, and most of them have serious side effects. The human body does not have urate oxidase, and uric acid is secreted via the kidney or the intestine. Reduction through kidney secretion is often the cause of hyperuricemia. We hypothesized that the intestine secretion could be enhanced when a recombinant urate-degrading bacterium was introduced into the gut. We engineered an Escherichia coli Nissle 1917 strain with a plasmid containing a gene cassette that encoded two proteins PucL and PucM for urate metabolism from Bacillus subtilis, the urate importer YgfU and catalase KatG from E. coli, and the bacterial hemoglobin Vhb from Vitreoscilla sp. The recombinant E. coli strain effectively degraded uric acid under hypoxic conditions. A new method to induce hyperuricemia in mice was developed by intravenously injecting uric acid. The engineered Escherichia coli strain significantly lowered the serum uric acid when introduced into the gut or directly injected into the blood vessel. The results support the use of urate-degrading bacteria in the gut to treat hyperuricemia. Direct injecting bacteria into blood vessels to treat metabolic diseases is proof of concept, and it has been tried to treat solid tumors.

Uricase-deficient rats with similarly stable serum uric acid to human's are sensitive model animals for studying hyperuricemia.

The aim of this study was to provide a sensitive model animal for studying hyperuricemia. Male uricase-deficient rats, named Kunming-DY rats, were raised for 130 days, or orally administered with purines and other chemicals. Serum uric acid (SUA) in the animals was assayed, and the UA level in their organs and their 24-h excretion was determined. Genes in the jejunum, ileum, kidney and liver related to UA synthesis and transportation were detected by quantitative RNA sequencing. Uricase-deficient rats have a high level of SUA and are sensitive to xanthine, adenosine, inosine, allopurinol, and alcohol. Besides, the high level of SUA in male uricase-deficient rats was stable, much higher than that in wild-type rats but similar to that in men. The distribution pattern of UA in uricase-deficient rats' organs was different from that in wild-type rats. The kidney, liver, and small intestine were the top three organs where UA distributed, but the UA in the small intestine, colon, lung, thymus, and brain was less affected by uricase deficiency, indicating that these organs are constitutive distribution organs in UA. The 24-h UA excreted by a uricase-deficient rat was about five times higher than that excreted by a wild-type rat. However, the 24-h UA excreted through feces was not significantly changed. Both the urine volume and UA in uricase-deficient rats significantly increased, and more than 90% of UA was excreted via urine. The expression of xanthine dehydrogenase was not upregulated. Some genes of transporter associated with uric acid excretion in the kidney were significantly regulated, though not sufficient to explain the increase in SUA. In conclusion, male uricase-deficient rats' UA metabolism is similar to that of men. The elevation of SUA in uricase-deficient rats is caused by uricase deficiency, and uricase-deficient rats are a sensitive model for studying hyperuricemia.

Xanthine Oxidoreductase Inhibitors Suppress the Onset of Exercise-Induced AKI in High HPRT Activity Urat1-Uox Double Knockout Mice.

BACKGROUND: Hereditary renal hypouricemia type 1 (RHUC1) is caused by URAT1/SLC22A12 dysfunction, resulting in urolithiasis and exercise-induced AKI (EIAKI). However, because there is no useful experimental RHUC1 animal model, the precise pathophysiologic mechanisms underlying EIAKI have yet to be elucidated. We established a high HPRT activity Urat1-Uox double knockout (DKO) mouse as a novel RHUC1 animal model for investigating the cause of EIAKI and the potential therapeutic effect of xanthine oxidoreductase inhibitors (XOIs). METHODS: The novel Urat1-Uox DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. RESULTS: Urat1-Uox DKO mice had uricosuric effects and elevated levels of plasma creatinine and BUN as renal injury markers, and decreased creatinine clearance observed in a forced swimming test. In addition, Urat1-Uox DKO mice had increased NLRP3 inflammasome activity and downregulated levels of Na+-K+-ATPase protein in the kidney, as Western blot analysis showed. Finally, we demonstrated that topiroxostat and allopurinol, XOIs, improved renal injury and functional parameters of EIAKI. CONCLUSIONS: Urat1-Uox DKO mice are a useful experimental animal model for human RHUC1. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1ß via NLRP3 inflammasome signaling and Na+-K+-ATPase dysfunction associated with excessive urinary urate excretion. In addition, XOIs appear to be a promising therapeutic agent for the treatment of EIAKI.

Uric acid: from a biological advantage to a potential danger. A focus on cardiovascular effects.

Non-communicable diseases represent nowadays the most common cause of death worldwide, having largely overcome infectious diseases. Among them, cardiovascular diseases constitute the majority. Given these premise, great efforts have been made by scientific societies to emphasize the fundamental role of cardiovascular prevention and risk factors control. In addition to classical cardiovascular risk factors such as smoking, arterial hypertension, hypercholesterolemia and male gender, new risk factors are emerging from international literature. Among them, uric acid is the protagonist. Several evidences show a direct role of hyperuricemia in the determinism of metabolic and vascular disorders. From the other hand, some researchers have demonstrated that uric acid is only a marker of cardiovascular damage and not a risk factor for its development. Aim of this review is to evaluate the scientific evidences on the role of uric acid in cardiovascular diseases in order to shed light on this confusing topic.

Hypoxia enhances H2O2-mediated upregulation of hepcidin: Evidence for NOX4-mediated iron regulation.

The exact regulation of the liver-secreted peptide hepcidin, the key regulator of systemic iron homeostasis, is still poorly understood. It is potently induced by iron, inflammation, cytokines or H2O2 but conflicting results have been reported on hypoxia. In our current study, we first show that pronounced (1%) and mild (5%) hypoxia strongly induces hepcidin in human Huh7 hepatoma and primary liver cells predominantly at the transcriptional level via STAT3 using two hypoxia systems (hypoxia chamber and enzymatic hypoxia by the GOX/CAT system). SiRNA silencing of JAK1, STAT3 and NOX4 diminished the hypoxia-mediated effect while a role of HIF1α could be clearly ruled out by the response to hypoxia-mimetics and competition experiments with a plasmid harboring the oxygen-dependent degradation domain of HIF1α. Specifically, hypoxia drastically enhances the H2O2-mediated induction of hepcidin strongly pointing towards an oxidase as powerful upstream control of hepcidin. We finally provide evidences for an efficient regulation of hepcidin expression by NADPH-dependent oxidase 4 (NOX4) in liver cells. In summary, our data demonstrate that hypoxia strongly potentiates the peroxide-mediated induction of hepcidin via STAT3 signaling pathway. Moreover, oxidases such as NOX4 or artificially overexpressed urate oxidase (UOX) can induce hepcidin. It remains to be studied whether the peroxide-STAT3-hepcidin axis simply acts to continuously compensate for oxygen fluctuations or is directly involved in iron sensing per se.

Testosterone-induced modulation of peroxisomal morphology and peroxisome-related gene expression in brown trout (Salmo trutta f. fario) primary hepatocytes.

Disruption of androgenic signaling has been linked to possible cross-modulation with other hormone-mediated pathways. Therefore, our objective was to explore effects caused by testosterone - T (1, 10 and 50µM) in peroxisomal signaling of brown trout hepatocytes. To study the underlying paths involved, several co-exposure conditions were tested, with flutamide - F (anti-androgen) and ICI 182,780 - ICI (anti-estrogen). Molecular and morphological approaches were both evaluated. Peroxisome proliferator-activated receptor alpha (PPARα), catalase and urate oxidase were the selected targets for gene expression analysis. The vitellogenin A gene was also included as a biomarker of estrogenicity. Peroxisome relative volumes were estimated by immunofluorescence, and transmission electron microscopy was used for qualitative morphological control. The single exposures of T caused a significant down-regulation of urate oxidase (10 and 50µM) and a general up-regulation of vitellogenin. A significant reduction of peroxisome relative volumes and smaller peroxisome profiles were observed at 50µM. Co-administration of T and ICI reversed the morphological modifications and vitellogenin levels. The simultaneous exposure of T and F caused a significant and concentration-dependent diminishing in vitellogenin expression. Together, the findings suggest that in the tested model, T acted via both androgen and estrogen receptors to shape the peroxisomal related targets.

Urat1-Uox double knockout mice are experimental animal models of renal hypouricemia and exercise-induced acute kidney injury.

Renal hypouricemia (RHUC) is a hereditary disease characterized by a low level of plasma urate but with normal urinary urate excretion. RHUC type 1 is caused by mutations of the urate transporter URAT1 gene (SLC22A12). However, the plasma urate levels of URAT1 knockout mice are no different from those of wild-type mice. In the present study, a double knockout mouse, in which the URAT1 and uricase (Uox) genes were deleted (Urat1-Uox-DKO), were used as an experimental animal model of RHUC type 1 to investigate RHUC and excise-induced acute kidney injury (EIAKI). Mice were given a variable content of allopurinol for one week followed by HPLC measurement of urate and creatinine concentrations in spot urine and blood from the tail. The urinary excretion of urate in Urat1-Uox-DKO mice was approximately 25 times higher than those of humans. With allopurinol, the plasma urate levels of Urat1-Uox-DKO mice were lower than those of Uox-KO mice. There were no differences in the urinary urate excretions between Urat1-Uox-DKO and Uox-KO mice administered with 9 mg allopurinol /100 g feed. In the absence of allopurinol, plasma creatinine levels of some Urat1-Uox-DKO mice were higher than those of Uox-KO mice. Consequently, hypouricemia and normouricosuria may indicate that the Urat1-Uox-DKO mouse administered with allopurinol may represent a suitable animal model of RHUC type 1. Urat1-Uox-DKO mice without allopurinol exhibited acute kidney injury, thus providing additional benefit as a potential animal model for EIAKI. Finally, our data indicate that allopurinol appears to provide prophylactic effects for EIAKI.

Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme.

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,1 as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2 while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system.

Coevolution of URAT1 and Uricase during Primate Evolution: Implications for Serum Urate Homeostasis and Gout.

Uric acid is the highly insoluble end-product of purine metabolism in humans. Serum levels exceeding the solubility threshold can trigger formation of urate crystals resulting in gouty arthritis. Uric acid is primarily excreted through the kidneys with 90% reabsorbed back into the bloodstream through the uric acid transporter URAT1. This reabsorption process is essential for the high serum uric acid levels found in humans. We discovered that URAT1 proteins from humans and baboons have higher affinity for uric acid compared with transporters from rats and mice. This difference in transport kinetics of URAT1 orthologs, along with inability of modern apes to oxidize uric acid due to loss of the uricase enzyme, prompted us to ask whether these events occurred concomitantly during primate evolution. Ancestral URAT1 sequences were computationally inferred and ancient transporters were resurrected and assayed, revealing that affinity for uric acid was increased during the evolution of primates. This molecular fine-tuning occurred between the origins of simians and their diversification into New- and Old-World monkey and ape lineages. Remarkably, it was driven in large-part by only a few amino acid replacements within the transporter. This alteration in primate URAT1 coincided with changes in uricase that greatly diminished the enzymatic activity and took place 27-77 Ma. These results suggest that the modifications to URAT1 transporters were potentially adaptive and that maintaining more constant, high levels of serum uric acid may have provided an advantage to our primate ancestors.

Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity.

Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two ß-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼ 20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cß distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins.

Cloning, Expression, and Purification of Recombinant Uricase Enzyme from Pseudomonas aeruginosa Ps43 Using Escherichia coli.

Uricase is an important microbial enzyme that can be used in the clinical treatment of gout, hyperuricemia, and tumor lysis syndrome. A total of 127 clinical isolates of Pseudomonas aeruginosa were tested for uricase production. A Pseudomonas strain named Ps43 showed the highest level of native uricase enzyme expression. The open reading frame of the uricase enzyme was amplified from Ps43 and cloned into the expression vector pRSET-B. Uricase was expressed using E. coli BL21 (DE3). The ORF was sequenced and assigned GenBank Accession No. KJ718888. The nucleotide sequence analysis was identical to the coding sequence of uricase gene puuD of P. aeruginosa PAO1. We report the successful expression of P. aeruginosa uricase in Escherichia coli. E. coli showed an induced protein with a molecular mass of about 58 kDa that was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. We also established efficient protein purification using the Ni-Sepharose column with activity of the purified enzyme of 2.16 IU and a 2-fold increase in the specific activity of the pure enzyme compared with the crude enzyme.

Uric acid-dependent inhibition of AMP kinase induces hepatic glucose production in diabetes and starvation: evolutionary implications of the uricase loss in hominids.

Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.

Evolutionary history and metabolic insights of ancient mammalian uricases.

Uricase is an enzyme involved in purine catabolism and is found in all three domains of life. Curiously, uricase is not functional in some organisms despite its role in converting highly insoluble uric acid into 5-hydroxyisourate. Of particular interest is the observation that apes, including humans, cannot oxidize uric acid, and it appears that multiple, independent evolutionary events led to the silencing or pseudogenization of the uricase gene in ancestral apes. Various arguments have been made to suggest why natural selection would allow the accumulation of uric acid despite the physiological consequences of crystallized monosodium urate acutely causing liver/kidney damage or chronically causing gout. We have applied evolutionary models to understand the history of primate uricases by resurrecting ancestral mammalian intermediates before the pseudogenization events of this gene family. Resurrected proteins reveal that ancestral uricases have steadily decreased in activity since the last common ancestor of mammals gave rise to descendent primate lineages. We were also able to determine the 3D distribution of amino acid replacements as they accumulated during evolutionary history by crystallizing a mammalian uricase protein. Further, ancient and modern uricases were stably transfected into HepG2 liver cells to test one hypothesis that uricase pseudogenization allowed ancient frugivorous apes to rapidly convert fructose into fat. Finally, pharmacokinetics of an ancient uricase injected in rodents suggest that our integrated approach provides the foundation for an evolutionarily-engineered enzyme capable of treating gout and preventing tumor lysis syndrome in human patients.

Strengthening the stability of a tunnel-shaped homotetramer protein with nanogels.

Urate oxidase (UOX, EC 1.7.3.3) is effective for the treatment of gout and hyperuricaemia associated with tumor lysis syndrome. The inherent poor stability of UOX to temperature, proteolysis, and acidic environments is known to limit its efficacy. Herein, we encapsulated UOX into spherical and porous nanogels with diameters of 20-40 nm via a two-step in situ polymerization in the presence of oxonic acid potassium salt, an inhibitor of UOX. The UOX nanogel retained 70% of the initial activity but showed an expanded pH spectrum from pH 6-10 to 3-10 and an extended half-life at 37 °C from 5 min to 3 h. The enhanced pH stability, thermal stability, and enzyme resistance of the UOX nanogels were also confirmed by using fluorescence spectroscopy and enzymatic digestion. A molecular dynamics simulation was performed as a way to probe the mechanism underlying the formation of UOX nanogels as well as the strengthened stability against harsh conditions. It was shown that the encapsulation into the polyacrylamide network reinforced the intersubunit hydrogen bonding, shielded the hydrolytic reaction site, and thus protected the tertiary and quaternary structure of UOX. The UOX nanogel with enhanced stability provided a stable enzyme model that enables the exploration of UOX in the diagnosis and therapy of disorders associated with altered purine metabolism.