Background and objectives: Hepcidin is a key regulator of iron homeostasis, but its study in the setting of chronic kidney disease (CKD) has been hampered by the lack of validated serum assays.Design, setting, participants, & measurements: This study reports the first measurements of bioactive serum hepcidin using a novel competitive ELISA in 48 pediatric (PCKD2-4) and 32 adult (ACKD2-4) patients with stages 2 to 4 CKD along with 26 pediatric patients with stage 5 CKD (PCKD5D) on peritoneal dialysis.Results: When compared with their respective controls (pediatric median ‫؍‬ 25.3 ng/ml, adult ‫؍‬ 72.9 ng/ml), hepcidin was significantly increased in PCKD2-4 (127.3 ng/ml), ACKD2-4 (269.9 ng/ml), and PCKD5D (652.4 ng/ml). Multivariate regression analysis was used to assess the relationship between hepcidin and indicators of anemia, iron status, inflammation, and renal function. In PCKD2-4 (R 2 ‫؍‬ 0.57), only ferritin correlated with hepcidin. In ACKD2-4 (R 2 ‫؍‬ 0.78), ferritin and soluble transferrin receptor were associated with hepcidin, whereas GFR was inversely correlated. In PCKD5D (R 2 ‫؍‬ 0.52), percent iron saturation and ferritin were predictors of hepcidin. In a multivariate analysis that incorporated all three groups (R 2 ‫؍‬ 0.6), hepcidin was predicted by ferritin, C-reactive protein, and whether the patient had stage 5D versus stages 2 to 4 CKD.Conclusions: These findings suggest that increased hepcidin across the spectrum of CKD may contribute to abnormal iron regulation and erythropoiesis and may be a novel biomarker of iron status and erythropoietin resistance.

The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-Dglucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.Vitamin C (L-ascorbic acid) is well known as an important antioxidant and enzyme cofactor in animals (1, 2) and in plants (3). Apparently, all plants are able to produce vitamin C and mutants completely deficient in synthesis have not been described, suggesting an essential role of ascorbate biosynthesis in these organisms (4). Although vertebrate vitamin C synthesis is restricted to one organ (liver in mammals and kidney in fish, amphibians, and reptiles) (5, 6), virtually all cells in plants can form ascorbate (4). In the few vertebrate species, such as humans, which lack ascorbate biosynthesis, loss of the pathway is compensated by dietary intake, particularly from plants.Different pathways of ascorbate synthesis have evolved in animals and plants. In animals, ascorbate is formed from UDP-D-glucuronate in a pathway involving D-glucuronate formation, reduction and lactonization of D-glucuronate to L-gulonolactone and oxidation of the latter to L-ascorbate (reviewed in Ref. 7). Deficiency of the enzyme catalyzing this last step (L-gulonolactone oxidase) is responsible for the loss of ascorbate synthesis in the vitamin C-requiring vertebrates (8). In plants, the ascorbate synthesis pathway has remained elusive until recently and alternative pathways may exist (9). The Smirnoff-Wheeler pathway (10) has garnered strong biochemical and genetic support (11-16) and appears to represent the major route to ascorbate biosynthesis. In this pathway, GDP-D-mannose, formed from D-mannose 1-phosphate, is successively converted to GDP-L-galactose, L-galactose 1-phosphate, L-galactose, L-galactono-1,4-lactone, and finally to L-ascorbate.Screens for ozone-sensitive (17) or ascorbate-deficient (18) mutants in Arabidopsis thaliana led to the identification of four loci (VTC1, VTC2, VTC3, and VTC4) involved in the maintenance of the vitamin C pool. Characterization of the vtc1 (19) and vtc4 (15) mutants, as well as biochemical studies (14), have allowed the identification of two of the enzymes required for L-ascorbic acid synthesis through the Smirnoff-Wheeler pathway. VTC1 and VTC4 encode GDP-mannose pyrophosphorylase (19) and L-Gal-1-P ...

We have shown that Rpl3, a protein of the large ribosomal subunit from baker's yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by topdown and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-L-[methyl-3 H]methionine. The results show that a ؉14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven ␤-strand methyltransferase, results in the loss of the ؉14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.The addition of methyl groups to proteins from the methyl donor S-adenosylmethionine is one of the most common posttranslational modifications, resulting in an expansion of the physico-chemical characteristics of amino acids and the potential to modulate protein function (1). Major sites of protein methylation are at lysine and arginine residues (2, 3), and less major sites include glutamate, glutamine, and histidine residues, as well as N-terminal amino and C-terminal carboxyl groups (4 -6). The extensive role of histone methylation in transcriptional control highlights the biological significance of this modification (7-10). Protein methylation is also important in the translational machinery. Indeed, many proteins involved in translation, including ribosomal proteins and various elongation and release factors, are subject to methylation in both prokaryotes and eukaryotes (11).Saccharomyces cerevisiae is an ideal organism to investigate the methylation of ribosomal proteins; its genome is well annotated and single open reading frame gene deletion mutants are available. High-resolution intact mass spectrometry suggested that six proteins of the large ribosomal subunit may be methylated: Rpl1ab, Rpl3, Rpl12ab, Rpl23ab, Rpl42ab, and Rpl43ab (12).2 This study, however, did not identify the sites of methylation in these proteins nor did it identify the corresponding methyltransferases. In our laboratory, we have been interested in characterizing these modifications and identifying the methyltransferases involved in an effort to understand their physiological significance in trans...

Using OPTN/UNOS database, we identified risk factors for development of NODM. Some of these factors are potentially modifiable, including obesity, HCV infection, and the use of tacrolimus. Clinical trials are needed to assess whether modifying these "modifiable risk factors" will indeed prevent NODM.

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.

We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation on Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.

Cellular metabolism converts available nutrients into usable energy and biomass precursors. The process is regulated to facilitate efficient nutrient use and metabolic homeostasis. Feedback inhibition of the first committed step of a pathway by its final product is a classical means of controlling biosynthesis1–4. In a canonical example, the first committed enzyme in the pyrimidine pathway in Escherichia coli is allosterically inhibited by cytidine triphosphate1,4,5. The physiological consequences of disrupting this regulation, however, have not been previously explored. Here we identify an alternative regulatory strategy that enables precise control of pyrimidine pathway end-product levels, even in the presence of dysregulated biosynthetic flux. The mechanism involves cooperative feedback regulation of the near-terminal pathway enzyme uridine monophosphate kinase6. Such feedback leads to build-up of the pathway intermediate uridine monophosphate, which is in turn degraded by a conserved phosphatase, here termed UmpH, with previously unknown physiological function7,8. Such directed overflow metabolism allows homeostasis of uridine triphosphate and cytidine triphosphate levels at the expense of uracil excretion and slower growth during energy limitation. Disruption of the directed overflow regulatory mechanism impairs growth in pyrimidine-rich environments. Thus, pyrimidine homeostasis involves dual regulatory strategies, with classical feedback inhibition enhancing metabolic efficiency and directed overflow metabolism ensuring end-product homeostasis.

Despite the use of erythropoiesis-stimulating agents (ESAs), the anemia of chronic kidney disease (CKD) can be resistant to therapy. Both absolute and functional iron deficiency along with inflammation can contribute to ESA resistance and can be difficult to identify with current-day markers of iron storage. Hepcidin, a small peptide produced by the liver, is a recently discovered key regulator of iron homeostasis. Via regulation of ferroportin, hepcidin inhibits intestinal iron absorption and iron release from macrophages and hepatocytes. Because of its renal elimination and regulation by inflammation, it is possible that progressive renal insufficiency leads to altered hepcidin metabolism, subsequently affecting enteric absorption of iron and the availability of iron stores. Thus, hepcidin likely plays a major role in the anemia of CKD as well as ESA resistance. This article discusses the biologic actions and regulation of hepcidin along with reviewing studies of hepcidin in CKD.