HDHD1, which is often deleted in X-linked ichthyosis, encodes a pseudouridine-5'-phosphatase.

Pseudouridine, the fifth-most abundant nucleoside in RNA, is not metabolized in mammals, but is excreted intact in urine. The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5'-phosphate, a potential intermediate in RNA degradation. We show that human erythrocytes contain a pseudouridine-5'-phosphatase displaying a Km ≤ 1 µM for its substrate. The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate, suggesting that it belonged to the 'haloacid dehalogenase' family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant HDHD1 dephosphorylated pseudouridine 5'-phosphate with a kcat of 1.6 s-1, a Km of 0.3 µM and a catalytic efficiency at least 1000-fold higher than that on which it acted on other phosphate esters, including 5'-UMP. The molecular identity of pseudouridine-5'-phosphatase was confirmed by the finding that its activity was negligible (<10% of controls) in extracts of B-cell lymphoblasts or erythrocytes from X-linked ichthyosis patients harbouring a combined deletion of the STS gene (the X-linked ichthyosis gene) and the HDHD1 gene. Furthermore, pseudouridine-5'-phosphatase activity was 1.5-fold higher in erythrocytes from women compared with men, in agreement with the HDHD1 gene undergoing only partial inactivation in females. In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RNA.

Cloning of neuropeptide-like precursor 1 in the gray flesh fly and peptide identification and expression.

The neuropeptide-like precursor 1 (NPLP1) was first identified in a peptidomics experiment on Drosophila melanogaster. Limited data on this novel neuropeptide precursor suggest a role in the regulation of ecdysis in holometabolous larvae. In this study, we characterized the NPLP1 precursor in the gray flesh fly, Neobellieria bullata, which is an excellent model for physiological assays and hence to discover the role of the NPLP1 peptides. Antisera against three of the D. melanogaster NPLP1 neuropeptides stained an identical set of neurons in the central nervous system of N. bullata compared to D. melanogaster. A novel approach was applied to identify the N. bullata NPLP1 orthologs. Using a combination of affinity chromatography, mass spectrometry, cDNA cloning and RACE experiments, we obtained almost the complete coding sequence of the NPLP1 mRNA. Three encoded NPLP1 peptides were identified in central nervous system extracts by mass spectrometry. Neither doses of 25-250pmol of synthetic Neb-MGYamide and Neb-PQNamide peptides, nor the NPLP1 antisera did affect the speed of retraction, contraction and tanning in the pupariation bioassay.

Molecular identification of pseudouridine-metabolizing enzymes.

Pseudouridine, a non-classical nucleoside present in human urine as a degradation product of RNAs, is one of the few molecules that has a glycosidic C-C bond. Through a data base mining approach involving transcriptomic data, we have molecularly identified two enzymes that are involved in the metabolism of pseudouridine in uropathogenic Escherichia coli, the principal agent of urinary tract infections in humans. The first enzyme, coded by the gene yeiC, specifically phosphorylates pseudouridine to pseudouridine 5'-phosphate. Accordingly, yeiC(-) mutants are unable to metabolize pseudouridine, in contrast to wild-type E. coli UTI89. The second enzyme, encoded by the gene yeiN belonging to the same operon as yeiC, catalyzes the conversion of pseudouridine 5'-phosphate to uracil and ribose 5-phosphate in a divalent cation-dependent manner. Remarkably, the glycosidic C-C bond of pseudouridine is cleaved in the course of this reaction, indicating that YeiN is the first molecularly identified enzyme able to hydrolyze a glycosidic C-C bond. Though this reaction is easily reversible, the association of YeiN with pseudouridine kinase indicates that it serves physiologically to metabolize pseudouridine 5'-phosphate rather than to form it. YeiN is homologous to Thermotoga maritima IndA, a protein with a new fold, which we now show to act also as a pseudouridine-5'-phosphate glycosidase. Data base mining indicates that most eukaryotes possess homologues of pseudouridine kinase and pseudouridine-5'-phosphate glycosidase and that these are most often associated in a single bifunctional protein. The gene encoding this bifunctional protein is absent from the genomes of man and other mammals, indicating that the capacity for metabolizing pseudouridine has been lost late in evolution.