Toru Sengoku

UTX and UTY Demonstrate Histone Demethylase-Independent Function in Mouse Embryonic Development

UTX (KDM6A) and UTY are homologous X and Y chromosome members of the Histone H3 Lysine 27 (H3K27) demethylase gene family. UTX can demethylate H3K27; however, in vitro assays suggest that human UTY has lost enzymatic activity due to sequence divergence. We produced mouse mutations in both Utx and Uty. Homozygous Utx mutant female embryos are mid-gestational lethal with defects in neural tube, yolk sac, and cardiac development. We demonstrate that mouse UTY is devoid of in vivo demethylase activity, so hemizygous XUtx− Y+ mutant male embryos should phenocopy homozygous XUtx− XUtx− females. However, XUtx− Y+ mutant male embryos develop to term; although runted, approximately 25% survive postnatally reaching adulthood. Hemizygous X+ YUty− mutant males are viable. In contrast, compound hemizygous XUtx− YUty− males phenocopy homozygous XUtx− XUtx− females. Therefore, despite divergence of UTX and UTY in catalyzing H3K27 demethylation, they maintain functional redundancy during embryonic development. Our data suggest that UTX and UTY are able to regulate gene activity through demethylase independent mechanisms. We conclude that UTX H3K27 demethylation is non-essential for embryonic viability.

A short peptide insertion crucial for angiostatic activity of human tryptophanyl-tRNA synthetase

Human tryptophanyl-tRNA synthetase (TrpRS) is secreted into the extracellular region of vascular endothelial cells. The splice variant form (mini TrpRS) functions in vascular endothelial cell apoptosis as an angiostatic cytokine. In contrast, the closely related human tyrosyl-tRNA synthetase (TyrRS) functions as an angiogenic cytokine in its truncated form (mini TyrRS). Here, we determined the crystal structure of human mini TrpRS at a resolution of 2.3 Å and compared the structure with those of prokaryotic TrpRS and human mini TyrRS. Deletion of the tRNA anticodon-binding (TAB) domain insertion, consisting of eight residues in the human TrpRS, abolished the enzymes apoptotic activity for endothelial cells, whereas its translational catalysis and cell-binding activities remained unchanged. Thus, we have identified the inserted peptide motif that activates the angiostatic signaling.

Crystal Structure of tRNA Adenosine Deaminase (TadA) from Aquifex aeolicus

The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-2. This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the β4 and β5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop.

Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the “editing” mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-βN) from the archaeon, Pyrococcus horikoshii, at 1.94-Å resolution. PheRS-βN includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe, indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNAPhe deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNAPhe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

Substrate tRNA Recognition Mechanism of a Multisite-specific tRNA Methyltransferase, Aquifex aeolicus Trm1, Based on the X-ray Crystal Structure

Archaeal and eukaryotic tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) produces N2,N2-dimethylguanine at position 26 in tRNA. In contrast, Trm1 from Aquifex aeolicus, a hyper-thermophilic eubacterium, modifies G27 as well as G26. Here, a gel mobility shift assay revealed that the T-arm in tRNA is the binding site of A. aeolicus Trm1. To address the multisite specificity, we performed an x-ray crystal structure study. The overall structure of A. aeolicus Trm1 is similar to that of archaeal Trm1, although there is a zinc-cysteine cluster in the C-terminal domain of A. aeolicus Trm1. The N-terminal domain is a typical catalytic domain of S-adenosyl-l-methionine-dependent methyltransferases. On the basis of the crystal structure and amino acid sequence alignment, we prepared 30 mutant Trm1 proteins. These mutant proteins clarified residues important for S-adenosyl-l-methionine binding and enabled us to propose a hypothetical reaction mechanism. Furthermore, the tRNA-binding site was also elucidated by methyl transfer assay and gel mobility shift assay. The electrostatic potential surface models of A. aeolicus and archaeal Trm1 proteins demonstrated that the distribution of positive charges differs between the two proteins. We constructed a tRNA-docking model, in which the T-arm structure was placed onto the large area of positive charge, which is the expected tRNA-binding site, of A. aeolicus Trm1. In this model, the target G26 base can be placed near the catalytic pocket; however, the nucleotide at position 27 gains closer access to the pocket. Thus, this docking model introduces a rational explanation of the multisite specificity of A. aeolicus Trm1.

Characterization and Structure of the Aquifex aeolicus Protein DUF752 A BACTERIAL tRNA-METHYLTRANSFERASE (MnmC2) FUNCTIONING WITHOUT THE USUALLY FUSED OXIDASE DOMAIN (MnmC1)

Background: Escherichia coli encodes a bifunctional oxidase/methyltransferase catalyzing the final steps of methylaminomethyluridine (mnm5U) formation in tRNA wobble positions. Results: Aquifex aeolicus encodes only a monofunctional aminomethyluridine-dependent methyltransferase, lacking the oxidase domain. Conclusion: An alternative pathway exists for mnm5U biogenesis. Significance: Information about how an organism modifies the wobble base of its tRNA is important for understanding the emergence of the genetic code. Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm5U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm5U34) by the MnmE-MnmG enzyme complex. The cmnm5U34 is further modified to mnm5U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm5U into 5-aminomethyluridine (nm5U34), and this reaction is immediately followed by the methylation of the free amino group into mnm5U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm5U in vitro, to form mnm5U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm5U34 in tRNAs.

Crystal structure of the bifunctional tRNA modification enzyme MnmC from Escherichia coli.

Post‐transcriptional modifications of bases within the transfer RNAs (tRNA) anticodon significantly affect the decoding system. In bacteria and eukaryotes, uridines at the wobble position (U34) of some tRNAs are modified to 5‐methyluridine derivatives (xm5U). These xm5U34‐containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box. In Escherichia coli (E.coli), the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5‐carboxymethylaminomethyl uridine (cmnm5U) to 5‐methylaminomethyl uridine (mnm5U). The C‐terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)‐dependent deacetylation of cmnm5U to 5‐aminomethyl uridine (nm5U), whereas the N‐terminal domain (MnmC2) catalyzes the subsequent S‐adenosyl‐L‐methionine‐dependent methylation of nm5U, leading to the final product, mnm5U34. Here, we determined the crystal structure of E.coli MnmC containing FAD, at 3.0 Å resolution. The structure of the MnmC1 domain can be classified in the FAD‐dependent glutathione reductase 2 structural family, including the glycine oxidase ThiO, whereas the MnmC2 domain adopts the canonical class I methyltransferase fold. A structural comparison with ThiO revealed the residues that may be involved in cmnm5U recognition, supporting previous mutational analyses. The catalytic sites of the two reactions are both surrounded by conserved basic residues for possible anticodon binding, and are located far away from each other, on opposite sides of the protein. These results suggest that, although the MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner.

Crystallization and preliminary X-ray analysis of the helicase domains of Vasa complexed with RNA and an ATP analogue

The helicase fragment of Vasa was purified and its RNA-binding activity was examined by a UV cross-linking assay. The fragment was crystallized in complex with poly(U) RNA (U(10)) and a non-hydrolyzable analogue of ATP. The crystal belonged to space group P2(1), with unit-cell parameters a = 71.06, b = 142.35, c = 130.47 A, beta = 90.86 degrees. The cryocooled crystal diffracted to about 2.2 A using synchrotron radiation from station BL41XU at SPring-8.

Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity

The ten–eleven translocation (TET) protein family, consisting of three isoforms (TET1/2/3), have been found in mammalian cells and have a crucial role in 5‐methylcytosine demethylation in genomic DNA through the catalysis of oxidation reactions assisted by 2‐oxoglutarate (2OG). DNA methylation/demethylation contributes to the regulation of gene expression at the transcriptional level, and recent studies have revealed that TET1 is highly elevated in malignant cells of various diseases and related to malignant alteration. TET1 inhibitors based on a scaffold of thioether macrocyclic peptides, which have been discovered by the random nonstandard peptide integrated discovery (RaPID) system, are reported. The affinity‐based selection was performed against the TET1 compact catalytic domain (TET1CCD) to yield thioether macrocyclic peptides. These peptides exhibited inhibitory activity of the TET1 catalytic domain (TET1CD), with an IC50 value as low as 1.1 μm. One of the peptides, TiP1, was also able to inhibit TET1CD over TET2CD with tenfold selectivity, although it was likely to target the 2OG binding site; this provides a good starting point to develop more selective inhibitors.