Motoaki Wakiyama

Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein

The TAR RNA‐binding Protein (TRBP) is a double‐stranded RNA (dsRNA)‐binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA‐binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α−β−β−β−α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)‐binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full‐length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation‐π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.

A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo

A pyrrolo-pyrimidine kinase inhibitor, RK-20449, eliminates chemotherapy-resistant human primary AML stem cells in vivo. Taking AML Head-On Like the mythic Lernaean Hydra, acute myeloid leukemia (AML) is hard to kill. It seems like every time one head is cut off, another two—meaner—grow back in its place. Leukemia stem cells (LSCs) are thought to contribute to this resilience; they may survive conventional chemotherapy and increase the risk of relapse. However, it has been difficult to specifically target these cells without also hitting the normal hematopoietic stem cells (HSCs) required for maintaining healthy blood cells. Now, Saito et al. find a new candidate drug that can specifically target LSCs. The authors performed a chemical library screen to target hematopoietic cell kinase (HCK), which they had previously found to be differentially expressed in human LSCs compared with HSCs. They found a candidate HCK inhibitor, RK-20449, which is a pyrrolo-pyrimidine derivative that could bind the active pocket of HCK. In a mouse xenograft of aggressive human AML, RK-20449 greatly reduced LSC burden. If these studies hold true in patients, RK-20449 could accomplish the Herculean task of decreasing the risk of relapse in AML. Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rgnull mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

Genetic encoding of non‐natural amino acids in Drosophila melanogaster Schneider 2 cells

Insect cells are useful for the high‐yield production of recombinant proteins including chemokines and membrane proteins. In this study, we developed an insect cell‐based system for incorporating non‐natural amino acids into proteins at specific sites. Three types of promoter systems were constructed, and their efficiencies were compared for the expression of the prokaryotic amber suppressor tRNATyr in Drosophila melanogaster Schneider 2 cells. When paired with a variant of Escherichia coli tyrosyl‐tRNA synthetase specific for 3‐iodo‐L‐tyrosine, the suppressor tRNA transcribed from the U6 promoter most efficiently incorporated the amino acid into proteins in the cells. The transient and stable introductions of these prokaryotic molecules into the insect cells were then compared in terms of the yield of proteins containing non‐natural amino acids, and the “transient” method generated a sevenfold higher yield. By this method, 4‐azido‐L‐phenylalanine was incorporated into human interleukin‐8 at a specific site. The yield of the azido‐containing IL‐8 was 1 μg/1 mL cell culture, and the recombinant protein was successfully labeled with a fluorescent probe by the Staudinger–Bertozzi reaction.

MRNA ENCODING THE TRANSLATION INITIATION FACTOR EIF-4E IS EXPRESSED EARLY IN XENOPUS EMBRYOGENESIS

The translation initiation factor eIF‐4E plays an important role in regulating the overall rate of translation in eukaryotic cells. To investigate the expression of eIF‐4E itseIF, we characterized the eIF‐4E mRNA expressed in Xenopus embryos. 5′‐RACE was performed to determine the 5′‐end of the mRNA and the result predicts isoforms differing at the amino‐terminal end. Expression of the eIF‐4E mRNA in Xenopus oocytes and embryos was examined by RT‐PCR. Xenopus eIF‐4E mRNA is produced during oogenesis and persists during the early stages of embryogenesis as a maternal mRNA.

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)+ and poly(A)- mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease untreated rabbit reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs, stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.

Tetrameric Interaction of the Ectoenzyme CD38 on the Cell Surface Enables Its Catalytic and Raft-Association Activities

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

High-level expression of porcine muscle adenylate kinase in Escherichia coli: Effects of the copy number of the gene and the translational initiation signals

Porcine muscle adenylate kinase (ADK) was overproduced in Escherichia coli using the expression plasmid with double A-T-G codon at the translational starting site and the Shine-Dalgarno (SD) sequence 10 bp apart from the first A-T-G. We used the expression vectors pKK223-3 and pMK2. pMK2 is about 10-20 times larger in copy number than pK223-3. For both vectors, duplication of A-T-G was effective and the quantity of the expressed ADK from the double A-T-G plasmid was 2 approximately 4-fold more than that achieved when only one A-T-G was present. The amount of the produced ADK was maximum in the case of using pMK2 with double A-T-G. The overproduced ADK formed inclusion bodies in E. coli. It was solubilized in 6 M guanidine hydrochloride and refolded. Through two steps of column chromatography, ADK was purified. It has the same amino acid composition and grossly the same activity as that reported by Schirmer et al. (1970). Its amino acid sequence of the NH2-terminal region was identical with that deduced from the cDNA sequence including the NH2-terminal methionine.

Structural basis for the specific recognition of the major antigenic peptide from the Japanese cedar pollen allergen Cry j 1 by HLA-DP5

The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

Anaphase‐promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell‐cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C‐Cdc20, and functions as a cytostatic factor that causes long‐term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc‐binding region (ZBR) domain of Emi2 inhibits cell‐cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin‐RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain‐initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double‐faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin‐RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C‐terminal RL residues [the post‐ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2‐specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.