Hirotaka Narita

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291–1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.

Nonagonistic Dectin-1 ligand transforms CpG into a multitask nanoparticulate TLR9 agonist

Significance CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 ligand, is a promising immunotherapeutic agent; however, developing an IFN-inducing CpG ODN forming a stable nanoparticle without aggregation has been unsuccessful. Here we generated a nanoparticulate CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. K3-SPG stimulates human peripheral blood mononuclear cells to produce large amounts of both type I and II IFN. K3-SPG thus became a potent adjuvant, especially for cytotoxic T-lymphocyte (CTL) induction to coadministered protein antigens without conjugation, which is attributable to its nanoparticulate nature rather than to targeting Dectin-1. Protective potency of K3-SPG as an influenza vaccine adjuvant was demonstrated in both murine and nonhuman primate models. K3-SPG may be used as an IFN inducer as well as a CTL inducer for immunotherapeutic applications. CpG DNA, a ligand for Toll-like receptor 9 (TLR9), has been one of the most promising immunotherapeutic agents. Although there are several types of potent humanized CpG oligodeoxynucleotide (ODN), developing “all-in-one” CpG ODNs activating both B cells and plasmacytoid dendritic cells forming a stable nanoparticle without aggregation has not been successful. In this study, we generated a novel nanoparticulate K CpG ODN (K3) wrapped by the nonagonistic Dectin-1 ligand schizophyllan (SPG), K3-SPG. In sharp contrast to K3 alone, K3-SPG stimulates human peripheral blood mononuclear cells to produce a large amount of both type I and type II IFN, targeting the same endosome where IFN-inducing D CpG ODN resides without losing its K-type activity. K3-SPG thus became a potent adjuvant for induction of both humoral and cellular immune responses, particularly CTL induction, to coadministered protein antigens without conjugation. Such potent adjuvant activity of K3-SPG is attributed to its nature of being a nanoparticle rather than targeting Dectin-1 by SPG, accumulating and activating antigen-bearing macrophages and dendritic cells in the draining lymph node. K3-SPG acting as an influenza vaccine adjuvant was demonstrated in vivo in both murine and nonhuman primate models. Taken together, K3-SPG may be useful for immunotherapeutic applications that require type I and type II IFN as well as CTL induction.

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions.

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.

Lipocalin 2 Bolsters Innate and Adaptive Immune Responses to Blood-Stage Malaria Infection by Reinforcing Host Iron Metabolism

Plasmodium parasites multiply within host erythrocytes, which contain high levels of iron, and parasite egress from these cells results in iron release and host anemia. Although Plasmodium requires host iron for replication, how host iron homeostasis and responses to these fluxes affect Plasmodium infection are incompletely understood. We determined that Lipocalin 2 (Lcn2), a host protein that sequesters iron, is abundantly secreted during human (P. vivax) and mouse (P. yoeliiNL) blood-stage malaria infections and is essential to control P. yoeliiNL parasitemia, anemia, and host survival. During infection, Lcn2 bolsters both host macrophage function and granulocyte recruitment and limits reticulocytosis, or the expansion of immature erythrocytes, which are the preferred target cell of P. yoeliiNL. Additionally, a chronic iron imbalance due to Lcn2 deficiency results in impaired adaptive immune responses against Plasmodium parasites. Thus, Lcn2 exerts antiparasitic effects by maintaining iron homeostasis and promoting innate and adaptive immune responses.

Two crystal modifications of (Pro-Pro-Gly)4-Hyp-Hyp-Gly-(Pro-Pro-Gly)4 reveal the puckering preference of Hyp(X) in the Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs in collagen helices.

Two crystal modifications of a collagen model peptide, (Pro-Pro-Gly)(4)-Hyp-Hyp-Gly-(Pro-Pro-Gly)(4) [where Hyp is (4R,2S)-L-hydroxyproline], showed very similar unit-cell parameters and belonged to the same space group P2(1). Both crystals exhibited pseudo-merohedral twinning. The main difference was in their molecular-packing arrangements. One modification showed pseudo-hexagonal packing, while the other showed pseudo-tetragonal packing. Despite their different packing arrangements, no significant differences were observed in the hydration states of these modifications. The peptide in the pseudo-tetragonal crystal showed a cyclic fluctuation of helical twists with a period of 20 A, while that in the pseudo-hexagonal crystal did not. In these modifications, the puckering conformations of four of the 12 Hyp residues at the X position of the Hyp(X)-Hyp(Y)-Gly sequence were in the opposite conformations to the previous hypothesis that Hyp(X) residues involved in Hyp(X):Hyp(Y) and Hyp(X):Pro(Y) stacking pairs prefer up-puckering and down-puckering conformations, respectively. Detailed investigation of the molecular interactions between Hyp(X) and adjacent molecules revealed that these opposite conformations appeared because the puckering conformation, which follows the hypothesis, is subject to steric hindrance from the adjacent molecule.

Crystal structure of afadin PDZ domain–nectin‐3 complex shows the structural plasticity of the ligand‐binding site

Afadin, a scaffold protein localized in adherens junctions (AJs), links nectins to the actin cytoskeleton. Nectins are the major cell adhesion molecules of AJs. At the initial stage of cell–cell junction formation, the nectin–afadin interaction plays an indispensable role in AJ biogenesis via recruiting and tethering other components. The afadin PDZ domain (AFPDZ) is responsible for binding the cytoplasmic C‐terminus of nectins. AFPDZ is a class II PDZ domain member, which prefers ligands containing a class II PDZ‐binding motif, X‐Φ‐X‐Φ (Φ, hydrophobic residues); both nectins and other physiological AFPDZ targets contain this class II motif. Here, we report the first crystal structure of the AFPDZ in complex with the nectin‐3 C‐terminal peptide containing the class II motif. We engineered the nectin‐3 C‐terminal peptide and AFPDZ to produce an AFPDZ–nectin‐3 fusion protein and succeeded in obtaining crystals of this complex as a dimer. This novel dimer interface was created by forming an antiparallel β sheet between β2 strands. A major structural change compared with the known AFPDZ structures was observed in the α2 helix. We found an approximately 2.5 Å‐wider ligand‐binding groove, which allows the PDZ to accept bulky class II ligands. Apparently, the last three amino acids of the nectin‐3 C‐terminus were sufficient to bind AFPDZ, in which the two hydrophobic residues are important.

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

Significance Each retinal rod and cone photoreceptor cell subtype expresses a distinct set of genes including the genes encoding phototransduction components such as opsins and transducins. This subtype-specific gene expression is essential for achieving proper function in each photoreceptor cell. While the various cone and rod photoreceptor cell gene expressions are regulated by multiple transcription factors, whether epigenetic regulation plays an important role for photoreceptor cell-specific gene expression has been unclear. In the current study, we found that a photoreceptor-specific protein, Samd7, functions as a component of the epigenetic gene-silencing complex and is essential for establishing rod photoreceptor cell identity and function by silencing nonrod gene expression in developing rod photoreceptor cells. Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7-null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7-deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.

Purification and characterization of recombinant sugarcane sucrose phosphate synthase expressed in E. coli and insect Sf9 cells: An importance of the N-terminal domain for an allosteric regulatory property

Sucrose phosphate synthase (SPS) catalyses the transfer of glycosyl group of uridine diphosphate glucose to fructose-6-phosphate to form sucrose-6-phosphate. Plant SPS plays a key role in photosynthetic carbon metabolisms, which activity is modulated by an allosteric activator glucose-6-phosphate (G6P). We produced recombinant sugarcane SPS using Escherichia coli and Sf9 insect cells to investigate its structure-function relationship. When expressed in E. coli, two forms of SPS with different sizes appeared; the larger was comparable in size with the authentic plant enzyme and the shorter was trimmed the N-terminal 20 kDa region off. In the insect cells, only enzyme with the authentic size was produced. We purified the trimmed SPS and the full size enzyme from insect cells and found their enzymatic properties differed significantly; the full size enzyme was activated allosterically by G6P, while the trimmed one showed a high activity even without G6P. We further introduced a series of N-terminal truncations up to 171 residue and found G6P-independent activity was enhanced by the truncation. These combined results indicated that the N-terminal region of sugarcane SPS is crucial for the allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

Development of Nonaggregating Poly-A Tailed Immunostimulatory A/D Type CpG Oligodeoxynucleotides Applicable for Clinical Use

Immunostimulatory CpG ODNs have been developed and utilized as TLR9-dependent innate immune activators and vaccine adjuvants. Four different types of immunostimulatory CpG ODNs (A/D, B/K, C, and P type) have been reported. A/D type ODNs are characterized by high IFN-α production but intrinsically form aggregates, hindering its good manufacturing practice grade preparation. In this study, we developed several D35-derived ODNs (a commonly used A/D type ODN), which were modified with the addition of a phosphorothioate polynucleotide tail (such as dAs40), and examined their physical properties, solubility in saline, immunostimulatory activity on human PBMCs, and vaccine adjuvant potential in monkeys. We found that two modified ODNs including D35-dAs40 and D35core-dAs40 were immunostimulatory, similar to original D35 in human PBMCs, resulting in high IFN-α secretion in a dose-dependent manner. Physical property analysis by dynamic light scattering revealed that both D35-dAs40 and D35core-dAs40 did not form aggregates in saline, which is currently impossible for the original D35. Furthermore, D35-dAs40 and D35core-dAs40 worked as better vaccine adjuvant in monkeys. These results suggested that D35-dAs40 and D35core-dAs40 are two promising prototypes of nonaggregating A/D type ODN with advantages of ease of drug preparation for clinical applications as vaccine adjuvants or IFN-α inducing immunomodifiers.

Refolding, crystallization and preliminary X-ray crystallographic study of the whole extracellular regions of nectins

The whole extracellular regions of nectin-1 (nectin-1-EC) and nectin-2 (nectin-2-EC) were expressed in E. coli as inclusion bodies, solubilized in 8 M urea and then refolded by rapid dilution. Refolded nectin-1-EC and nectin-2-EC were subsequently purified using three chromatographic steps and crystallized by the hanging-drop vapour-diffusion method.