The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.45-Å resolution. The structure showed a 'closed umbrella' shape with a long helix consisting of the cytoplasmic coiled coil and the voltage-sensing helix, S4, and featured a wide inner-accessible vestibule. Two out of three arginines in S4 were located below the phenylalanine constituting the gating charge-transfer center. The extracellular region of each protomer coordinated a Zn(2+), thus suggesting that Zn(2+) stabilizes the resting state of Hv1 by competing for acidic residues that otherwise form salt bridges with voltage-sensing positive charges on S4. These findings provide a platform for understanding the general principles of voltage sensing and proton permeation.

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.

Interleukin-6 (IL-6) is a multifunctional cytokine produced by various cells to regulate hematopoiesis, inflammation, immune responses, and bone homeostasis. IL-6 is also known to modulate the differentiation of osteoblasts and osteoclasts. IL-6 is believed to play a positive regulatory role in osteoclast differentiation by inducing the expression of receptor activator of NF-B ligand (RANKL) on the surface of osteoblasts: RANKL then interacts with RANK expressed on osteoclast progenitors, inducing osteoclast differentiation via the RANK signaling pathway, which involves NF-B, JNK, and p38. In this report, we demonstrate that IL-6 can also directly act on osteoclast progenitors to suppress their differentiation via an inhibition of RANK signaling pathways. IL-6 specifically suppressed RANK-mediated IB degradation and JNK activation. Microarray analysis revealed that costimulation with IL-6 and RANKL up-regulates the transcription of MKP1 and MKP7, which encode enzymes that dephosphorylate JNK, and down-regulates the transcription of Senp2 and Cul4A, which are related to the ubiquitin pathway. Thus, IL-6 directly acts on osteoclast progenitors and suppresses their differentiation by regulating the transcription of specific genes related to MAPK phosphatases and the ubiquitin pathway.Bone tissue is continuously remodeled under physiological conditions (1), but dysregulation of the balance between bone formation and resorption induces pathological conditions, including osteoporosis and osteosclerosis. Bone remodeling is maintained by two key cell populations, osteoblasts and osteoclasts, which are regulated by cytokines, hormones, and growth factors (2, 3). Among the cytokines, the interleukin-6 (IL-6) 3 family cytokines are known to modulate both osteoblast and osteoclast differentiation (3-5).IL-6 is a multifunctional cytokine produced by various cell types that regulates hematopoiesis, acute phase reactions, immune responses, and bone homeostasis (6 -8). The receptor for IL-6 consists of a ligand-binding subunit and a common signal-transducing subunit, gp130 (9). gp130 contains a number of tyrosine residues in its cytoplasmic region, as well as four copies of the YXXQ motif, which is required for the tyrosine phosphorylation of STAT3. Activated STAT3 dimerizes, enters the nucleus, and regulates the transcription of various genes that regulate cell survival, proliferation, and differentiation in a cell-specific manner (6, 8). Tyr-759 of gp130 is required for the tyrosine phosphorylation of Src homology 2 domain-containing tyrosine phosphatase (SHP)-2, which activates the MAPK ERK via a complex comprising SHP2, Gab1/2, and phosphatidylinositol 3-kinase p85 (10 -12).It has been reported that IL-6 together with soluble IL-6 receptor acts to induce the expression of receptor activator of NF-B ligand (RANKL) on the surface of osteoblasts. RANKL interacts with RANK, which is expressed on the surface of osteoclast progenitors. Coculture experiments have demonstrated that this interaction is indispensable for the differen...

Triple-helical structures of (Pro-Hyp-Gly)n (n = 10, 11) at 100 K and room temperature (RT) were analyzed at 1.26 A resolution by using synchrotron radiation data. Totals of 49 and 42 water molecules per seven triplets in an asymmetric unit were found for the structures at 100 K and RT, respectively. These water molecules were classified into two groups, those in the first and second hydration shells. Although there was no significant difference between water molecules in the first shell at 100 K and those at RT, a significant difference between those in the second shell was observed. That is, the number of water molecules at RT decreased to one half and the average distance from peptide chains at RT became longer by about 0.3 A. On the other hand, of seven triplets in an asymmetric unit, three proline residues at the X position at 100 K clearly showed an up-puckering conformation, as opposed to the recent propensity-based hypothesis for the stabilization and destabilization of triple-helical structures by proline hydroxylation. This puckering was attributed to the interaction between proline rings and the surrounding water molecules at 100 K, which is much weaker at RT, as shown by longer average distance from peptide chains.

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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.innate immunity | two-photon microscopy | MARCO | Siglec-1 | β-glucan

No abstract

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 cisdimerization of nectins through the first Ig-like domains.