Structure of the whole cytosolic region of ATP-dependent protease FtsH

An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA+ ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA+ domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA+ domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.     

Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRα enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor γ coactivator 1 α and β (PGC-1α and -β) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRα is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRα-associated proteins in HeLa cells stably expressing ERRα. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1α-dependent transactivation of ERRα by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRα and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRα. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRα by associating with both acetylated histone H3 and HDAC1.     

Selectivity of neuronal adaptation does not match response selectivity: a single-cell study of the FMRI adaptation paradigm

fMRI-based adaptation paradigms (fMR-A) have been used to infer neuronal stimulus selectivities in humans. Inferring neuronal selectivities from fMR-A, however, requires an understanding of the relationship between the stimulus selectivity of neuronal adaptation and responses. We studied this relationship by recording single cells in macaque inferior temporal (IT) cortex, an area that shows fMRI adaptation. Repetition of identical object images reduced the responsiveness of single IT neurons. Presentation of an image to which the neuron was unresponsive did not alter the response to a subsequent image that activated the neuron. Successive presentation of two different images to which the neuron responded similarly produced adaptation, but less so than the repeated presentation of an image. The neuronal adaptation at the single-cell level showed a greater degree of stimulus selectivity than the responses. This complicates the interpretation of fMR-A paradigms when inferring neuronal selectivity.     

Evaluation of characteristic aroma compounds of Citrus natsudaidai Hayata (Natsudaidai) cold-pressed peel oil

The characteristic aroma compounds of Citrus natsudaidai Hayata essential oil were evaluated by a combination of instrumental and sensory methods. Sixty compounds were identified and quantified, accounting for 94.08% of the total peel oil constituents. Limonene was the most abundant compound (80.68%), followed by gamma-terpinene (5.30%), myrcene (2.25%) and alpha-pinene (1.30%). Nineteen compounds which could not be identified in the original oil were identified in the oxygenated fraction. Myrcene, linalool, alpha-pinene, beta-pinene, limonene, nonanal, gamma-terpinene, germacrene D, and perillyl alcohol were the active aroma components (FD-factor > 3(6)), whereas beta-copaene, cis-sabinene hydrate and 1-octanol were suggested as characteristic aroma compounds, having a Natsudaidai-like aroma in the GC effluent. Three other compounds, heptyl acetate, (E)-limonene oxide and 2,3-butanediol, which each showed a high RFA value (>35) were considered to be important in the reconstruction of the original Natsudaidai oil from pure odor chemicals. The results indicate that 1-octanol was the aroma impact compound of C. natsudaidai Hayata peel oil.     

Volatile profiles in cold-pressed peel oil from Korean and Japanese Shiranui (Citrus unshiu Marcov. x C. sinensis Osbeck x C. reticulata Blanco)

A comparison of the volatile profiles between Korean and Japanese Shiranui cold-pressed peel oil was performed by GC and GC-MS. Limonene was the most abundant in the Japanese (91.8%) and Korean (86.4%) oil. Alcohols accounted for 1.8% in the Korean oil, and 0.2% in the Japanese oil, in which the respective linalool levels were 1.2% and 0.1%. The level of aldehydes was also higher in the Korean oil (1.6%) than in the Japanese oil (0.7%).     

Superior protective and therapeutic effects of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma

Fusion vaccine of dendritic cells (DCs) and tumor cells has the advantage of inducing an immune response against multiple tumor Ags, including unknown tumor Ags. Using the liver metastasis model of C1300 neuroblastoma cells, we assessed the protective and therapeutic effects of fusion cells transduced with the IL-12 gene and/or the IL-18 gene. Improving the fusion method by combining polyethylene glycol and electroporation increased loading efficiency. In the A/J mice vaccinated with fusion cells modified with the LacZ gene (fusion/LacZ), IFN-gamma production and CTL activity increased significantly compared with that of DCs/LacZ, C1300/LacZ, or a mixture of the two (mixture/LacZ). With the transduction of IL-12 and IL-18 genes into the fusion cells (fusion/IL-12/IL-18), the level of IFN-gamma increased more than five times that of other fusion groups. In addition, NK cell activity and CTL activity increased significantly compared with that of mixture/LacZ, fusion/LacZ, DC/LacZ, or C1300/LacZ. In the protective and therapeutic studies of fusion cell vaccine, mice vaccinated with fusion/LacZ, fusion/IL-12, fusion/IL-18, or fusion/IL-12/IL-18 showed a significant decrease in liver metastasis and a significant increase in survival compared with mice given a mixture/LacZ, DCs/LacZ, or C1300/LacZ. In particular, the mice receiving fusion/IL-12/IL-18 vaccine showed a complete protective effect and the highest therapeutic effects. The present study investigates the improved loading efficiency of fusion cells and suggests that the introduction of IL-12 and IL-18 genes can induce extremely strong protective and therapeutic effects on liver metastasis of neuroblastoma.     

Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C

The chitin-binding domain of Streptomyces griseus chitinase C (ChBD_ChiC) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD_ChiC, in contrast to three such residues on CBD_Cel5 in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD_ChiC, site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD_ChiC with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD_ChiC binds to chitin via a mechanism significantly different from CBD_Cel5, where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD_ChiC-type CBMs are dominant in CBM family 5.     

Establishing interspecific mosaic genome lines between Drosophila ananassae and Drosophila pallidosa by means of parthenogenesis

Strong sexual isolation exists between the closely related species Drosophila ananassae and D. pallidosa, but there is no obvious post-mating isolation; both sexes of the hybrids and their descendants appear to be completely viable and fertile. Strains exhibiting parthenogenesis have been derived from wild populations of both species. We intercrossed such strains and established iso-female lines after the second generation of parthenogenesis. These lines are clones, carrying homozygous chromosomes that are interspecific recombinants. We established 266 such isogenic lines and determined their genetic constitution by using chromosomal and molecular markers. Strong pseudo-linkage was seen between loci on the left arm of chromosome 2 and on the right arm of chromosome 3; the frequency of inheriting the two chromosome regions from the same species was significantly larger than expected. One possible cause of pseudo-linkage is female meiotic bias, so that chromosomes of the same species origin tend to be distributed to the same gamete. But this possibility is ruled out; backcross analysis indicated that the two chromosome regions segregated independently in female hybrids. The remaining possibility is elimination of low-fitness flies carrying the two chromosome regions from different species. Thus, genetic incompatibility was detected in the species pair for which no hybrid breakdown had previously been indicated. The 'interspecific mosaic genome' lines reported here will be useful for future research to identify genes involved in speciation and phenotypic evolution.     

Localization of mRNAs for phosphatidylinositol phosphate kinases in the mouse brain during development

The gene expression for seven phosphatidylinositol phosphate kinases (PIPKs)-types Ialpha, Ibeta, Igamma, types IIalpha, IIbeta, IIgamma, and type III-was examined using in situ hybridization histochemistry, in the mouse brain during normal development. In the embryonic mouse brain, positive expression signals were detected only for the genes encoding PIPK Igamma and PIPK IIbeta in both the cerebral ventricular and mantle zones, with weaker signals in the former zone. On the other hand, the genes encoding all PIPKs were essentially detected in the external granule cell layer which represents the germinal zone for the neuronal granule cells. In the postnatal brain, among the seven PIPKs, the expression for genes encoding PIPK Igamma and IIbeta is evident in most gray matter, while the expression for the other five types was weak in the cortical gray matter and negligible in most non-cortical gray matter such as the diencephalon and brain stem nuclei. While the expression for most PIPKs in the mature hippocampus was distinct, the expression in the CA3 and the dentate gyrus was less definite for the genes encoding PIPK Ialpha and IIgamma, respectively. The distinct expression for the gene encoding PIPK IIalpha was detected in the postnatal white matter such as the cerebellar medulla, the corpus callosum, the hippocampal fimbriae, and the internal capsule.     

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.