Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

Regulation of excitation energy in Nannochloropsis photosystem II

Photosynthesis Research - Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res...     

Comparative analysis of strategies to prepare electron sinks in aquatic photoautotrophs

Photosynthesis Research - While subject to illumination, photosystem I (PSI) has the potential to produce reactive oxygen species (ROS) that can cause photo-oxidative damage in oxygenic...     

Schizosaccharomyces pombe RanGAP homolog, SpRna1, is required for centromeric silencing and chromosome segregation

We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

Synthesis of amide compounds of ferulic acid,and their stimulatory effects on insulin secretion in vitro

We prepared amide compounds which were derived from ferulic acid using various amines, and investigated their stimulatory effects on insulin secretion using rat pancreatic RIN-5F cells. Most of these compounds exhibited significant promotion of the insulin-release at a concentration of 10 microM and in particular, the amides having n-butyl, n-pentyl, pyrrolidine, and piperidine groups showed high activity.     

Targeting apoptotic tumor cells to Fc gamma R provides efficient and versatile vaccination against tumors by dendritic cells

Dendritic cells (DCs) loaded with tumor-associated Ags (TAAs) act as potent adjuvant that initiates antitumor immune responses in vivo. However, TAA-based DC vaccination requires prior identification of TAAs. Apoptotic tumor cells (ATCs) can be an excellent source for DC loading because their potential uncharacterized Ags would be efficiently presented to T cells without any prior characterization and isolation of these Ags. However, ATCs alone are considered to be inefficient for activating antitumor immunity, possibly because of their inability to induce DC maturation. In this study, the aim was to enhance antitumor immune response by taking advantage of ATCs that have been opsonized with IgG (ATC-immune complexes, ATC-ICs) so as to target them to FcR for IgG (FcgammaRs) on DCs. It was found that when compared with ATCs, ATC-ICs were efficiently internalized by DCs via FcgammaRs, and this process induced maturation of DCs, which was more efficient than that of ATCs. Importantly, ATC-IC loading was shown to be more efficient than ATCs alone in its capacity for inducing antitumor immunity in vivo, in terms of cytotoxic T cell induction and tumor rejection. These results show that using ATC-ICs may overcome the limitations and may enhance the immune response of current ATC-based DC vaccination therapy.     

Sclerostin is a novel secreted osteoclast-derived bone morphogenetic protein antagonist with unique ligand specificity

Sclerosteosis is a progressive sclerosing bone dysplasia. Sclerostin (the SOST gene) was originally identified as the sclerosteosis-causing gene. However, the physiological role of sclerostin remains to be elucidated. Sclerostin was intensely expressed in developing bones of mouse embryos. Punctuated expression of sclerostin was localized on the surfaces of both intramembranously forming skull bones and endochondrally forming long bones. Sclerostin-positive cells were identified as osteoclasts. Recombinant sclerostin protein produced in cultured cells was efficiently secreted as a monomer. We examined effects of sclerostin on the activity of BMP2, BMP4, BMP6, and BMP7 for mouse preosteoblastic MC3T3-E1 cells. Sclerostin inhibited the BMP6 and BMP7 activity but not the BMP2 and BMP4 activity. Sclerostin bound to BMP6 and BMP7 with high affinity but bound to BMP2 and BMP4 with lower affinity. In conclusion, sclerostin is a novel secreted osteoclast-derived BMP antagonist with unique ligand specificity. We suggest that sclerostin negatively regulates the formation of bone by repressing the differentiation and/or function of osteoblasts induced by BMPs. Since sclerostin expression is confined to the bone-resorbing osteoclast, it provides a mechanism whereby bone apposition is inhibited in the vicinity of resorption. Our findings indicate that sclerostin plays an important role in bone remodeling and links bone resorption and bone apposition.     

Functional domains essential for Gs activity in prostaglandin EP2 and EP3 receptors

The interaction of cell surface hormone receptors with heterotrimeric G proteins is crucial for hormonal actions. The domains of the receptor, which interact with and activate G protein, have been extensively studied. However, precise molecular mechanisms underlying regulation of the receptor-induced G protein activation are still poorly understood. Prostaglandin E(2) (PGE(2)) receptors comprise of four subtypes, EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 couple to Gs and EP3 to Gi. To assess the functional domains essential for Gs activation in prostanoid receptors, EP2, EP3beta and each intracellular loop- (IC-) interchanged EP2/EP3 chimeras were tested for agonist binding and functional responses. In EP2 receptor, substitution of IC1 or IC3 resulted in loss of binding activity, while substitution of IC2, N- (IC2N) or C-terminal half region of IC2 (IC2C) had no effects on the binding activity. Wild-type EP2 and IC2C-substituted EP2 showed agonist-induced Gs activity, but IC2- and IC2N-substituted EP2 failed to elicit Gs activity upon agonist stimulation. On the other hand, in EP3 receptor substitution of IC1 resulted in loss of PGE(2) binding, while substitution of IC2, IC3, IC2N or IC2C had no effects on binding activity. Wild-type EP3beta, IC3- or IC2C-substituted EP3 failed to show Gs activity upon agonist stimulation, but IC2- or IC2N-substituted EP3 chimera showed agonist-dependent Gs activity. These results indicated that the second intracellular loop of the EP2 plays an essential role in activation of Gs.     

Decrease in the efficiency of the electron donation to tyrosine Z of photosystem II in an SQDG-deficient mutant of Chlamydomonas

Photosystem (PS) II activity of a sulfoquinovosyl diacylglycerol (SQDG)-deficient mutant (hf-2) of Chlamydomonas was partially decreased compared with that of wild-type. The susceptibility to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modified in the mutant. Photometric measurements in the isolated thylakoid membranes of hf-2 revealed that the lowered activity in the mutant was derived from a decrease in the efficiency of the electron donation from water to tyrosine Z, not from the efficiency of the electron transport from Q(A) to Q(B). This result was confirmed by the decay kinetics of chlorophyll fluorescence determined in vivo. We conclude that SQDG contributes to maintaining the conformation of PSII complexes, particularly that of D1 polypeptides, which are necessary for maximum activities in Chlamydomonas.