Shoji Ohkuma

Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog of mot-2 (mortalin)/mthsp70/Grp75

The Caenorhabditis elegans homolog of mortalin/mthsp70/Grp75 (called mot‐2 hereafter) was isolated by screening of a nematode cDNA library with mouse mot‐2 cDNA. The isolated clone matched to hsp70F of C. elegans. Analysis with two of the antibodies raised against hsp70F revealed that unlike mammalian mot‐2, it is heat inducible. Transient induction of hsp70F by heat shock led to a slight (<13%) extension in the C. elegans life span. The transgenic worms that constitutively over‐expressed hsp70F predominantly in muscle showed life span extension (∼43% for mean and ∼45% for maximum life span) as compared to the wild‐type and green fluorescent protein‐transgenic worms. Life span extension of human cells was obtained by over‐expression of mot‐2 [Kaul et al. (2000) FEBS Lett. 474, 159–164]. Our results show, for the first time, that this member of the hsp70 family governs the longevity of worms and thus there are common pathways that determine mammalian and worm longevity.

Evidence for rotation of V1-ATPase

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells.

Autophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells. Addendum to: Quantitative Monitoring of Autophagic Degradation Akinori Kawai, Syuichi Takano, Nobuhiro Nakamura and Shoji Ohkuma Biochem Biophys Res Commun 2006; 351:71-7

Knockdown of Mitochondrial Heat Shock Protein 70 Promotes Progeria-like Phenotypes in Caenorhabditis elegans

Mitochondrial heat shock protein 70 (mthsp70) functions as a mitochondrial import motor and is essential in mitochondrial biogenesis and energy generation in eukaryotic cells. HSP-6 (hsp70F) is a nematode orthologue of mthsp70. Knockdown of HSP-6 by RNA interference in young adult nematodes caused a reduction in the levels of ATP-2, HSP-60 and CLK-1, leading to abnormal mitochondrial morphology and lower ATP levels. As a result, RNA interference-treated worms had lower motility, defects in oogenesis, earlier accumulation of autofluorescent material, and a shorter life span. These are the major phenotypes observed during the aging of worms, suggesting that the reduction of HSP-6 causes early aging or progeria-like phenotypes. The amount of HSP-6 became dramatically reduced at the expected mean life span in not only wild-type but also in long and short life span mutant worms (wild-type, daf-2, and daf-16). Mitochondrial HSP-60 and ATP-2 were also reduced following the reduction of HSP-6 during aging. These results suggest that the reduction of HSP-6 causes defects in mitochondrial function at the final stage of aging, leading to mortality.

Convergence of Cell Cycle Regulation and Growth Factor Signals on GRASP65

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

Bafilomycin A1 induces apoptosis in the human pancreatic cancer cell line Capan‐1

Bafilomycin A1, a specific inhibitor of vacuolar type H+‐ATPase, can inhibit the growth of a variety of cultured cells in a dose‐dependent manner, but its mechanism is unclear. The aim of this study was to examine whether bafilomycin A1 inhibits the growth of Capan‐1 human pancreatic cancer cells through apoptosis. The effect of bafilomycin A1 on tumour growth in vitroand in vivowas examined using an MTT assay and an in vivotumour model. The presence or absence of apoptosis was determined by morphology and DNA analysis of tumour cells. The concentration of bafilomycin A1 for 50 per cent inhibition of cell viability during 72 h by the MTT assay was 5 nm. In DNA analysis, a ladder of fragmented DNA was detected in Capan‐1 cells treated with bafilomycin A1 at concentrations greater than 10 nm for 24 h. Nude mice bearing a xenografted Capan‐1 cell line tumour received 4 weeks of bafilomycin A1 (1·0 mg/kg per day). This treatment significantly inhibited tumour growth compared with controls after 21 days (P<0·05). Histopathological examination of tumour cells in the treated group demonstrated signs of apoptosis with chromatin condensation and cell shrinkage. These observations suggest that bafilomycin A1 inhibits the growth of Capan‐1 human pancreatic cancer cells through apoptosis.

Prodigiosin 25-C uncouples vacuolar type H+-ATPase, inhibits vacuolar acidification and affects glycoprotein processing

Prodigiosin 25‐C inhibited the accumulation of 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine and acridine orange in the acidic compartments of baby hamster kidney cells with little perturbation of cellular ATP levels. In rat liver lysosomes, prodigiosin 25‐C inhibited the proton pump activity with an IC50 of approximately 30 nM, but did not affect ATPase activity up to 1 μM. It also delayed the transport of vesicular stomatitis virus G protein and induced a drastic swelling of Golgi apparatus and mitochondria. These results indicate that prodigiosin 25‐C raises the pH of acidic compartments through inhibition of the proton pump activity of vacuolar type H+‐ATPase, thereby causing the functional and morphological changes to the Golgi apparatus.

Contribution of lysosomes to the subcellular distribution of basic drugs in the rat liver

AbstractPurpose. We examined the subcellular distribution of the basic drugs, chlorpromazine (CPZ), imipramine (IMP) and biperiden (BP), in rat liver, and evaluated the contribution of lysosome (Lys) to their intracellular distribution in comparison with that of mitochondria (Mit). Methods. In an in vivo distribution, the concentrations of CPZ, IMP and BP in the liver subcellular fractions were determined. In an in vitro study, uptake of [3H]IMP into Lys and Mit fractions was determined in the presence or absence of several agents. Results. The distribution of these drugs 10 min after administration was quite similar. However, the relative specific contents (the drug concentration per protein of each fraction divided by that of the total homogenate) in Lys were 7.3, 9.6 and 4.2, respectively for CPZ, IMP and BP, whereas those in the other organella were only 0.4 ~ 1.7. In an in vitro uptake study, the dose response of IMP uptake into Lys was biphasic, while that into Mit fractions was monophasic. The binding of IMP to the high affinity sites of Lys was pH dependent and disappeared in 50 mM NH4C1 or 50 µM CPZ, both of which increased the intralysosomal pH. the low affinity sites were not affected by these drugs. Conclusions. The results indicated that Lys has the highest affinity for the basic drugs in the liver and that its contribution to their subcellular distribution depends on the intralysosomal pH, which is also affected by these drugs. The importance of these effects may become significant in combination therapy using various basic drugs.

Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase

SummaryBafilomycin A1, a potent selective inhibitor of vacuolar H+-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A1 for 50% inhibition of cell growth ranged from 10 to 50 nM. The dose response was nearly parallel with that of the bafilomycin A1-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A1 was similar to that produced by NH4Cl in which little difference was recognized in effect among cell types.

BE-18591 as a new H+/Cl− symport ionophore that inhibits immunoproliferation and gastritis

In our previous papers [e.g. Sato et al., J. Biol. Chem. 273 (1998) 21455–21462], we have shown that prodigiosins can uncouple various H+‐ATPases through their H+/Cl− symport activity. BE‐18591 is an enamine of 4‐methoxy‐2,2′‐bipyrrole‐5‐carboxyaldehyde (tambjamine group antibiotics) which resembles the prodigiosins. We found that BE‐18591 was a new group of antibiotics that uncouples various H+‐ATPases: it inhibited proton pump activities with IC50s of about 1–2 nM (about 20 pmol/mg protein) for submitochondrial particles as well as gastric vesicles and of 230 nM (about 230 pmol/mg protein) for lysosomes, but it had little effect on their ATP hydrolyses (up to 10 μM), a property of H+/Cl− symport activity. At low concentrations (<1 μM), BE‐18591 inhibited immunoproliferation, the IC50 of lipopolysaccharide‐stimulated mouse splenocytes was 38 nM, that of Concanavalin A‐stimulated cells was 230 nM. Gastritis of rabbits was also inhibited. At higher concentrations (>1 μM), BE‐18591 induced neurite outgrowth (15% induction in 48 h at 4 μM), inhibited bone resorption (∼35% in 48 h at 10 μM) and caused cell death (∼30% in 48 h at 4 μM) but with little apoptosis.