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Mohammad Abu Yousuf Fumika Mi-ichi Kumiko Nakada-Tsukui Tomoyoshi Nozaki 《Eukaryotic cell》2010,9(6):926-933
Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.Pyridine nucleotide transhydrogenase (PNT) participates in the bioenergetic processes of the cell. PNT generally resides on the cytoplasmic membranes of bacteria and the inner membrane of mammalian mitochondria (3, 16) and utilizes the electrochemical proton gradient across the membrane to drive NADPH formation from NADH (14, 15, 39) according to the reaction H+out + NADH + NADP+↔H+in + NAD+ + NADPH, where “out” and “in” denote the cytosol and the matrix of the mitochondria, or the periplasmic space and the cytosol of bacteria, respectively.PNT has been identified in several protozoan parasites, including Entamoeba histolytica (8, 51), Eimeria tenella (17, 47), Mastigamoeba balamuthi (11) Plasmodium falciparum (10), Plasmodium yoelii (6), and Plasmodium berghei (12). In general, PNT contains conserved structural units consisting of three domains, the NAD(H)-binding domain (domain I [dI]) and the NADP(H)-binding domain (domain III [dIII]), both of which face the matrix side of the eukaryotic mitochondria or the cytoplasmic side in bacteria, and the hydrophobic domain (domain II [dII]), containing 11 to 13 transmembrane regions. PNT from E. tenella and E. histolytica exists as a single polypeptide in an unusual configuration consisting of dIIb-dIII-dI-dIIa, with a 38-amino-acid-long linker region between dIII and dI (48).E. histolytica, previously considered an “amitochondriate” protist, is currently considered to possess a mitochondrion-related organelle with reduced and divergent functions, the mitosome (1, 21, 23a, 26, 42). Our recent proteomic study of isolated mitosomes identified about 20 new constituents (26), together with four proteins previously demonstrated in E. histolytica mitosomes: Cpn60 (8, 19, 21, 42), Cpn10 (46), mitochondrial Hsp70 (2, 44), and mitochondrion carrier family (MCF) (ADP/ATP transporter) (7). Despite the early presumption of PNT being localized in mitosomes (8), based on the amino-terminal region rich in hydroxylated (five serines and threonines) and acidic (three glutamates) amino acids, which slightly resembles known mitochondrion- and hydrogenosome-targeting sequences (8, 35), PNT was not discovered in the mitosome proteome. We also doubted this premise because PNT was one of the major proteins identified in isolated phagosomes (32, 33). Thus, the intracellular localization and trafficking of PNT remain unknown.In this report, we showed that E. histolytica PNT (EhPNT) is localized to various vesicles and vacuoles, including lysosomes and phagosomes, using wild-type amoebae and antiserum raised against recombinant EhPNT and an E. histolytica line expressing EhPNT with a carboxyl-terminal hemagglutinin (HA) epitope tag and anti-HA antibody. We also showed that all domains of EhPNT are required for its trafficking to the acidic compartment by using amoeba transformants expressing the HA tag or green fluorescent protein (GFP) fused with a region containing various domains of EhPNT. 相似文献
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Ohwada T Ishikawa S Mine Y Inami K Yanagimoto T Karaki F Kabasawa Y Otani Y Mochizuki M 《Bioorganic & medicinal chemistry》2011,19(8):2726-2741
Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications. 相似文献
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Ishima Y Yoshida F Kragh-Hansen U Watanabe K Katayama N Nakajou K Akaike T Kai T Maruyama T Otagiri M 《Free radical research》2011,45(10):1196-1206
Endogenous S-nitrosated human serum albumin (E-Mono-SNO-HSA) is a large molecular weight nitric oxide (NO) carrier in human plasma, which has shown many beneficial effects in different animal models. To construct more efficient SNO-HSA preparations, SNO-HSA with many conjugated SNO groups has been prepared using chemical modification (CM-Poly-SNO-HSA). We have compared the properties of such a preparation to those of E-Mono-SNO-HSA. Cellular uptake of NO from E-Mono-SNO-HSA partly takes place via low molecular weight thiol, and it results in cytoprotective effects by induction of heme oxygenase-1. By contrast, transfer of NO from CM-Poly-SNO-HSA into the cells is faster and more pronounced. The influx mainly takes place by cell-surface protein disulfide isomerase. The considerable NO inflow results in apoptotic cell death by ROS induction and caspase-3 activation. Thus, increasing the number of SNO groups on HSA does not simply intensify the cellular responses to the product but can also result in very different effects. 相似文献
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Yujiro Umezaki Sean E. Hayley Michelle L. Chu Hanna W. Seo Prasun Shah Fumika N. Hamada 《Current biology : CB》2018,28(5):779-787.e3
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Kei Okatsu Fumika Koyano Mayumi Kimura Hidetaka Kosako Yasushi Saeki Keiji Tanaka Noriyuki Matsuda 《The Journal of cell biology》2015,209(1):111-128
PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria. 相似文献
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Kobayashi T Sato S Takamiya S Komaki-Yasuda K Yano K Hirata A Onitsuka I Hata M Mi-ichi F Tanaka T Hase T Miyajima A Kawazu S Watanabe Y Kita K 《Mitochondrion》2007,7(1-2):125-132
The mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp. is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques--Percoll density gradient centrifugation and fluorescence-activated organelle sorting--for improving the purity of mitochondria isolated from the crude organelle preparation of Plasmodium falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium. 相似文献
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Nobutaka Hanagata Akihide Ito Haruko Uehara Fumika Asari Toshifumi Takeuchi Isao Karube 《Journal of biotechnology》1993,30(3)
The formation processes of Carthamus tinctorius cell aggregates in a growth medium and the correlation of red pigment formation with cell aggregate sizes were investigated. About 80% of cell aggregates in the growth medium were > 1.00 mm in size. The growth rate of large cell aggregates was more rapid than that of small cell aggregates. Most cell aggregates > 0.50 mm in size became larger or smaller than their original sizes during the culture. A high level of red pigment formation was observed when cell aggregates obtained by the preculture using cell aggregates < 1.00 mm in size were cultured in the production medium. 相似文献