[Show abstract][Hide abstract] ABSTRACT: Continuous cell lines that originate from mammalian tissues serve as not only invaluable tools for life sciences, but also important animal cell substrates for the production of various types of biological pharmaceuticals. Vero cells are susceptible to various types of microbes and toxins and have widely contributed to not only microbiology, but also the production of vaccines for human use. We here showed the genome landscape of a Vero cell line, in which 25,877 putative protein-coding genes were identified in the 2.97-Gb genome sequence. A homozygous ∼9-Mb deletion on chromosome 12 caused the loss of the type I interferon gene cluster and cyclin-dependent kinase inhibitor genes in Vero cells. In addition, an ∼59-Mb loss of heterozygosity around this deleted region suggested that the homozygosity of the deletion was established by a large-scale conversion. Moreover, a genomic analysis of Vero cells revealed a female Chlorocebus sabaeus origin and proviral variations of the endogenous simian type D retrovirus. These results revealed the genomic basis for the non-tumourigenic permanent Vero cell lineage susceptible to various pathogens and will be useful for generating new sub-lines and developing new tools in the quality control of Vero cells.
DNA research: an international journal for rapid publication of reports on genes and genomes 09/2014; · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L-929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogen-infection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets.
Microbes and Infection 09/2014; · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A nucleolar protein, PICT1, regulates the tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, while the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fails to repress PICT1 degradation. In addition, the 20S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, while the same mutant underwent in vitro degradation by the 20S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.
[Show abstract][Hide abstract] ABSTRACT: The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We here showed that the phosphorylation of CERT at serine-315 (S315), which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at S315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine rich motif; SRM) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the SRM and the phosphorylation of S315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.
Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sphingolipids are essential components in eukaryotes and have various cellular functions. Recent developments in genome-editing technologies have facilitated gene disruption in various organisms and cell lines. We here show the disruption of various sphingolipid metabolic genes in human cervical carcinoma HeLa cells by using transcription activator-like effector nucleases (TALENs). A TALEN pair targeting the human CERT gene (alternative name COL4A3BP) encoding a ceramide transport protein induced a loss-of-function phenotype in more than 60% of HeLa cells even though the cell line has a pseudo-triploid karyotype. We have isolated several loss-of-function mutant clones for CERT, UGCG (encoding glucosylceramide synthase), and B4GalT5 (encoding the major lactosylceramide synthase), and also a CERT/UGCG double-deficient clone. Characterization of these clones supported previous proposals that CERT primarily contributes to the synthesis of SM but not GlcCer, and that B4GalT5 is the major LacCer synthase. These newly established sphingolipid-deficient HeLa cell mutants together with our previously established stable transfectants provide a 'sphingolipid-modified HeLa cell panel,' which will be useful to elucidate the functions of various sphingolipid species against essentially the same genomic background.
PLoS ONE 01/2014; 9(2):e88124. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.
PLoS ONE 01/2014; 9(10):e109886. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is critical for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires the yeast oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control the late stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.
Journal of Cell Science 11/2013; · 5.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the β isoform of class II PI3K (PI3K-C2β) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2β abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2β substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2β plays an indispensable role in the HCV replication cycle through the binding to HCV core protein.
Biochemical and Biophysical Research Communications 09/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Life creates many varieties of lipids. The choline-containing sphingophospholipid sphingomyelin (SM) exists ubiquitously or widely in vertebrates and lower animals, but is absent or rare in bacteria, fungi, protists, and plants. In the biosynthesis of SM, ceramide, which is synthesized in the endoplasmic reticulum, is transported to the Golgi region by the ceramide transport protein CERT, probably in a non-vesicular manner, and is then converted to SM by SM synthase, which catalyzes the reaction of phosphocholine transfer from phosphatidylcholine (PtdCho) to ceramide. Recent advances in genomics and lipidomics indicate that the phylogenetic occurrence of CERT and its orthologs is nearly parallel to that of SM. Based on the chemistry of lipids together with evolutionary aspects of SM and CERT, several concepts are here proposed. SM may serve as a chemically inert and robust, but non-covalently interactive lipid class at the outer leaflet of the plasma membrane. The functional domains and peptidic motifs of CERT are separated by exon units, suggesting an exon-shuffling mechanism for the generation of an ancestral CERT gene. CERT may have co-evolved with SM to bypass a competing metabolic reaction at the bifurcated point in the anabolism of ceramide. Human CERT is identical to the splicing variant of human Goodpasture antigen-binding protein (GPBP) annotated as an extracellular non-canonical serine/threonine protein kinase. The relationship between CERT and GPBP has also been discussed from an evolutionary aspect. Moreover, using an analogy of "compatible (or osmoprotective) solutes" that can accumulate to very high concentrations in the cytosol without denaturing proteins, choline phospholipids such as PtdCho and SM may act as compatible phospholipids in biomembranes. This article is part of a Special Issue entitled New frontiers in sphingolipid biology.
Biochimica et Biophysica Acta 07/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal infectious neurodegenerative disorders. Their causative agents are prions which are composed of disease-associated forms of prion protein (PrP(Sc)). Naturally occurring cases of TSEs are found in several mammalian species including humans, sheep, goats, minks, cattle and deer. Prions are also experimentally transmissible to other mammals such as mice, hamsters and monkeys, but interspecies transmission is often inefficient due to the 'species-barrier'. Studies have suggested that the barrier is not simply determined by differences in amino acid sequences of cellular prion protein (PrP(C)) among animal species, but also by prion strains which are closely associated with conformational properties of PrP(Sc) aggregates. Although the conformational properties of PrP(Sc) remain largely unknown, recent investigation of local structures of PrP(C) and, in particular, structural modeling of PrP(Sc) aggregates have provided molecular insight into this field. In this review, we discuss the species-barrier phenomenon in terms of the protein science.
Journal of Biochemistry 01/2013; · 3.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.
PLoS ONE 01/2013; 8(11):e80583. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ceramide transport from the endoplasmic reticulum to the Golgi apparatus is crucial in sphingolipid biosynthesis, and the process relies on the ceramide trafficking protein (CERT), which contains pleckstrin homology (PH) and StAR-related lipid transfer domains. The CERT PH domain specifically recognizes phosphatidylinositol 4-monophosphate (PtdIns(4)P), a characteristic phosphoinositide in the Golgi membrane, and is indispensable for the endoplasmic reticulum-to-Golgi transport of ceramide by CERT. In this study, we determined the three-dimensional structure of the CERT PH domain by using solution NMR techniques. The structure revealed the presence of a characteristic basic groove near the canonical PtdIns(4)P recognition site. An extensive interaction study using NMR and other biophysical techniques revealed that the basic groove coordinates the CERT PH domain for efficient PtdIns(4)P recognition and localization in the Golgi apparatus. The notion was also supported by Golgi mislocalization of the CERT mutants in living cells. The distinctive binding modes reflect the functions of PH domains, as the basic groove is conserved only in the PH domains involved with the PtdIns(4)P-dependent lipid transport activity but not in those with the signal transduction activity.
Journal of Biological Chemistry 08/2012; 287(40):33706-18. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are characterized by the replicative propagation of disease-associated forms of prion protein (PrP(Sc); PrP refers to prion protein). The propagation is believed to proceed via two steps; the initial binding of the normal form of PrP (PrP(C)) to PrP(Sc) and the subsequent conversion of PrP(C) to PrP(Sc). We have explored the two-step model in prion-infected mouse neuroblastoma (ScN2a) cells by focusing on the mouse PrP (MoPrP) segment 92-GGTHNQWNKPSKPKTN-107, which is within a region previously suggested to be part of the binding interface or shown to differ in its accessibility to anti-PrP antibodies between PrP(C) and PrP(Sc). Exchanging the MoPrP segment with the corresponding chicken PrP segment (106-GGSYHNQKPWKPPKTN-121) revealed the necessity of MoPrP residues 99 to 104 for the chimeras to achieve the PrP(Sc) state, while segment 95 to 98 was replaceable with the chicken sequence. An alanine substitution at position 100, 102, 103, or 104 of MoPrP gave rise to nonconvertible mutants that associated with MoPrP(Sc) and interfered with the conversion of endogenous MoPrP(C). The interference was not evoked by a chimera (designated MCM2) in which MoPrP segment 95 to 104 was changed to the chicken sequence, though MCM2 associated with MoPrP(Sc). Incubation of the cells with a synthetic peptide composed of MoPrP residues 93 to 107 or alanine-substituted cognates did not inhibit the conversion, whereas an anti-P8 antibody recognizing the above sequence in PrP(C) reduced the accumulation of PrP(Sc) after 10 days of incubation of the cells. These results suggest the segment 100 to 104 of MoPrP(C) plays a key role in conversion after binding to MoPrP(Sc).
Journal of Virology 03/2012; 86(10):5626-36. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intracellular transport of lipids from the sites of their synthesis to their appropriate destination is a critical step for lipid metabolism. One well-defined inter-organelle lipid movement is the transport of ceramide by ceramide transport protein (CERT). Ceramide, a key intermediate for both sphingomyelin and glycosphingolipids, is synthesized at the endoplasmic reticulum and delivered to the Golgi apparatus to be converted to sphingomyelin. CERT delivers ceramide from the ER to the Golgi apparatus in a non-vesicular and ATP-dependent manner. This chapter describes a reconstitution assay system for ceramide transport with semi-intact cells, which is useful for the study of the CERT-mediated inter-organelle transport of ceramide.
Methods in cell biology 01/2012; 108:117-29. · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.
Journal of Biological Chemistry 11/2010; 285(46):35505-18. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are fatal neurodegenerative disorders that accompany an accumulation of the disease-associated form(s) of prion protein (PrPSc) in the central nervous system. The neuropathological changes in the brain begin with focal deposits of PrPSc, followed by pathomorphological abnormalities of axon terminal degeneration, synaptic loss, atrophy of dendritic trees, and eventual neuronal cell death in the lesions. However, the underlying molecular basis for these neuropathogenic abnormalities is not fully understood.
In a proteomic analysis of soluble proteins in the brains of mice challenged intracerebrally with scrapie prion (Obihiro I strain), we found that the amount of the full-length form of collapsin response mediator protein-2 (CRMP-2; 61 kDa) decreased in the late stages of the disease, while the amount of its truncated form (56 kDa) increased to comparable levels observed for the full-length form. Detailed analysis by liquid chromatography-electrospray ionization-tandem mass spectrometry showed that the 56-kDa form (named CRMP-2-ΔC) lacked the sequence from serine518 to the C-terminus, including the C-terminal phosphorylation sites important for the regulation of axonal growth and axon-dendrite specification in developing neurons. The invariable size of the mRNA transcript in Northern blot analysis suggested that the truncation was due to post-translational proteolysis. By overexpression of CRMP-2-ΔC in primary cultured neurons, we observed the augmentation of the development of neurite branch tips to the same levels as for CRMP-2T514A/T555A, a non-phosphorylated mimic of the full-length protein. This suggests that the increased level of CRMP-2-ΔC in the brain modulates the integrity of neurons, and may be involved in the pathogenesis of the neuronal abnormalities observed in the late stages of the disease.
We identified the presence of CRMP-2-ΔC in the brain of a murine model of prion disease. Of note, C-terminal truncations of CRMP-2 have been recently observed in models for neurodegenerative disorders such as ischemia, traumatic brain injury, and Wallerian degeneration. While the structural identity of CRMP-2-ΔC in those models remains unknown, the present study should provide clues to the molecular pathology of degenerating neurons in prion diseases in connection with other neurodegenerative disorders.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that yeast glycosylphosphatidylinositol-anchored proteins (GPI-APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI-APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI-APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER-to-Golgi transport of GPI-APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI-APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI-APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI-APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are characterized by the accumulation of a pathological form of prion protein (PrP(Sc)), which behaves as an infectious agent. Here we developed an in vitro co-culture system to analyze the PrP(Sc) transmission from ScN2a cell, which persistently retains PrP(Sc), to naïve N2a cell. In this cell-to-cell transmission system, PrP(Sc) transmitted to recipient N2a cell was able to be detected within 5-7days. Further characterization showed that higher cell density greatly facilitated the transmission of PrP(Sc). This improved in vitro transmission method may become a useful tool for unveiling the molecular mechanism of PrP(Sc) transmission.
Biochemical and Biophysical Research Communications 07/2010; 397(3):505-8. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena.