Seok-Yong Choi

High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis

Fusion of vesicles into target membranes during many types of regulated exocytosis requires both SNARE-complex proteins and fusogenic lipids, such as phosphatidic acid. Mitochondrial fusion is less well understood but distinct, as it is mediated instead by the protein Mitofusin (Mfn). Here, we identify an ancestral member of the phospholipase D (PLD) superfamily of lipid-modifying enzymes that is required for mitochondrial fusion. Mitochondrial PLD (MitoPLD) targets to the external face of mitochondria and promotes trans-mitochondrial membrane adherence in a Mfn-dependent manner by hydrolysing cardiolipin to generate phosphatidic acid. These findings reveal that although mitochondrial fusion and regulated exocytic fusion are mediated by distinct sets of protein machinery, the underlying processes are unexpectedly linked by the generation of a common fusogenic lipid. Moreover, our findings suggest a novel basis for the mitochondrial fragmentation observed during apoptosis.

piRNA-Associated Germline Nuage Formation and Spermatogenesis Require MitoPLD Profusogenic Mitochondrial-Surface Lipid Signaling

The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD(-/-) mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates nuage components critical for piRNA production.

Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.

Inverse agonist of estrogen-related receptor γ controls Salmonella typhimurium infection by modulating host iron homeostasis

In response to microbial infection, expression of the defensin-like peptide hepcidin (encoded by Hamp) is induced in hepatocytes to decrease iron release from macrophages. To elucidate the mechanism by which Salmonella enterica var. Typhimurium (S. typhimurium), an intramacrophage bacterium, alters host iron metabolism for its own survival, we examined the role of nuclear receptor family members belonging to the NR3B subfamily in mouse hepatocytes. Here, we report that estrogen-related receptor γ (ERRγ, encoded by Esrrg) modulates the intramacrophage proliferation of S. typhimurium by altering host iron homeostasis, and we demonstrate an antimicrobial effect of an ERRγ inverse agonist. Hepatic ERRγ expression was induced by S. typhimurium–stimulated interleukin-6 signaling, resulting in an induction of hepcidin and eventual hypoferremia in mice. Conversely, ablation of ERRγ mRNA expression in liver attenuated the S. typhimurium–mediated induction of hepcidin and normalized the hypoferremia caused by S. typhimurium infection. An inverse agonist of ERRγ ameliorated S. typhimurium–mediated hypoferremia through reduction of ERRγ–mediated hepcidin mRNA expression and exerted a potent antimicrobial effect on the S. typhimurium infection, thereby improving host survival. Taken together, these findings suggest an alternative approach to control multidrug-resistant intracellular bacteria by modulating host iron homeostasis.

Helicobacter pylori and interleukin-8 in gastric cancer

Helicobacter pylori (H. pylori) is a major etiological factor in the development of gastric cancer. Large-scale epidemiological studies have confirmed the strong association between H. pylori infection and both cancer development and progression. Interleukin-8 (IL-8) is overexpressed in gastric mucosa exposed to H. pylori. The expression of IL-8 directly correlates with a poor prognosis in gastric cancer. IL-8 is multifunctional. In addition to its potent chemotactic activity, it can induce proliferation and migration of cancer cells. In this review, we focus on recent insights into the mechanisms of IL-8 signaling associated with gastric cancer. The relationship between IL-8 and H. pylori is discussed. We also summarize the current therapeutics against IL-8 in gastric cancer.

Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D–generated signaling lipid

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)–triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld null flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

A novel zebrafish human tumor xenograft model validated for anti-cancer drug screening

The development of a relatively simple, reliant and cost-effective animal test will greatly facilitate drug development. In this study, our goal was the establishment of a rapid, simple, sensitive and reproducible zebrafish xenograft model for anti-cancer drug screening. We optimized the conditions for the cancer cell xenograft in terms of injected cell numbers, incubation temperature and time. A range of human carcinoma cell types were stained with a fluorescent dye prior to injection into the fish larvae. Subsequent cancer cell dissemination was observed under fluorescent microscopy. Differences in injected cell numbers were reflected in the rate of dissemination from the xenograft site. Paclitaxel, known as a microtubule stabilizer, dose-dependently inhibited cancer cell dissemination in our zebrafish xenograft model. An anti-migratory drug, LY294002 (phosphatidylinositol 3-kinase inhibitor) also decreased the cancer cell dissemination. Chemical modifications to increase cancer drug pharmacokinetics, such as increased solubility (17-DMAG compared to geldanamycin) could also be assessed in our xenograft model. In addition to testing our new model using known anti-cancer drugs, we carried out further validation by screening a tagged triazine library. Two novel anti-cancer drug candidates were discovered. Therefore, our zebrafish xenograft model provides a vertebrate animal system for the rapid screening and pre-clinical testing of novel anti-cancer agents, prior to the requirement for testing in mammals. Our model system should greatly facilitate drug development for cancer therapy because of its speed, simplicity and reproducibility.

A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators

BackgroundMitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques.ResultsIn order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction.ConclusionsThe adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions.See Commentary: http://www.biomedcentral.com/1741-7007/8/99

Real-time imaging of mitochondria in transgenic zebrafish expressing mitochondrially targeted GFP

Mitochondria maintain a web-shaped network in cells through a balance between fusion and fission. Under certain physiological and pathological conditions, this balance is breached, and as a result, change in mitochondrial morphology ensues. Real-time monitoring of such change is of significant importance for studying mitochondrial physiology and pathology, such as apoptosis, aging, and neurodegeneration. Numerous studies have been conducted in animal cell culture systems concerning mitochondrial morphology change. However, very little is known to date about the real-time changes in mitochondrial morphology at the organism level due to difficulties in observation and administration of mitochondria-disrupting drugs. Here we report the generation of transgenic zebrafish (Danio rerio) expressing mitochondrially targeted green fluorescent protein (GFP). The transparency of transgenic zebrafish embryos make it possible to monitor mitochondrial morphology in real time and in vivo. Since zebrafish inhabit fresh water, incubating zebrafish in drug-dissolved water sufficed to administer drugs to the zebrafish. We observed real-time and in vivo fragmentation of mitochondria in the transgenic embryos upon incubation in water with the following apoptosis-inducing drugs: valinomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and staurosporine. Thus, the transgenic zebrafish we generated could provide a platform for research on apoptosis and mitochondrial physiology and a screen for apoptosis-modulating drugs. It could also facilitate study of the pathogenesis of apoptosis-related diseases.