Higd-1a interacts with Opa1 and is required for the morphological and functional integrity of mitochondria.

The activity and morphology of mitochondria are maintained by dynamic fusion and fission processes regulated by a group of proteins residing in, or attached to, their inner and outer membranes. Hypoxia-induced gene domain protein-1a (Higd-1a)/HIMP1-a/HIG1, a mitochondrial inner membrane protein, plays a role in cell survival under hypoxic conditions. In the present study, we showed that Higd-1a depletion resulted in mitochondrial fission, depletion of mtDNA, disorganization of cristae, and growth retardation. We demonstrated that Higd-1a functions by specifically binding to Optic atrophy 1 (Opa1), a key element in fusion of the inner membrane. In the absence of Higd-1a, Opa1 was cleaved, resulting in the loss of its long isoforms and accumulation of small soluble forms. The small forms of Opa1 do not interact with Higd-1a, suggesting that a part of Opa1 in or proximal to the membrane is required for that interaction. Opa1 cleavage, mitochondrial fission, and cell death induced by dissipation of the mitochondrial membrane potential were significantly inhibited by ectopic expression of Higd-1a. Furthermore, growth inhibition due to Higd-1a depletion could be overcome by overexpression of a noncleavable form of Opa1. Collectively, our observations demonstrate that Higd-1a inhibits Opa1 cleavage and is required for mitochondrial fusion by virtue of its interaction with Opa1.

The BTB/POZ-ZF transcription factor dPLZF is involved in Ras/ERK signaling during Drosophila wing development.

In Drosophila, broad complex, tramtrack, bric à brac (BTB)/poxvirus and zinc finger (POZ) transcription factors are essential regulators of development. We searched the Drosophila genome for BTB/POZ-ZF domains and discovered an unknown Drosophila gene, dPLZF, which encodes an orthologue of human PLZF. We then characterized the biological function of the dPLZF via genetic interaction analysis. Ectopic expression of dPLZF in the wing induced extra vein formation during wing development in Drosophila. Genetic interactions between dPLZF and Ras or extracellular signal-regulated kinase (ERK) significantly enhanced the formation of vein cells. On the other hand, loss-of-function mutations in dPLZF resulted in a dramatic suppression of the extra and ectopic vein formation induced by elevated Ras/ERK signaling. Moreover, dPLZF activity upregulated the expression of rhomboid (rho) and spitz, which perform crucial functions in vein cell formation in the developing wing. These results indicate that dPLZF is a transcription factor controlled by the Ras/ERK signaling pathway, which is a prominent regulator of vein cell formation during wing development in Drosophila.

Hypoxia-induced SM22α in A549 cells activates the IGF1R/PI3K/Akt pathway, conferring cellular resistance against chemo- and radiation therapy.

Chemo- or radiation-resistance in tumors caused by hypoxia often undermines efficacy of cancer therapy. Thus, therapies that overcome cellular resistance during hypoxia are necessary. SM22α is an actin-binding protein found in smooth muscle, fibroblasts, and some epithelium. We demonstrate that SM22α is induced in A549 non-small cell lung carcinoma cells by hypoxia and its overexpression increased chemo- and radiation-resistance. Hypoxia-mediated induction of SM22α expression is hypoxia-inducible factor-independent. Moreover, SM22α overexpression enhances tumor cell growth and activates the IGF1R/PI3K/Akt pathway via direct interaction with IGF1Rß. Our results suggest SM22α as a novel regulator of hypoxic survival pathway of A549 NSCLC cells.

Elevated fibroblast growth factor-inducible 14 expression promotes gastric cancer growth via nuclear factor-κB and is associated with poor patient outcome.

The fibroblast growth factor-inducible 14 (Fn14) gene encodes a type I transmembrane protein that belongs to the tumor necrosis factor receptor superfamily and regulates multiple cellular processes in diverse physiological and pathological conditions, including cancer. Here, we describe an important role for Fn14 in regulating the growth of gastric cancer cells. Previous gene expression data analysis demonstrated that Fn14 was up-regulated in various tumor tissues, including gastric cancer. Using qRT-PCR, we showed that Fn14 was overexpressed in gastric tumor tissues compared to normal tissues. Furthermore, Fn14 expression levels were inversely correlated with gastric cancer patient survival. Using ectopic overexpression and shRNA-mediated knockdown of Fn14, we demonstrated that the expression level of Fn14 affected cell growth in gastric cancer. The effect of Fn14 on cell growth was mediated by the NF-κB activity and eventually by the transcriptional regulation of the anti-apoptotic Bcl-2 family gene (Bcl-xL). These results suggest that Fn14 may play an important role in gastric tumor growth by regulating NF-κB-mediated anti-apoptosis and that Fn14 may be a useful prognostic marker for gastric cancer.

Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea.

A recombinant protein-based enzyme-linked immunosorbent assay (RP ELISA) exists for the detection of antibodies to foot-and-mouth disease virus (FMDV) type A. In this study, the efficacy of the RP ELISA was compared to that of other current tests by examining sera collected in the field during an FMD type A outbreak in South Korea in 2010. The RP ELISA detected early antibodies to FMDV with the same sensitivity as the liquid-phase blocking ELISA (LPB ELISA), identifying FMD farm outbreaks correctly on a herd basis. In addition, the two assays exhibited a high correlation coefficient (γ(2)=0.83) when testing thirty seven sera from one outbreak farm exhibiting various antibody titers. The sensitivity and specificity of the RP ELISA relative to the LPB ELISA were 84% and 97%, respectively, and excellent agreement (kappa=0.82) was observed between the two tests. Taken together, the RP ELISA should be a useful alternative to the LPB ELISA for the detection of early antibodies to FMDV type A during an outbreak.

The survival effect of mitochondrial Higd-1a is associated with suppression of cytochrome C release and prevention of caspase activation.

Higd-1a (hypoxia induced gene domain family-1a) is a mitochondrial inner membrane protein with a conformation of N-terminal outside-C-terminal outside and loop inside. There are four Higd genes, Higd-1a, -1b, -1c and -2a, in the mouse. Higd-1a and -2a are expressed primarily in the brain, heart, kidney and leukocytes. HIF (hypoxia-inducible factor) overexpression induced the endogenous expression and promoter activity of Higd-1a. Mutation of the HRE (hypoxia-response element) site at -32bp in the Higd-1a promoter reduced the promoter activity, suggesting that transcription of Higd-1a is regulated by binding of the transcription factor HIF to the HRE. Higd-1a promoted cell survival under hypoxia. RAW264.7 cells stably transfected with Higd-1a underwent less apoptosis than control cells in a hypoxic condition, and hypoxia-induced apoptosis was strongly enhanced when endogenous Higd-1a was silenced by siRNA. The survival effect of Higd-1a was completely abolished by deletion of the 26 N-terminal amino acids, and we showed that Higd-1a increased survival by inhibiting cytochrome C release and reducing the activities of caspases. However, expression of Bcl-2, Bax, Bad, and BNIP3 and translocation of AIF were unaffected under the same conditions. Higd-2a also enhanced cell survival under hypoxia. Cells transfected with Higd-2a underwent less apoptosis than control cells in hypoxic conditions, and hypoxia-induced apoptosis increased when endogenous Higd-2a was depleted. Together these observations indicate that Higd-1a is induced by hypoxia in a HIF-dependent manner and its anti-apoptotic effect results from inhibiting cytochrome C release and reducing caspase activities.

Silencing of BNIP3 results from promoter methylation by DNA methyltransferase 1 induced by the mitogen-activated protein kinase pathway.

We have previously shown that Ras mediates NO-induced BNIP3 expression via the MEK-E RK-HIF-1 pathway i n mouse macrophages, and that NO-induced death results at least in part from the induction of BNIP3. In the present study, we describe another aspect of Ras regulation of BNIP3 expression in pancreatic cancer cells. Human BNIP3 promoter-driven luciferase activity was efficiently induced by activated Ras in AsPC-1, Miapaca-2, PK-1 and PANC-1 cells. However, expression of endogenous BNIP3 was not induced, and BNIP3 up-regulation by hypoxia was also inhibited. Treatment of the cells with the DNMT inhibitor, 5-aza-2-deoxycytidine, restored BNIP3 induction, indicating that DNA methylation of the BNIP3 promoter was responsible for the inhibition of BNIP3 induction. Furthermore, inhibition of the MEK pathway with U0126 reduced DNMT1 expression, but not that of DNMT3a and 3b, and restored the hypoxia-inducibility of BNIP3, suggesting that the DNA methylation of the BNIP3 promoter was mediated by DNMT1 via the MEK pathway.

Crystallographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation.

CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser(132) loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.

Aberrant up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer.

The LAMB3 and LAMC2 genes encode the laminin-5 ß3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.

Identification of three positive regulators in the geldanamycin PKS gene cluster of Streptomyces hygroscopicus JCM4427.

In the Streptomyces hygroscopicus JCM4427 geldanamycin biosynthetic gene cluster, Five putative regulatory genes were identified by protein homology searching. Among three of those genes, gel14, gel17, and gel19, are located downstream of polyketide synthase genes. Gel14 and Gel17 are members of the LAL family of transcriptional regulators, including an ATP/GTP-binding domain at the N-terminus and a DNA binding helix-turn-helix domain at the C-terminus. Gel19 is a member of the TetR family transcriptional regulators, which generally act to repress transcription. To verify the biological significance of the putative regulators in geldanamycin production, they were individually characterized by gene disruption, genetic complementation and transcriptional analyses. All three genes were confirmed as positive regulators of geldanamycin production. Specifically, Gel17 and Gel19 are required for gel14 as well as gelA gene expression.

Comparative genomic analysis of the false killer whale (Pseudorca crassidens) LMBR1 locus.

The sequencing and comparative genomic analysis of LMBR1 loci in mammals or other species, including human, would be very important in understanding evolutionary genetic changes underlying the evolution of limb development. In this regard, comparative genomic annotation of the false killer whale LMBR1 locus could shed new light on the evolution of limb development. We sequenced two false killer whale BAC clones, corresponding to 156 kb and 144 kb, respectively, harboring the tightly linked RNF32, LMBR1, and NOM1 genes. Our annotation of the false killer whale LMBR1 gene showed that it consists of 17 exons (1473 bp), in contrast to 18 exons (1596 bp) in human, and it displays 93.1% and 95.6% nucleotide and amino acid sequence similarity, respectively, compared with the human gene. In particular, we discovered that exon 10, deleted in the false killer whale LMBR1 gene, is present only in primates, and this fact strongly implies that exon 10 might be crucial in determining primate-specific limb development. ZRS and TFBS sequences have been well conserved across 11 species, suggesting that these regions could be involved in an important function of limb development and limb patterning. The neighboring gene RNF32 showed several lineage-conserved exons, such as exons 2 through 9 conserved in eutherian mammals, exons 3 through 9 conserved in mammals, and exons 5 through 9 conserved in vertebrates. The other neighboring gene, NOM1, had undergone a substitution (ATG→GTA) at the start codon, giving rise to a 36 bp shorter N-terminal sequence compared with the human sequence. Our comparative analysis of the false killer whale LMBR1 genomic locus provides important clues regarding the genetic regions that may play crucial roles in limb development and patterning.

SM22α-induced activation of p16INK4a/retinoblastoma pathway promotes cellular senescence caused by a subclinical dose of γ-radiation and doxorubicin in HepG2 cells.

Smooth muscle protein 22-alpha (SM22α) is known as a transformation- and shape change-sensitive actin cross-linking protein found in smooth muscle tissue and fibroblasts; however, its functional role remains uncertain. We reported previously that SM22α overexpression confers resistance against anti-cancer drugs or radiation via induction of metallothionein (MT) isozymes in HepG2 cells. In this study, we demonstrate that SM22α overexpression leads cells to a growth arrest state and promotes cellular senescence caused by treatment with a subclinical dose of γ-radiation (0.05 and 0.1 Gy) or doxorubicin (0.01 and 0.05 µg/ml), compared to control cells. Senescence growth arrest is known to be controlled by p53 phosphorylation/p21(WAF1/Cip1) induction or p16(INK4a)/retinoblastoma protein (pRB) activation. SM22α overexpression in HepG2 cells elevated p16(INK4a) followed by pRB activation, but did not activate the p53/p21(WAF1/Cip1) pathway. Moreover, MT-1G, which is induced by SM22α overexpression, was involved in the activation of the p16(INK4a)/pRB pathway, which led to a growth arrest state and promoted cellular senescence caused by damaging agents. Our findings provide the first demonstration that SM22α modulates cellular senescence caused by damaging agents via regulation of the p16(INK4a)/pRB pathway in HepG2 cells and that these effects of SM22α are partially mediated by MT-1G.

Identification of autoantibody against fatty acid synthase in hepatocellular carcinoma mouse model and its application to diagnosis of HCC.

Autoantibodies, which are generated by immune system recognizing the presence of the abnormal tumor-associated antigens, are promising biomarkers for early detection of tumors. Recently, we established a B cell hybridoma pool derived from H-ras12V transgenic mouse, a typical hepatocellular carcinoma model, as a source of tumor-associated autoantibodies without using any extracellular antigens and have characterized the specific target antigens against them. K1 autoantibody, one of them, was investigated in this study and its target antigen was identified by mass spectrometric analysis as fatty acid synthase (FASN), an important oncogenic protein. Moreover, a specific mimotope against K1 autoantibody was screened from the cyclic random hepta-peptide phage library and, using it as a coating antigen for ELISA, we could distinguish patients with hepatocellular carcinoma (HCC) vs. normal subjects with 96.55% sensitivity and 100% specificity. These results imply that anti-FASN autoantibody is induced in patients with HCC and detection of anti-FASN autoantibody can be used for the diagnosis of HCC.

Use of a baculovirus-expressed structural protein for the detection of antibodies to foot-and-mouth disease virus type A by a blocking enzyme-linked immunosorbent assay.

A blocking enzyme-linked immunosorbent assay (ELISA) with a baculovirus-expressed structural protein was developed for the detection of antibodies to foot-and-mouth disease virus type A. It exhibited 99% specificity with a cutoff of 53% inhibition. Its sensitivity was comparable to the sensitivities of the virus neutralization test and the liquid-phase blocking ELISA, indicating its potential as an alternative assay.

Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer.

Toll-like receptor 2 (TLR2) initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. We have determined the crystal structures of TLR2-TLR6-diacylated lipopeptide, TLR2-lipoteichoic acid, and TLR2-PE-DTPA complexes. PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, is a synthetic phospholipid derivative. Two major factors contribute to the ligand specificity of TLR2-TLR1 or TLR2-TLR6 heterodimers. First, the lipid channel of TLR6 is blocked by two phenylalanines. Simultaneous mutation of these phenylalanines made TLR2-TLR6 fully responsive not only to diacylated but also to triacylated lipopeptides. Second, the hydrophobic dimerization interface of TLR2-TLR6 is increased by 80%, which compensates for the lack of amide lipid interaction between the lipopeptide and TLR2-TLR6. The structures of the TLR2-lipoteichoic acid and the TLR2-PE-DTPA complexes demonstrate that a precise interaction pattern of the head group is essential for a robust immune response by TLR2 heterodimers.

SM22alpha inhibits cell proliferation and protects against anticancer drugs and gamma-radiation in HepG2 cells: involvement of metallothioneins.

Smooth muscle protein 22-alpha (SM22alpha) has been postulated to affect the structure and function of the actin filament. In this study, we report on the significant induction of SM22alpha by cytotoxic agents in HepG2 cells. SM22alpha-overexpression inhibited the activation of IGF-1Rbeta/Akt and Erk, consequently suppressing cell proliferation. On the other hand, SM22alpha-overexpressing cells became resistant to apoptotic cell death caused by cytotoxic agents, in which metallothionein (MT) isoforms, especially MT1G, were significantly induced. MT1G-overexpression also conferred cellular resistance, and SM22alpha regulated the expression of MT1G at a transcriptional level. This study provides the first demonstration of SM22alpha-induced blockage of cell proliferation and cellular resistance to overcome the detrimental effects of damaging agents.

Rational biosynthetic engineering for optimization of geldanamycin analogues.

Tailor made: We report the rational biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound. A rational biosynthetic engineering approach was applied to the optimization of the pharmacological properties of the benzoquinone ansamycin, geldanamycin. Geldanamycin and its natural or semisynthetic derivatives have the potential to serve as anticancer chemotherapeutic agents. However, these first-generation Hsp90 inhibitors share an unfavorable structural feature that causes both reduced efficacy and toxicity during clinical evaluation. We report the rationally designed biosynthesis of C15 hydroxylated non-quinone geldanamycin analogues by site-directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post-PKS tailoring genes. A 15-hydroxyl-17-demethoxy non-quinone analogue, DHQ3, exhibited stronger inhibition of Hsp90 ATPase activity (4.6-fold) than geldanamycin. Taken together, the results of the present study indicate that rational biosynthetic engineering allows the generation of derivatives of geldanamycin with superior pharmacological properties.

Inhibitory effect of a phosphatidyl ethanolamine derivative on LPS-induced sepsis.

Sepsis is the leading cause of death in critically ill patients. Today, around 60% of all cases of sepsis are caused by Gram-negative bacteria. The cell wall component lipopoly-saccharide (LPS) is the main initiator of the cascade of cellular reactions in Gram-negative infections. The core receptors for LPS are toll-like receptor 4 (TLR4), MD-2 and CD14. Attempts have been made to antagonize the toxic effect of endotoxin using monoclonal antibodies against CD14 and synthetic lipopolysaccharides but there is as yet no effective treatment for septic syndrome. Here, we describe an inhibitory effect of a phosphatidylethanolamine derivative, PE-DTPA (phosphatidylethanolamine diethyl-enetriaminepentaacetate) on LPS recognition. PE-DTPA bound strongly to CD14 (K ( d ), 9.52 x 10(-8) M). It dose dependency inhibited LPS-mediated activation of human myeloid cells, mouse macrophage cells and human whole blood as measured by the production of tumor necrosis factor-a (TNF-alpha) and nitric oxide, whereas other phospho-lipids including phosphatidylserine and phosphatidylethanolamine had little effect. PE-DTPA also inhibited transcription dependent on NF-kappaB activation when it was added together with LPS, and it rescued LPS-primed mice from septic death. These results suggest that PE-DTPA is a potent antagonist of LPS, and that it acts by competing for binding to CD14.

Induction of Mac-2BP by nerve growth factor is regulated by the PI3K/Akt/NF-kappaB-dependent pathway in the HEK293 cell line.

Mac-2BP is a ligand of the galectin family that has been suggested to affect tumor proliferation and metastasis formation. We assessed Mac-2BP expression at the transcriptional and translational levels to evaluate nerve growth factor (NGF)-induced Mac-2BP expression. A time kinetic analysis using reverse transcription-polymerase chain reaction showed that NGF-induced Mac-2BP transcript levels were 4-5 times higher than in controls. Mac-2BP enzyme-linked immunosorbent assay and immunofluorescence staining showed a 2-3-fold increase in intracellular and secreted Mac-2BP as a result of NGF stimulation. This increase was regulated by Akt activation and NF-kappaB binding. p65 and p50-NF-kappaB are major transcriptional factors in the Mac-2BP promoter region, and were shown to be regulated in accordance with the Akt activation states. Collectively, these results suggest that NGF induces Mac-2BP expression via the PI3K/Akt/NF-kappaB pathway.

Characterization of tailoring genes involved in the modification of geldanamycin polyketide in Streptomyces hygroscopicus JCM4427.

Geldanamycin and its analogs are important anticancer agents that inhibit the newly targeted, heat-shock protein (Hsp) 90, which is a chaperone protein in eukaryotic cells. To resolve which geldanamycin biosynthetic genes are responsible for particular post-polyketide synthase (PKS) processing steps and in which order the reactions occur, we individually inactivated candidate genes in Streptomyces hygroscopicus subsp. duamyceticus JCM4427, and isolated and elucidated the structures of intermediates from each mutant. The results indicated that gel7 governs at least one of the benzoquinone ring oxidation steps. In addition, gel16 was found to be involved in double-bond formation between C-4 and C-5 of 4,5-dihydrogeldanamycin, which confirmed our previous findings that this double bond reduced during the post-PKS modification of the polyketide assembly. In addition, pro-geldanamycin, which does not possess a double bond at C-4/5, was purified from the gel7 and 8 double-gene-inactivated mutant.