Atsushi Fukuoh

Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA.

ABSTRACT Mitochondrial transcription factor A (TFAM), a transcription factor for mitochondrial DNA (mtDNA) that also possesses the property of nonspecific DNA binding, is essential for maintenance of mtDNA. To clarify the role of TFAM, we repressed the expression of endogenous TFAM in HeLa cells by RNA interference. The amount of TFAM decreased maximally to about 15% of the normal level at day 3 after RNA interference and then recovered gradually. The amount of mtDNA changed closely in parallel with the daily change in TFAM while in organello transcription of mtDNA at day 3 was maintained at about 50% of the normal level. TFAM lacking its C-terminal 25 amino acids (TFAM-ΔC) marginally activated transcription in vitro. When TFAM-ΔC was expressed at levels comparable to those of endogenous TFAM in HeLa cells, mtDNA increased twofold, suggesting that TFAM-ΔC is as competent in maintaining mtDNA as endogenous TFAM under these conditions. The in organello transcription of TFAM-ΔC-expressing cells was no more than that in the control. Thus, the mtDNA amount is finely correlated with the amount of TFAM but not with the transcription level. We discuss an architectural role for TFAM in the maintenance of mtDNA in addition to its role in transcription activation.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Gene and cytokine profile analysis of macrolide-resistant Mycoplasma pneumoniae infection in Fukuoka, Japan

BackgroundRecent epidemiologic data suggest that the prevalence of macrolide resistant Mycoplasma pneumoniae (MR-M. pneumoniae) is increasing rapidly worldwide. This study assessed the present status of M. pneumoniae infection in Japan and clinical end-points to distinguish children with MR-M. pneumoniae.MethodsDuring an outbreak of M. pneumoniae infections in Fukuoka, Japan in 2010–11, a total of 105 children with clinically suspected M. pneumoniae infection were enrolled. M. pneumoniae was analyzed for macrolide resistance in domain V of the 23S rRNA gene. Sixty -five patients with PCR positive for M. pneumoniae were analyzed with regard to clinical symptoms, efficacy of several antimicrobial agents and several laboratory data.ResultsCausative pathogens were detected in 81.0% (85 of 105) and M. pneumoniae was identified 61.9% (65 of 105). The resistance rate of M. pneumoniae was 89.2% (58 of 65) in this general pediatric outpatient setting. Patients infected with MR-M. pneumoniae showed longer times to resolution of fever and required frequent changes of the initially prescribed macrolide to another antimicrobial agent. We observed three different genotypes of M. pneumoniae including the rarely reported A2063T mutation (A2063G: 31 strains, A2063T: 27 strains, no mutation: 7 strains). Drug susceptibility testing showed different antimicrobial susceptibility profiles for each genotype. Serum IFN-gamma, IL-6 and IP-10 levels were higher in patients with MR-genotypes than in those infected with no-mutation strains (p < 0.001).ConclusionsMacrolide resistance is more common than previously thought and a small epidemic of rarely reported A2063T mutation was observed in Fukuoka, Japan. Furthermore our results reveal the possibility that levels of certain inflammatory cytokines may be a candidate to predict MR-M.pneumoniae infection.

DNA conformation‐dependent activities of human mitochondrial RNA polymerase

Mitochondrial RNA polymerase (POLRMT) is a core protein for mitochondrial DNA (mtDNA) transcription. In addition, POLRMT is assumed to be involved in replication, although its exact role is not yet clearly elucidated. We have found novel properties of human POLRMT using a reconstituted transcription system. Various lengths of RNA molecules were synthesized from templates even without a defined promoter sequence, when we used supercoiled circular double‐stranded DNA as a template. This promoter‐independent activity was as strong as the promoter‐dependent one. Promoter‐independent DNA conformation‐dependent transcription required TFB2M. On supercoiled templates, the promoter‐independent activity was strongly suppressed by a putatively physiological amount of TFAM, while promoter‐dependent transcription was inhibited to a lesser extent. These different inhibition patterns by TFAM may be important for prevention of random RNA synthesis in vivo. Promoter‐independent activity was also observed on relaxed circular single‐stranded DNA, where its activity no longer required TFB2M. RNA synthesis on single‐stranded DNA was weakly suppressed by a putatively physiological amount of TFAM but restored by the addition of mitochondrial single‐stranded DNA binding protein. We suggest that these properties of POLRMT could explain the characteristic features of mammalian mtDNA transcription and replication.

Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

Effects of DNA lesions on the transcription reaction of mitochondrial RNA polymerase: implications for bypass RNA synthesis on oxidative DNA lesions

Oxidative DNA lesions inhibit the transcription of RNA polymerase II, but in the presence of transcription elongation factors, the transcription can bypass the lesions. Single-subunit mitochondrial RNA polymerase (mtRNAP) catalyses the synthesis of essential transcripts in mitochondria where reactive oxidative species (ROS) are generated as by-products. The occurrence of RNA synthesis by mtRNAP at oxidative DNA lesions remains unknown. Purified mtRNAP and a complex of RNA primer/DNA template containing a single DNA lesion, such as ROS-induced 8-oxoguanine (8-oxoG), two isomeric thymine glycols (5R-Tg or 5S-Tg), the UV-induced cis-syn cyclobutane pyrimidine dimer (CPD) and the pyrimidine(6-4)pyrimidone photoproduct (6-4pp), or a spontaneous common DNA lesion, a base-loss-induced apurinic/apyrimidinic (AP) site, were used for in vitro RNA synthesis assays. In this report, we show that mtRNAP bypassed the oxidative DNA lesions of non-bulky 8-oxoG and 5R-Tg and 5S-Tg with pausing sites but did not bypass the UV-induced DNA lesions and the AP site. The bacteriophage T7 phage RNA polymerase, which is homologous to mtRNAP, bypassed 8-oxoG but stalled at 5R-Tg and 5S-Tg. As expected, although translesion RNA synthesis in 8-oxoG on the DNA templates generated incorrect transcripts with a G:C to T:A transversion, the synthesis in Tg could lead to the correct transcripts with no transcriptional mutagenesis. Collectively, these data suggest that mtRNAP may tolerate the mitochondrial genome containing oxidative DNA lesions induced by ROS from the side effects of an ATP generation reaction.

Inhibitory effect of coenzyme Q1 on eukaryotic DNA polymerase γ and DNA topoisomerase II activities on the growth of a human cancer cell line

Coenzyme Q (CoQ) is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ having shorter isoprenoid chains, especially CoQ1 and CoQ2, selectively inhibited the in vitro activity of eukaryotic DNA polymerase (pol) γ, which is a mitochondrial pol. These compounds did not influence the activities of nuclear DNA replicative pols such as α, δ and ɛ, and nuclear DNA repair‐related pols such as β, η, ι, κ and λ. CoQ also inhibited DNA topoisomerase II (topo II) activity, although the enzymatic characteristics, including modes of action, amino acid sequences and three‐dimensional structures, were markedly different from those of pol γ. These compounds did not inhibit the activities of procaryotic pols such as Escherichia coli pol I, and other DNA metabolic enzymes such as human immunodeficiency virus reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. CoQ1, which has the shortest isoprenoid chains, had the strongest inhibitory effect on pol γ and topo II activities among CoQ1–CoQ10, with 50% inhibitory concentration (IC50) values of 12.2 and 15.5 µM, respectively. CoQ1 could prevent the growth of human promyelocytic leukemia cells, HL‐60, and the 50% lethal dose (LD50) value was 14.0 µM. The cells were halted at S phase and G1 phase in the cell cycle, and suppressed mitochondrial proliferation. From these results, the relationship between the inhibition of pol γ/topo II and cancer cell growth by CoQ is discussed. (Cancer Sci 2006; 97: 716–723)

Methods for Assessing Binding of Mitochondrial Transcription Factor A (TFAM) to DNA

It is now recognized that mammalian mitochondrial DNA forms a higher structure called the nucleoid, corresponding to the nucleosome of nuclear DNA. Mitochondrial transcription factor A (TFAM), which was cloned as a transcription factor for mitochondrial DNA, is essential for the maintenance of mitochondrial DNA. In fact, TFAM markedly enhances the promoter-specific transcription of mitochondrial DNA. In addition, TFAM has an ability to bind to DNA in a sequence-independent manner and is abundant enough to cover an entire region of mitochondrial DNA. Over-expression of human TFAM in cells increases the amount of mitochondrial DNA almost in parallel with the TFAM. TFAM may stabilize mitochondrial DNA by packaging and regulate (or titrate) the amount of mitochondrial DNA. Thus, TFAM may play a crucial role in maintaining mitochondrial DNA as a main component of the nucleoid (or more appropriately mitochromosome).

Scavenger receptor CL-P1 mediates endocytosis by associating with AP-2μ2

BACKGROUND Scavenger receptor CL-P1 (collectin placenta 1) has been found recently as a first membrane-type collectin which is mainly expressed in vascular endothelial cells. CL-P1 can endocytose OxLDL as well as microbes but in general, the endocytosis mechanism of a scavenger receptor is not well elucidated. METHODS We screened a placental cDNA library using a yeast two-hybrid system to detect molecules associated with the cytoplasmic domain of CL-P1. We analyzed the binding and endocytosis of several ligands in CL-P1 transfectants and performed the inhibition study using tyrphostin A23 which is a specific inhibitor of tyrosine kinase, especially in μ2-dependent endocytosis and the site-directed mutagenesis in the endocytosis YXXΦ motif in CL-P1 cytoplasmic region. Furthermore, the SiRNA study of clathrin, adaptor AP-2 and dynamin-2 during the endocytosis of OxLDL in CL-P1 transfectant cells was carried out. RESULTS We identified μ2 subunit of the AP-2 adaptor complex as a molecule associated with the cytoplasmic region of CL-P1. We demonstrated that AP-2μ2 was essential for CL-P1 mediated endocytosis of OxLDL in CL-P1 transfectant cells and its endocytosis was also mediated by clathrin, dynamin and adaptin complex molecules. CONCLUSIONS Tyrosine-based YXXΦ sequences play an important role in CL-P1-mediated OxLDL endocytosis associated with AP-2μ2. GENERAL SIGNIFICANCE This might be the first finding of the clear endocytosis mechanism in scavenger receptor CL-P1.