Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

A genomic approach to understanding the cause and effect of annual ryegrass toxicity

Annual Ryegrass Toxicity (ARGT) is a potentially lethal disease affecting livestock grazing on pastures or consuming fodder that include annual ryegrass (Lolium rigidum) contaminated with corynetoxins. The corynetoxins (CTs), among the most lethal toxins produced in nature, are produced by the bacterium Rathayibacter toxicus that uses a nematode vector to attach to and infect the seedheads of L.rigidum. There is little known of the factors that control toxin production. Several studies have speculated that a bacteriophage specific to R.toxicus may be implicated in CT production. We have developed a PCR-based assay to test for both bacterium and phage in ryegrass material and results indicate that there is a correlation between phage and bacterial presence in all toxic ryegrass samples tested so far. This PCR-based technique may ultimately allow for a rapid, high-throughput screening assay to identify potentially toxic pastures and feed in the field. Currently, ~80% of the 45 Kb genome has been sequenced an investigation to further elucidate its potential role in toxin production.Furthermore, specific alterations in gene expression as a result of exposure to CTs or the closely related tunicamycins (TMs), which are commercially available and considered biologically indistinguishable from CTs, will be evaluated for use as biomarkers of exposure. The effects of both toxins will be analysed in vitro using a rat hepatocyte cell line and screened on a low-density DNA micro array “CT-Chip” that contains <100 selected rat hepatic genes. The results are expected to further define the bioequivalence of CTs and TMs and to identify levels of exposure that are related to specific toxic effects or have no adverse effect on livestock.

Cell cycle sensing of oxidative stress in saccharomyces cerevisiae by oxidation of a specific cysteine residue in the transcription factor Swi6p

Yeast cells begin to bud and enter S phase when growth conditions are favourable during G1 phase. When subjected to some oxidative stresses, cells delay entry at G1 allowing repair of cellular damage. Hence, oxidative stress sensing is coordinated with the regulation of cell cycle. We identified a novel function of the cell-cycle regulator of Saccharomyces cerevisiae, Swi6p, as a redox sensor through its cysteine residue at position 404. When alanine was substituted at this position, the resultant mutant, C404A, was sensitive to several reactive oxygen species and oxidants including linoleic acid hydroperoxide, the superoxide anion and diamide. This mutant lost the ability to arrest in G1 phase upon treatment with lipid hydroperoxide. The Cys404 residue of Swi6p in wild-type cells was oxidised to a sulfenic acid when cells were subjected to linoleic acid hydroperoxide. Mutation of Cys404 to Ala abolished the down-regulation of expression of the G1 cyclin genes CLN1, CLN2, PCL1 and PCL2 that occurred when cells of the wild type were exposed to the lipid hydroperoxide. In conclusion, oxidative stress signaling for cell-cycle regulation occurs through oxidation of the G1/S-speicific transcription factor Swi6p and consequently leads to suppression of the expression of G1-cyclins and delay in cells entering the cell cycle.

Biosynthesis, localization, and macromolecular arrangement of the plasmodium falciparum translocon of exported proteins (PTEX)

To survive within its host erythrocyte, Plasmodium falciparum must export hundreds of proteins across both its parasite plasma membrane and surrounding parasitophorous vacuole membrane, most of which are likely to use a protein complex known as PTEX (Plasmodium translocon of exported proteins). PTEX is a putative protein trafficking machinery responsible for the export of hundreds of proteins across the parasitophorous vacuole membrane and into the human host cell. Five proteins are known to comprise the PTEX complex, and in this study, three of the major stoichiometric components are investigated including HSP101 (a AAA+ ATPase), a protein of no known function termed PTEX150, and the apparent membrane component EXP2. We show that these proteins are synthesized in the preceding schizont stage (PTEX150 and HSP101) or even earlier in the life cycle (EXP2), and before invasion these components reside within the dense granules of invasive merozoites. From these apical organelles, the protein complex is released into the host cell where it resides with little turnover in the parasitophorous vacuole membrane for most of the remainder of the following cell cycle. At this membrane, PTEX is arranged in a stable macromolecular complex of >1230 kDa that includes an ∼600-kDa apparently homo-oligomeric complex of EXP2 that can be separated from the remainder of the PTEX complex using non-ionic detergents. Two different biochemical methods undertaken here suggest that PTEX components associate as EXP2-PTEX150-HSP101, with EXP2 associating with the vacuolar membrane. Collectively, these data support the hypothesis that EXP2 oligomerizes and potentially forms the putative membrane-spanning pore to which the remainder of the PTEX complex is attached.

X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells

It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations.

Biosynthesis and expression of a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats-15

Background
ADAMTS proteoglycanases show proteolytic activity toward versican and other proteoglycans.

Results
ADAMTS15, which cleaves versican, is expressed during early cardiac development and during musculoskeletal development.

Conclusion
With unique and overlapping biological properties, ADAMTS15 is likely to have cooperative roles with other members of the ADAMTS proteoglycanase clade.

Significance
Versican cleavage has profound effects on developmental morphogenesis and regulates cancer cell behavior.

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2–3× slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis is only weakly inhibited by the presence of oxygen (Rock, J. D., Thomson, M. J., Read, R. C., and Moir, J. W. (2007) J. Bacteriol. 189, 1138–1144). NmFNR binds to DNA containing a consensus FNR box sequence, and this binding stabilizes the iron-sulfur cluster in the presence of oxygen. Partial degradation of the 4Fe-4S cluster to a 3Fe-4S occurs, and this form remains bound to the DNA. The 3Fe-4S cluster is converted spontaneously back to a 4Fe-4S cluster under subsequent anaerobic reducing conditions in the presence of ferrous iron. The finding that binding to DNA stabilizes FNR in the presence of oxygen such that it has a half-life of ∼30 min on the DNA has implications for our appreciation of how oxygen switches off FNR activatable genes in vivo.