The Impact of the C-Terminal Domain on the Interaction of Human DNA Topoisomerase II α and β with DNA

<b>Background</b> Type II DNA topoisomerases are essential, ubiquitous enzymes that act to relieve topological problems arising in DNA from normal cellular activity. Their mechanism of action involves the ATP-dependent transport of one DNA duplex through a transient break in a second DNA duplex; metal ions are essential for strand passage. Humans have two isoforms, topoisomerase IIα and topoisomerase IIβ, that have distinct roles in the cell. The C-terminal domain has been linked to isoform specific differences in activity and DNA interaction. <b>Methodology/Principal Findings</b> We have investigated the role of the C-terminal domain in the binding of human topoisomerase IIα and topoisomerase IIβ to DNA in fluorescence anisotropy assays using full length and C-terminally truncated enzymes. We find that the C-terminal domain of topoisomerase IIβ but not topoisomerase IIα affects the binding of the enzyme to the DNA. The presence of metal ions has no effect on DNA binding. Additionally, we have examined strand passage of the full length and truncated enzymes in the presence of a number of supporting metal ions and find that there is no difference in relative decatenation between isoforms. We find that calcium and manganese, in addition to magnesium, can support strand passage by the human topoisomerase II enzymes. <b>Conclusions/Significance</b> The C-terminal domain of topoisomerase IIβ, but not that of topoisomerase IIα, alters the enzyme's KD for DNA binding. This is consistent with previous data and may be related to the differential modes of action of the two isoforms in vivo. We also show strand passage with different supporting metal ions for human topoisomerase IIα or topoisomerase IIβ, either full length or C-terminally truncated. They all show the same preferences, whereby Mg > Ca > Mn

Structure of the TPR Domain of AIP: Lack of Client Protein Interaction with the C-Terminal alpha-7 Helix of the TPR Domain of AIP Is Sufficient for Pituitary Adenoma Predisposition

PMCID: PMC3534021This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

The Immunophilin-Like Protein XAP2 Is a Negative Regulator of Estrogen Signaling through Interaction with Estrogen Receptor α

XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERβ-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events

The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs

Horizon Scanning to Predict and Prioritize Invasive Alien Species With the Potential to Threaten Human Health and Economies on Cyprus

Invasive alien species (IAS) are known to be a major threat to biodiversity and ecosystem function and there is increasing evidence of their impacts on human health and economies globally. We undertook horizon scanning using expert-elicitation to predict arrivals of IAS that could have adverse human health or economic impacts on the island of Cyprus. Three hundred and twenty five IAS comprising 89 plants, 37 freshwater animals, 61 terrestrial invertebrates, 93 terrestrial vertebrates, and 45 marine species, were assessed during a two-day workshop involving 39 participants to derive two ranked lists: (1) IAS with potential human health impacts (20 species ranked within two bands: 1–10 species or 11–20 species); and, (2) IAS with potential economic impacts (50 species ranked in three bands of 1–10, 11–20, and 21–50). Five species of mosquitoes (Aedes aegypti, Aedes albopictus, Aedes flavopictus, Aedes japonicus, and Culex quinquefasciatus) were considered a potential threat to both human health and economies. It was evident that the IAS identified through this process could potentially arrive through many pathways (25 and 23 pathways were noted for the top 20 IAS on the human health and economic impact lists respectively). The Convention on Biological Diversity Level II (subcategory) pathways Contaminant on plants, pet/aquarium/terrarium species (including live food for such species), hitchhikers in or on aeroplanes, hitchhikers in or on ship/boats, and vehicles were the main pathways that arose across both lists. We discuss the potential of horizon scanning lists to inform biosecurity policies and communication around IAS, highlighting the importance of increasing understanding amongst all stakeholders, including the public, to reduce the risks associated with predicted IAS arrivals

Octreotide and the mTOR inhibitor RAD001 (everolimus) block proliferation and interact with the Akt-mTOR-p70S6K pathway in a neuro-endocrine tumour cell Line.

BACKGROUND/AIM: The mode of action of the somatostatin analog octreotide on neuro-endocrine tumour proliferation is largely unknown. Overexpression of the proto-oncogene Akt/PKB (protein kinase B) has been demonstrated in certain neuro-endocrine tumours: Akt activates downstream proteins including mTOR and p70S6K, which play an important role in cell proliferation. RAD001 (everolimus) is a novel agent that is being trialled in the treatment of neuro-endocrine tumours, and is known to interact with mTOR. We explored the mechanism of action of octreotide, RAD001, and their combination on cell proliferation and kinase activation in a neuro-endocrine tumour cell line (rat insulinoma cell line, INS1). METHODS: Proliferation assays were used to determine the effects of octreotide, RAD001, and their combination on cell proliferation. Western blotting was used to characterize the expression of phosphorylated Akt, phosphorylated TSC2, phosphorylated mTOR, and phosphorylated 70S6K. RESULTS: Treatment with octreotide and RAD001 inhibited proliferation and attenuated phosphorylation of all downstream targets of Akt: TSC2, mTOR, and p70S6K. CONCLUSIONS: In this cell model, octreotide and RAD001 appear to act through a similar pathway and inhibit the Akt-mTOR-p70S6 kinase pathway downstream of Akt. There may be some overlapping effects of the two inhibitors on the mTOR pathway, although it is likely that other additional effects may differentiate the two agents