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Dataset for reprogramming the transcriptional response to hypoxia with a chromosomally encoded cyclic peptide HIF-1 inhibitor.
Full text linkDataset from:
Mistry, Ishna and Tavassoli, Ali (2016) Reprogramming the transcriptional response to hypoxia with a chromosomally encoded cyclic peptide HIF-1 inhibitor. ACS Synthetic Biology (doi: 10.1021/acssynbio.6b00219).
Array data from TaqMan human hypoxia array analysis of the engineered cell line +/- dox and +/- HIF-2a siRNA.</span
Reprogramming the cellular response to hypoxia
No full textHIF-1 is a heterodimeric transcription factor comprising HIF-1[alpha] and HIF-1[Beta] subunits. Cellular response to hypoxia relies on the dimerisation of HIF-1? and HIF-1? in the nucleus, forming HIF-1, which then initiates transcription of target genes. Both subunits are constitutively expressed, but the ? subunit is degraded in an oxygen-dependent manner, so it is only stabilised in hypoxia. HIF-1 expression is deregulated in many cancers and has been associated with resistance to chemotherapy. Therefore, understanding the molecular mechanisms of hypoxic response is key to developing cancer therapeutics.This thesis describes the construction and validation of a novel method to study the HIF-1 pathway by reprogramming cellular behaviour. This is achieved by targeting HIF-1[alpha]/HIF-1[Beta] dimerisation with an endogenously expressed molecular inhibitor. The compound, cyclo-CLLFVY, was previously identified through screening of a library of biologically synthesised cyclic peptides. In this study, the peptide sequence was genetically-encoded, between split Nostoc punctiforme DnaE inteins, onto the chromosome of a mammalian cell line, to allow inducible expression and in situ cyclisation of the peptide. The utility of this methodology was demonstrated via identification of genes specifically transactivated by HIF-1 or HIF-2 transcription factors in hypoxia, in the integrated cell line. Furthermore, cell viability assays showed that specific inhibition of HIF-1 dimerisation in these cells increased cellular sensitivity to glucose deprivation and to inhibition of glycolysis.In addition, the epigenetic regulation of HIF-1[alpha] was studied. HIF-1 transactivates the expression of its [alpha]-subunit resulting in positive autoregulation, which contributes to the build up of HIF-1[alpha] at the onset of hypoxia. This autoregulation is dependent on an unmethylated CpG site in the hypoxia response element (HRE) within the promoter of the HIF-1[alpha] gene. Given the key role of HIF-1 in the development of the mammalian embryo, the methylation status of the HIF-1[alpha] HRE in developing tissue was sought. The HIF-1[alpha] HRE was unmethylated in several embryonic tissues, suggesting that transactivation of HIF-1[alpha] plays a role in HIF-1-mediated gene expression during development
Post-translational control of protein function with light using a LOV-intein fusion protein
No full textMethods for the post-translational control of protein function with light hold much value as tools in cell biology. To this end, we report a fusion protein that consists of DnaE split-inteins, flanking the light sensitive LOV2 domain of Avena sativa. The resulting chimera combines the activities of these two unrelated proteins to enable controlled formation of a functional protein via upregulation of intein splicing with blue light in bacterial and human cells
Reprogramming the transcriptional response to hypoxia with a chromosomally encoded cyclic peptide HIF-1 inhibitor
No full textThe cellular response to hypoxia is orchestrated by HIF-1, a heterodimeric transcription factor composed of an and subunit that enables cell survival under low oxygen conditions by altering the transcription of over 300 genes. There is significant evidence that inhibition of HIF-1 would be beneficial for cancer therapy. We recently reported a cyclic hexapeptide that inhibits the HIF-1/HIF-1 protein-protein interaction in vitro and prevents HIF-1-mediated hypoxia-response signalling in cells. This cyclic peptide was identified from a library of 3.2 x 106 members generated using SICLOPPS split-intein mediated protein splicing. With a view to demonstrating the potential for encoding the production of a therapeutic agents in response to a disease marker, we have engineered human cells with an additional chromosomal control circuit that conditionally encodes the production of our cyclic peptide HIF-1 inhibitor. We demonstrate the conditional production of our HIF-1 inhibitor in response to hypoxia, and its inhibitory effect on HIF-1 dimerization and downstream hypoxia-response signalling. These engineered cells are used to illustrate the synthetic lethality of inhibiting HIF-1 dimerization and glycolysis in hypoxic cells. Our approach not only eliminates the need for the chemical synthesis and targeted delivery of our HIF-1 inhibitor to cells, it also demonstrates the wider possibility that the production machinery of other bioactive compounds may be incorporated onto the chromosome of human cells. This work demonstrates the potential of sentinel circuits that produce molecular modulators of cellular pathways in response to environmental, or cellular disease stimuli
Hypoxia drives the assembly of the multi-enzyme purinosome complex
No full textThe purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis was observed in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand
Probing the epigenetic regulation of HIF-1α transcription in developing tissue
No full textHIF-1 is the master regulator of cellular hypoxia response; the oxygen sensitive HIF-1α subunit transactivates its own expression in hypoxia via a hypoxia response element (HRE) in the promoter of the HIF-1α gene. This transactivation loop significantly contributes to the build up of HIF-1α at the onset of hypoxia, with the binding of HIF-1 to the HIF-1α promoter being dependent on the epigenetic status of a CpG dinucleotide in the upstream HRE. Given the central role played by HIF-1 in tissue development, we sought to probe the epigenetic status of the HIF-1α HRE and that of its downstream target EPO in embryonic tissue. Our data shows that the CpG dinucleotide in HIF-1α HRE is unmethylated in several embryonic tissue samples, suggesting that transactivation of HIF-1α plays a significant role in HIF-1 mediated hypoxia response during development
Dataset for: Hypoxia drives the assembly of the multi-enzyme purinosome complex
No full textDataset supporting the paper: Doigneaux, C., Pedley, A., Mistry, I. N., Papayova, M., Benkovic, S., & Tavassoli, A. (Accepted/In press). Hypoxia drives the assembly of the multi-enzyme purinosome complex. The Journal of Biological Chemistry. DOI:10.1074/jbc.RA119.012175</span
Fluorescent transmembrane anion transporters: shedding light on anionophoric activity in cells
No full textA series of fluorescent anion transporters consisting of a urea or thiourea group linked to a naphthalimide fluorophore have been synthesised and their anion transport properties studied. The compounds possess similar anion transport properties of (thio)urea-based anionophores that have previously been reported. Fluorescence studies in cells show all anionophores cross the plasma membrane and localise within the interior of cells. The most lipophilic, aromatic substituted transporters localise homogeneously throughout the cell and are toxic towards cancer cells with the highly fluorinated compound 6 being the most effective. The least lipophilic, alkyl substituted transporters localise in specific vesicles and are non-toxic to cells. This work provides new insight to the actions of anionophores in cells and may be useful in the design of novel antineoplastic agents.<br/
Dataset for Fluorescent transmembrane anion transporters: shedding light on anionophoric activity in cells
No full textA series of fluorescent anion transporters consisting of a urea or thiourea group linked to a naphthalimide fluorophore have been synthesised and their anion transport properties studied. The compounds possess similar anion transport properties of (thio)urea-based anionophores that have previously been reported. Fluorescence studies in cells show all anionophores cross the plasma membrane and localise within the interior of cells. The most lipophilic, aromatic substituted transporters localise homogeneously throughout the cell and are toxic towards cancer cells with the highly fluorinated compound 6 being the most effective. The least lipophilic, alkyl substituted transporters localise in specific vesicles and are non-toxic to cells. This work provides new insight to the actions of anionophores in cells and may be useful in the design of novel antineoplastic agents.</span
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