Correction: Hypoxia inducible factor (HIF) as a model for studying inhibition of protein-protein interactions.
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ABSTRACT:
SUBMITTER: Burslem GM
PROVIDER: S-EPMC6156820 | biostudies-literature | 2017 Jul
REPOSITORIES: biostudies-literature
Publications
Correction: Hypoxia inducible factor (HIF) as a model for studying inhibition of protein-protein interactions.
Burslem George M GM Kyle Hannah F HF Nelson Adam A Edwards Thomas A TA Wilson Andrew J AJ
Chemical science 20170606 7
[This corrects the article DOI: 10.1039/C7SC00388A.]. ...[more]
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Project description:[This corrects the article DOI: 10.1039/C7SC90030A.].
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Project description:[This corrects the article DOI: 10.1039/C7SC00388A.].
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Project description:The modulation of protein-protein interactions (PPIs) represents a major challenge in modern chemical biology. Current approaches (e.g. high-throughput screening, computer aided ligand design) are recognised as having limitations in terms of identification of hit matter. Considerable success has been achieved in terms of developing new approaches to PPI modulator discovery using the p53/hDM2 and Bcl-2 family of PPIs. However these important targets in oncology might be considered as "low-hanging-fruit". Hypoxia inducible factor (HIF) is an emerging, but not yet fully validated target for cancer chemotherapy. Its role is to regulate the hypoxic response and it does so through a plethora of protein-protein interactions of varying topology, topography and complexity: its modulation represents an attractive approach to prevent development of new vasculature by hypoxic tumours.
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Project description:Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that is composed of a hypoxia-inducible alpha subunit (HIF-1alpha and HIF-2alpha) and a constitutively expressed beta subunit (HIF-1beta). HIF mediates the adaptation of cells and tissues to low oxygen concentrations. It also plays an important role in tumorigenesis and constitutes an important therapeutic target in anti-tumor therapy. We have screened a number of reported HIF inhibitors for their effects on HIF-transcriptional activity and found that the DNA damage inducing agents camptothecin and mitomycin C produced the most robust effects. Camptothecin is a reported inhibitor of HIF-1alpha translation, while mitomycin C has been reported to induce p53-dependent HIF-1alpha degradation. In this study we demonstrate that the inhibitory effect of mitomycin C on HIF-1alpha protein expression is not dependent on p53 and protein degradation, but also involves HIF-1alpha translational regulation. Initiation of a DNA damage response with the small molecule p53 activator NSC-652287 (RITA) has been reported to inhibit HIF-1alpha protein synthesis by increasing the phosphorylation of eIF2alpha. However, we show here that even when eIF2alpha phosphorylation is prevented, the DNA damage inducing drugs mitomycin C, camptothecin and NSC-652287 still inhibit HIF-1alpha protein synthesis to the same extent. The inhibitory effects of camptothecin on HIF-1alpha expression but not that of mitomycin C and NSC-652287 were dependent on cyclin-dependent kinase activity. In conclusion, specific types of DNA damage can bring about selective inhibition of HIF-1alpha protein synthesis. Further characterization of the involved mechanisms may reveal important novel therapeutic targets.
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Project description:The ?-subunits of hypoxia-inducible factors (HIF1? and HIF2?) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (Spry2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation by a number of different mechanisms. Because of the seemingly opposite actions of HIF? subunits and Spry2 on cellular processes, we investigated whether Spry2 regulates the levels of HIF1? and HIF2? proteins. In cell lines from different types of tumors in which the decreased protein levels of Spry2 have been associated with poor prognosis, silencing of Spry2 elevated HIF1? protein levels. Increases in HIF1? and HIF2? protein levels due to silencing of Spry2 also up-regulated HIF? target genes. Using HIF1? as a prototype, we show that Spry2 decreases HIF1? stability and enhances the ubiquitylation of HIF1? by a von Hippel-Lindau protein (pVHL)-dependent mechanism. Spry2 also exists in a complex with HIF1?. Because Spry2 can also associate with pVHL, using a mutant form of Spry2 (3P/3A-Spry2) that binds HIF1?, but not pVHL, we show that WT-Spry2, but not the 3P/3A-Spry2 decreases HIF1? protein levels. In accordance, expression of WT-Spry2, but not 3P/3A-Spry2 results in a decrease in HIF1?-sensitive glucose uptake. Together our data suggest that Spry2 acts as a scaffold to bring more pVHL/associated E3 ligase in proximity of HIF1? and increase its ubiquitylation and degradation. This represents a novel action for Spry2 in modulating biological processes regulated by HIF? subunits.
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Project description:Hypoxia-inducible factor (HIF) controls an extensive range of adaptive responses to hypoxia. To better understand this transcriptional cascade we performed genome-wide chromatin immunoprecipitation using antibodies to two major HIF-alpha subunits, and correlated the results with genome-wide transcript profiling. Within a tiled promoter array we identified 546 and 143 sequences that bound, respectively, to HIF-1alpha or HIF-2alpha at high stringency. Analysis of these sequences confirmed an identical core binding motif for HIF-1alpha and HIF-2alpha (RCGTG) but demonstrated that binding to this motif was highly selective, with binding enriched at distinct regions both upstream and downstream of the transcriptional start. Comparison of HIF-promoter binding data with bidirectional HIF-dependent changes in transcript expression indicated that whereas a substantial proportion of positive responses (>20% across all significantly regulated genes) are direct, HIF-dependent gene suppression is almost entirely indirect. Comparison of HIF-1alpha- versus HIF-2alpha-binding sites revealed that whereas some loci bound HIF-1alpha in isolation, many bound both isoforms with similar affinity. Despite high-affinity binding to multiple promoters, HIF-2alpha contributed to few, if any, of the transcriptional responses to acute hypoxia at these loci. Given emerging evidence for biologically distinct functions of HIF-1alpha versus HIF-2alpha understanding the mechanisms restricting HIF-2alpha activity will be of interest.
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Project description:Hypoxia-inducible factor-1alpha (HIF-1alpha), a transcription factor, plays a critical role in adaption to hypoxia, which is a major feature of diseases, including cancer. Protein disulfide isomerase (PDI) is up-regulated in numerous cancers and leads to cancer progression. PDI, a member of the TRX superfamily, regulates the transcriptional activities of several transcription factors. To investigate the mechanisms by which PDI affects the function of HIF-1alpha, the overexpression or knockdown of PDI was performed. The overexpression of PDI decreased HIF-1alpha expression in the human hepatocarcinoma cell line, Hep3B, whereas the knockdown of endogenous PDI increased its expression. NH4Cl inhibited the decrease in HIF-1alpha expression by PDI overexpression, suggesting that HIF-1alpha was degraded by the lysosomal pathway. HIF-1alpha is transferred to lysosomal membranes by heat shock cognate 70 kDa protein (HSC70). The knockdown of HSC70 abolished the decrease, and PDI facilitated the interaction between HIF-1alpha and HSC70. HIF-1alpha directly interacted with PDI. PDI exists not only in the endoplasmic reticulum (ER), but also in the cytosol. Hypoxia increased cytosolic PDI. We also investigated changes in the redox state of HIF-1alpha using PEG-maleimide, which binds to thiols synthesized from disulfide bonds by reduction. An up-shift in the HIF-1alpha band by the overexpression of PDI was detected, suggesting that PDI formed disulfide bond in HIF-1alpha. HIF-1alpha oxidized by PDI was not degraded in HSC70-knockdown cells, indicating that the formation of disulfide bond in HIF-1alpha was important for decreases in HIF-1alpha expression. To the best of our knowledge, this is the first study to show the regulation of the expression and redox state of HIF-1alpha by PDI. We also demonstrated that PDI formed disulfide bonds in HIF-1alpha 1-245 aa and decreased its expression. In conclusion, the present results showed that PDI is a novel factor regulating HIF-1alpha through lysosome-dependent degradation by changes in its redox state.
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Project description:Factor Inhibiting HIF (FIH) catalyzes the ?-hydroxylation of asparagine residues in HIF-? transcription factors as well as ankyrin repeat domain (ARD) proteins such as Notch and Gankyrin. Although FIH-mediated hydroxylation of HIF-? is well characterized, ARDs were only recently identified as substrates, and less is known about their recognition and hydroxylation by FIH. We investigated the molecular determinants of FIH substrate recognition, with a focus on differences between HIF and ARD substrates. We show that for ARD proteins, structural context is an important determinant of FIH-recognition, but analyses of chimeric substrate proteins indicate that the ankyrin fold alone is not sufficient to explain the distinct substrate properties of the ARDs compared with HIF. For both substrates the kinetic parameters of hydroxylation are influenced by the amino acids proximal to the target asparagine. Although FIH tolerates a variety of chemically disparate residues proximal to the asparagine, we demonstrate that certain combinations of amino acids are not permissive to hydroxylation. Finally, we characterize a conserved RLL motif in HIF and demonstrate that it mediates a high affinity interaction with FIH in the presence of cell lysate or macromolecular crowding agents. Collectively, our data highlight the importance of residues proximal to the asparagine in determining hydroxylation, and identify additional substrate-specific elements that contribute to distinct properties of HIF and ARD proteins as substrates for FIH. These distinct features are likely to influence FIH substrate choice in vivo and, therefore, have important consequences for HIF regulation.
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Project description:During normal development or during disease, animal cells experience hypoxic (low oxygen) conditions, and the hypoxia-inducible factor (HIF) transcription factors implement most of the critical changes in gene expression that enable animals to adapt to this stress. Here, we examine the roles of HIF-1 in post-mitotic aging. We examined the effects of HIF-1 over-expression and of hif-1 loss-of-function mutations on longevity in C. elegans, a powerful genetic system in which adult somatic cells are post-mitotic. We constructed transgenic lines that expressed varying levels of HIF-1 protein and discovered a positive correlation between HIF-1 expression levels and lifespan. The data further showed that HIF-1 acted in parallel to the SKN-1/NRF and DAF-16/FOXO transcription factors to promote longevity. HIF-1 over-expression also conferred increased resistance to heat and oxidative stress. We isolated and characterized additional hif-1 mutations, and we found that each of 3 loss-of-function mutations conferred increased longevity in normal lab culture conditions, but, unlike HIF-1 over-expression, a hif-1 deletion mutation did not extend the lifespan of daf-16 or skn-1 mutants. We conclude that HIF-1 over-expression and hif-1 loss-of-function mutations promote longevity by different pathways. These data establish HIF-1 as one of the key stress-responsive transcription factors that modulate longevity in C. elegans and advance our understanding of the regulatory networks that link oxygen homeostasis and aging.
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Project description:Oxidative stress contributes to tissue injury in conditions ranging from cardiovascular disease to stroke, spinal cord injury, neurodegeneration, and perhaps even aging. Yet the efficacy of antioxidants in human disease has been mixed at best. We need a better understanding of the mechanisms by which established antioxidants combat oxidative stress. Iron chelators are well established inhibitors of oxidative death in both neural and non-neural tissues, but their precise mechanism of action remains elusive. The prevailing but not completely substantiated view is that iron chelators prevent oxidative injury by suppressing Fenton chemistry and the formation of highly reactive hydroxyl radicals. Here, we show that iron chelation protects, rather unexpectedly, by inhibiting the hypoxia-inducible factor prolyl 4-hydroxylase isoform 1 (PHD1), an iron and 2-oxoglutarate-dependent dioxygenase. PHD1 and its isoforms 2 and 3 are best known for stabilizing transcriptional regulators involved in hypoxic adaptation, such as HIF-1alpha and cAMP response element-binding protein (CREB). Yet we find that global hypoxia-inducible factor (HIF)-PHD inhibition protects neurons even when HIF-1alpha and CREB are directly suppressed. Moreover, two global HIF-PHD inhibitors continued to be neuroprotective even in the presence of diminished HIF-2alpha levels, which itself increases neuronal susceptibility to oxidative stress. Finally, RNA interference to PHD1 but not isoforms PHD2 or PHD3 prevents oxidative death, independent of HIF activation. Together, these studies suggest that iron chelators can prevent normoxic oxidative neuronal death through selective inhibition of PHD1 but independent of HIF-1alpha and CREB; and that HIF-2alpha, not HIF-1alpha, regulates susceptibility to normoxic oxidative neuronal death.
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