Dataset Information

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

ABSTRACT: FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.

SUBMITTER: Kerr E

PROVIDER: S-EPMC3392639 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

Kerr E E Holohan C C McLaughlin K M KM Majkut J J Dolan S S Redmond K K Riley J J McLaughlin K K Stasik I I Crudden M M Van Schaeybroeck S S Fenning C C O'Connor R R Kiely P P Sgobba M M Haigh D D Johnston P G PG Longley D B DB

Cell death and differentiation 20120210 8

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by th ...[more]

PMID: 22322857

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Project description:The prevention and treatment of malaria requires a multi-pronged approach, including the development of drugs that have novel modes of action. Histone deacetylases (HDACs), enzymes involved in post-translational protein modification, are potential new drug targets for malaria. However, the lack of recombinant P. falciparum HDACs and suitable activity assays, has made the investigation of compounds designed to target these enzymes challenging. Current approaches are indirect and include assessing total deacetylase activity and protein hyperacetylation via Western blot. These approaches either do not allow differential compound effects to be determined or suffer from low throughput. Here we investigated dot blot and ELISA methods as new, higher throughput assays to detect histone lysine acetylation changes in P. falciparum parasites. As the ELISA method was found to be superior to the dot blot assay using the control HDAC inhibitor vorinostat, it was used to evaluate the histone H3 and H4 lysine acetylation changes mediated by a panel of six HDAC inhibitors that were shown to inhibit P. falciparum deacetylase activity. Vorinostat, panobinostat, trichostatin A, romidepsin and entinostat all caused an ~3-fold increase in histone H4 acetylation using a tetra-acetyl lysine antibody. Tubastatin A, the only human HDAC6-specific inhibitor tested, also caused H4 hyperacetylation, but to a lesser extent than the other compounds. Further investigation revealed that all compounds, except tubastatin A, caused hyperacetylation of the individual N-terminal H4 lysines 5, 8, 12 and 16. These data indicate that tubastatin A impacts P. falciparum H4 acetylation differently to the other HDAC inhibitors tested. In contrast, all compounds caused hyperacetylation of histone H3. In summary, the ELISA developed in this study provides a higher throughput approach to assessing differential effects of antiplasmodial compounds on histone acetylation levels and is therefore a useful new tool in the investigation of HDAC inhibitors for malaria.

Project description:Histone acetylation, balanced by histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, affects dynamic transitions of chromatin structure to regulate transcriptional accessibility. However, little is known about the interplay between HAT and HDAC complexes in Fusarium graminearum, a causal agent of Fusarium Head Blight (FHB) that uniquely contains chromosomal regions enriched for house-keeping or infection-related genes. In this study, we identified the ortholog of the human inhibitor of growth (ING1) gene in F. graminearum (FNG1) and found that it specifically interacts with the FgEsa1 HAT of the NuA4 complex. Deletion of FNG1 led to severe growth defects and blocked conidiation, sexual reproduction, DON production, and plant infection. The fng1 mutant was normal in H3 acetylation but significantly reduced in H4 acetylation. A total of 34 spontaneous suppressors of fng1 with faster growth rate were isolated. Most of them were still defective in sexual reproduction and plant infection. Thirty two of them had mutations in orthologs of yeast RPD3, SIN3, and SDS3, three key components of the yeast Rpd3L HDAC complex. Four mutations in these three genes were verified to suppress the defects of fng1 mutant in growth and H4 acetylation. The rest two suppressor strains had a frameshift or nonsense mutation in a glutamine-rich hypothetical protein that may be a novel component of the FgRpd3 HDAC complex in filamentous fungi. FgRpd3, like Fng1, localized in euchromatin. Deletion of FgRPD3 resulted in severe growth defects and elevated H4 acetylation. In contract, the Fgsds3 deletion mutant had only a minor reduction in growth rate but FgSIN3 appeared to be an essential gene. RNA-seq analysis revealed that 48.1% and 54.2% of the genes with altered expression levels in the fng1 mutant were recovered to normal expression levels in two suppressor strains with mutations in FgRPD3 and FgSDS3, respectively. Taken together, our data showed that Fng1 is important for H4 acetylation as a component of the NuA4 complex and functionally related to the FgRpd3 HDAC complex for transcriptional regulation of genes important for growth, conidiation, sexual reproduction, and plant infection in F. graminearum.

Dataset Information

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

Publications

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

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