Gossypol Interferes with Both Type I and Type II Topoisomerase Activities Without Generating Strand Breaks

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks. © 2012 Springer Science+Business Media New York

GCN5 participates in ADA3 partnership as revealed with yeast hybrid

41st FEBS Congress on Molecular and Systems Biology for a Better Life -- SEP 03-08, 2016 -- Kusadasi, TURKEYWOS: 000383616900265FEB

Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes.

General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA- and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes

Che1/AATF interacts with subunits of the histone acetyltransferase core module of SAGA complexes

WOS: 000417698200025PubMed ID: 29232376General Control Non-derepressible 5 (GCN5) and Alteration/Deficiency in Activation 2 and 3 proteins (ADA2 and ADA3, respectively) are subunits of the Histone AcetylTransferase (HAT) module of SAGA-and ATAC-type co-activators. We previously reported four new interacting partners of human ADA3 identified by screening a human fetal brain cDNA library using yeast two hybrid technology. One of these partners was Apoptosis-Antagonizing Transcription Factor (AATF), also known as Che-1, an RNA polymerase II-binding protein with a number of roles in different cellular processes including regulation of transcription, cell proliferation, cell cycle control, DNA damage responses and apoptosis. Che-1/AATF is a potential therapeutic target for cancer treatments. In this study, we aimed to identify whether besides ADA3, other components of the HAT modules of SAGA and ATAC complexes, human ADA2 and GCN5 also interact with Che-1/AATF. Co-immunoprecipitation and co-localization experiments were used to demonstrate association of AATF both with two ADA2 isoforms, ADA2A and ADA2B and with GCN5 proteins in human cells and yeast two-hybrid assays to delineate domains in the ADA2 and GCN5 proteins required for these interactions. These findings provide new insights into the pathways regulated by ADA-containing protein complexes.Turkish Scientific and Technological Research Assembly [112T429]; Hungarian NRDIO [GINOP-2.3.2-15-2016-00020]This work was supported by the Turkish Scientific and Technological Research Assembly (112T429) (Dr. Sevil Zencir), and Hungarian NRDIO-GINOP-2.3.2-15-2016-00020 (Prof Imre M. Boros). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

The sequences of the primers used in the amplification of corresponding genes.

<p>The sequences of the primers used in the amplification of corresponding genes.</p

Co-localizations of ADA2A, ADA2B, GCN5 with AATF protein in HEK293 cells.

<p>Intracellular co-localization of CFP-conjugated ADA2A (A), ADA2B (B), GCN5 (C), empty vector expressing CFP alone (D) and YFP-conjugated AATF proteins (middle column). Yellow color in the merged image (last column) indicates co-localization. Live cell images were captured by confocal microscopy and pseudo-coloured red (CFP) and green (YFP). Insets show single transfections. Upper right corners of nuclei are marked with a curved line. The last column shows merged images. Scale bar is 5 μm.</p

Effect of co-expression of ADA2A, ADA2B, GCN5 with AATF on the expression of an MDM2 promoter-luciferase reporter gene.

<p>U2OS cells were transfected with a reporter plasmid encoding a luciferase gene under the control of the MDM2 promoter or co-transfected with the reporter plasmid and plasmids expressing ADA2A, ADA2B, GCN5 with AATF protein partner as indicated. Luciferase activity was measured 48 h post-transfection. Results represent the average ± SD from three independent assays. Co-expression of AATF with ADA2A, ADA2B or GCN5, significantly reduced the activation imparted by AATF on MDM2 promoter-directed transcription (<i>p</i> <0.01).</p