Abstract
Yeast two- and three-hybrid systems were used to screen cDNA libraries from HeLa cells and human brain tissue to identify novel protein partners of DNA Fragmentation Factor, the major apoptotic nuclease. The two-hybrid system revealed the DFF45 inhibitory subunit of the nuclease as the only identified partner of the DFF40 catalytic subunit. Similar analysis revealed several protein candidates that potentially interact with the DFF45 subunit: FBXO28, FOSL1, PGK1, PCNT, FHL1 and GFAP. Recombinant GFAP protected DFF45 against cleavage with caspase-3 and prevented activation of the DFF nuclease in vitro. In addition, three-hybrid system results revealed a putative novel protein partner of the DFF40-DFF45 heterodimer. The candidate cDNA contained two open reading frames that mapped to an intron of the GBF1 gene. Products of the candidate cDNA derived from a cell-free transcription/translation system inhibited DNA cleavage by recombinant caspase-activated DFF. This putative partner of DFF may have functional importance in regulating the apoptotic response because its RNAi silencing facilitated cleavage of the DFF45 inhibitor subunit and affected chromatin fragmentation in HeLa cells undergoing apoptosis. This hypothetical protein, named DRIG based on an acronym specifying its genomic location, could be a novel factor involved in regulation of DFF40 apoptotic nuclease.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.