Expression, mitochondrial localisation and isolation of human cell induced death effector - a (CIDEa)
J. Šantorová , K. Janouchová and P. Ježek
Dept.75 Membrane Transport Biophysics, Institute of Physiology, Prague, Czech Republic. E-mail: firstname.lastname@example.org; email@example.com
CIDEa, CIDEb proteins are related to both N terminals of the heterodimeric DNA fragmentation factor DFF, consisting of the 40-kDa caspase-3-activated nuclease (DFF40 or CAD), & its 45-kDa inhibitor (DFF45 or ICAD) . The DFF45&DFF40 complex is cleaved by caspase-3 and released nuclease then causes apoptotic DNA fragmentation. CIDE-induced apoptosis is not sensitive to caspase inhibitors but is inhibited by DFF45. The N-domain of CIDEa binds to the homologous domain on DFF45 opposing its inhibitory effect on DFF40. However, mitochondrial localization and CIDEb(a) dimerization is likely required for induction of apoptosis .
In this work we have confirmed mitochondrial localization of recombinant human CIDEa in W303 and JB516 yeast strains of S. cerevisiae and of human CIDEa or CIDEa-fused with a red fluorescent protein (RFP-CIDEa) in selected culture cells, such as embryonic kidney 293T cells, hepatocellular carcinoma HEPG2 cells, and insulinoma INS1-E cells. The CIDEa import into the inner membrane was proven by immunodetection of fractionated mitochondria and its identity was verified by Western blotting and by MALDI-TOF-assisted peptide mapping of the trypsinized samples of isolated yeast mitochondria. RFP-CIDEa transfected to cultured cells was co-localized with mitotrackers and with mitochondrially-targeted green fluorescent protein. Further we transformed Rosetta Competent cells with His-tag CIDEa and isolated protein on Ni –NTA agarose column. Pure protein was used for reconstitution and protein-protein interaction studies.
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