Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis.
DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.
Widlak
Piotr
P
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Lanuszewska
Joanna
J
Cary
Robert B
RB
Garrard
William T
WT
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D053938:DNA Fragmentation; D019281:Dimerization; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D021122:Protein Subunits; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; D005047:Etoposide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases