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1.
Oncogene ; 25(12): 1763-74, 2006 Mar 16.
Article in English | MEDLINE | ID: mdl-16278674

ABSTRACT

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which upon apoptosis induction translocates to the nucleus where it interacts with DNA by virtue of positive charges clustered on the AIF surface. Here we show that the AIF interactome, as determined by mass spectroscopy, contains a large panel of ribonucleoproteins, which apparently bind to AIF through the RNA moiety. However, AIF is devoid of any detectable RNAse activity both in vitro and in vivo. Recombinant AIF can directly bind to DNA as well as to RNA. This binding can be visualized by electron microscopy, revealing that AIF can condense DNA, showing a preferential binding to single-stranded over double-stranded DNA. AIF also binds and aggregates single-stranded and structured RNA in vitro. Single-stranded poly A, poly G and poly C, as well double-stranded A/T and G/C RNA competed with DNA for AIF binding with a similar efficiency, thus corroborating a computer-calculated molecular model in which the binding site within AIF is the same for distinct nucleic acid species, without a clear sequence specificity. Among the preferred electron donors and acceptors of AIF, nicotine adenine dinucleotide phosphate (NADP) was particularly efficient in enhancing the generation of higher-order AIF/DNA and AIF/RNA complexes. Altogether, these data support a model in which a direct interaction of AIF contributes to the compaction of nucleic acids within apoptotic cells.


Subject(s)
Apoptosis Inducing Factor/metabolism , Chromatin Assembly and Disassembly/physiology , DNA/metabolism , RNA/metabolism , Amino Acid Sequence , Apoptosis Inducing Factor/chemistry , Brain/metabolism , Chromatin Immunoprecipitation , DNA/chemistry , DNA/genetics , HeLa Cells , Humans , Mass Spectrometry , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Polymerase Chain Reaction , RNA/chemistry , RNA/genetics
2.
Cell Death Differ ; 12(6): 614-26, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15818416

ABSTRACT

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.


Subject(s)
Cardiolipins/metabolism , Carrier Proteins/pharmacology , Membrane Proteins/metabolism , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Adenosine Diphosphate/pharmacology , Animals , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Membrane Permeability , Cytochromes c/metabolism , Female , Intracellular Membranes/drug effects , Intracellular Membranes/metabolism , Male , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Mitochondria, Liver/drug effects , Oxidation-Reduction , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-bcl-2/deficiency , Proto-Oncogene Proteins c-bcl-2/genetics , bcl-2 Homologous Antagonist-Killer Protein , bcl-2-Associated X Protein , bcl-X Protein
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