Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo.
Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo.
Manara
Anna
A
Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy.
Lindsay
Jennefer
J
Marchioretto
Marta
M
Astegno
Alessandra
A
Gilmore
Andrew P
AP
Esposti
Mauro Degli
MD
Crimi
Massimo
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D053181:Caspase 8 / Q000378:metabolism; D002474:Cell-Free System; D045304:Cytochromes c / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism
D051023:BH3 Interacting Domain Death Agonist Protein; D050505:Mutant Proteins; D010743:Phospholipids; D050998:bcl-2 Homologous Antagonist-Killer Protein; D045304:Cytochromes c; D053181:Caspase 8