Abstract
The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family. BCL-2-associated X apoptosis regulator (BAX) plays a pivotal role in the initiation of mitochondria-mediated apoptosis as one of the factors causing mitochondrial outer-membrane permeabilization (MOMP). Of current interest are endogenous BAX ligands that inhibit its MOMP activity. Mitochondrial-derived peptides (MDPs) are a recently identified class of mitochondrial retrograde signaling molecules and are reported to be potent apoptosis inhibitors. Among them, humanin (HN) has been shown to suppress apoptosis by inhibiting BAX translocation to the mitochondrial outer membrane, but the molecular mechanism of this interaction is unknown. Here, using recombinant protein expression, along with light-scattering, CD, and fluorescence spectroscopy, we report that HN and BAX can form fibers together in vitro Results from negative stain EM experiments suggest that BAX undergoes secondary and tertiary structural rearrangements and incorporates into the fibers, and that its membrane-associating C-terminal helix is important for the fibrillation process. Additionally, HN mutations known to alter its anti-apoptotic activity affect fiber morphology. Our findings reveal for the first time a potential mechanism by which BAX can be sequestered by fibril formation, which can prevent it from initiating MOMP and committing the cell to apoptosis.
Highlights
The mitochondrial, or intrinsic, apoptosis pathway is regulated mainly by members of the B-cell lymphoma 2 (BCL-2) protein family
Our findings reveal for the first time a potential mechanism by which BCL-2–associated X apoptosis regulator (BAX) can be sequestered by fibril formation, which can prevent it from initiating mitochondrial outermembrane permeabilization (MOMP) and committing the cell to apoptosis
Increased scattering was not observed when HN was added into buffer alone or buffer was titrated into BAX
Summary
The interaction between BAX and HN was first interrogated using fluorescence spectroscopy. The aggregates were imaged as fibers and came in two major conformations: Single fibers and bundles (Fig. 1b). The latter contained multiple fibers organized in parallel and appeared to be highly ordered. The CD spectrum of HN peptide alone is characteristic of disordered protein, demonstrating that the protein and peptide are together required for -sheet fibrillation Their interaction instigates mutual conformational changes that result in fibers. The observation that salt inhibits the formation of parallel bundles and increases light scattering suggests that the BAX/HN interaction is enhanced by ionic conditions and that interactions between individual fibers might be mediated by electrostatic interactions
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