Humanin variants differentially regulate BID fibrilization and suppress BAX- and BID-dependent membrane permeabilization.

Humanin (HN), a mitochondria-derived peptide, exerts cytoprotective effects by modulating apoptosis through interactions with pro-apoptotic BCL-2 proteins, BAX and BID. Previous studies showed that HN induces β-sheet-rich fibrilization of BAX and truncated BID, suggesting a mechanism that may explain its cytoprotective function. Mutants of HN were shown to alter the morphology and fibrilization with BAX. In this study we tested whether the same HN variants can also influence fibrilization with BID, cleaved BID, or active truncated BID. We also evaluated the impact of fibrils formed between BAX or BID with HN on membrane morphology and permeability. The single Cysteine, C8, in HN is critical for fibrilization with all forms of BID. Whereas the central hydrophobic residues, L9 and L10, modulate fibril morphology with BID and cleaved BID; as they did with BAX. Interestingly, L12 has a differential effect on fibrilization with BID and BAX. Substitutions to the above residues of HN prevent formation of fibrils with truncated Bid. The HN fibril promotes vesicles to assemble as shown by electron microscopy, and this is moderated in the presence of BAX or BID. Vesicle leakage assays further demonstrated that HN reduced BAX- and tBID-mediated membrane permeabilization. Importantly, inhibition was strongly dependent on the order of sample addition. Finally, HN fibrils densely packed on isolated mitochondria in an organized parallel configuration. These findings extend the model of HN action by integrating BID activation-state specificity, HN residue-level determinants, and direct membrane association into a unified framework for HN-mediated regulation of mitochondrial apoptosis.