cytochrome-c-t and humanin

Humanin (HN) is a 24-residue peptide identified from the brain of a patient with Alzheimer's disease (AD). HN has been found to protect against neuronal insult caused by Aβ peptides or transfection of familial AD mutant genes. In order to elucidate the molecular mechanisms of HN neuroprotection, we explored the effects of HN on the association of Bax or Bid with lipid bilayers and their oligomerization in the membrane. By using single-molecule fluorescence and Förster resonance energy transfer techniques, we showed that Bax was mainly present as monomers, dimers and tetramers in lipid bilayers, while truncated Bid (tBid) enhanced the membrane association and tetramerization of Bax. HN (100 nmol/L) inhibited the self-association and tBid-activated association of Bax with the bilayers, and significantly decreased the proportion of Bax in tetramers. Furthermore, HN inhibited Bid translocation to lipid bilayers. HN could bind with Bax and Bid either in solution or in the membrane. However, HN could not pull the proteins out of the membrane. Based on these results, we propose that HN binds to Bax and cBid in solution and inhibits their translocation to the membrane. Meanwhile, HN interacts with the membrane-bound Bax and tBid, preventing the recruitment of cytosolic Bax and its oligomerization in the membrane. In this way, HN inhibits Bax pore formation in mitochondrial outer membrane and suppresses cytochrome c release and mitochondria-dependent apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Cytochromes c; Fluorescence Resonance Energy Transfer; Humans; Intracellular Signaling Peptides and Proteins; Lipid Bilayers; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Neuroprotective Agents; Protein Binding; Protein Multimerization; Protein Transport; Single Molecule Imaging

Mice wherein the wild-type mitochondrial DNA polymerase (pol gamma) is replaced by a proofreading-deficient version are born with mutation frequencies in mitochondrial DNA (mtDNA) much higher than are ever normally seen in old rodents or humans. These mice, however, are phenotypically normal at birth, raising the question regarding how the much lower frequencies observed in normal aging could possibly contribute to the aging process. In contrast, transgenic mice with cardiac-specific expression of a proofreading-deficient poly gamma from birth onwards accumulate mtDNA mutations to levels normally seen in aging. But these mice develop dilated cardiomyopathy suggesting that age-related mtDNA mutations are pathogenic. Using computer simulation, we show that both findings are predicted based on the hypotheses that (1) rare lethal mutations that cause apoptosis underlie the pathogenesis of mutagenesis in mtDNA and (2) most sporadic mtDNA mutations are phenotypically recessive and therefore nonpathogenic. Biochemical evidence is presented that mitochondria with mtDNA mutations generate a peptide that causes the release of cytochrome c, providing a mechanism for the increased apoptosis observed in aging. Simulation also predicts that normal, age-related accumulation of mtDNA mutations causes significant levels of cell death. These findings suggest that mtDNA mutations play an important role in the aging process and that their pathogenic mechanism is linked to apoptosis.

Topics: Animals; Apoptosis; Computer Simulation; Cytochromes c; DNA, Mitochondrial; Intracellular Signaling Peptides and Proteins; Mice; Mutation; Peptides

We showed that humanin (HN), an endogenous peptide against Alzheimer disease-related insults, was expressed in muscles of patients with chronic progressive external ophthalmoplegia (CPEO), a major mitochondrial disease. Because HN was recently found to block proapoptotic Bax function and exert its versatile cytoprotective effects in association with an increase in ATP levels, HN expression may thus reflect a physiological response against degenerative changes in the muscles of patients with CPEO. We found HN expression in all four patients examined, each of whom had different mitochondrial DNA mutations including two different single DNA deletions, multiple deletions, and no major mutations detected. We also found that HN expression was not linked to focal cytochrome c deficiency, strongly associated with the subtype of CPEO with single deletions. These results suggest that HN expression is more closely related to degenerative changes in all types of CPEO. Notably, HN was also expressed in non-degenerative muscle fibers of patients with CPEO or Leigh syndrome, who had the 8993T>G mutation in the mitochondrial ATPase 6 gene known to be associated with impaired ATP synthesis. Collectively, our findings suggest that HN may be specifically expressed in response to defects in energy production in muscles with mitochondrial abnormalities.

Topics: Adolescent; Child; Child, Preschool; Cytochromes c; DNA, Mitochondrial; Female; Gene Expression; Humans; Infant; Intracellular Signaling Peptides and Proteins; Leigh Disease; Male; Middle Aged; Mitochondrial Proton-Translocating ATPases; Muscle, Skeletal; Ophthalmoplegia, Chronic Progressive External; Oxidative Phosphorylation Coupling Factors; Point Mutation; Sequence Deletion

Humanin (HN) is a recently identified endogenous peptide that protects cells against cytotoxicity induced by various stimuli. Recently, we showed that HN binds to and inhibits Bax, a proapoptotic Bcl-2 family protein, suggesting a mechanism for HN action. In this study, we identified Bim, a Bcl-2 homology 3-only member of the Bcl-2/Bax family, as an additional HN target protein. Using in vitro protein binding, immunoprecipitation, and coimmunolocalization assays, we demonstrated that HN binds directly to the extra long isoform of Bim (BimEL) but not the long (BimL) or short (BimS) isoforms. HN also protects cells against apoptosis induced by BimEL but not BimL and BimS in gene transfection studies. In contrast, mutants of HN which failed to bind BimEL failed to protect from BimEL-induced cell death. Moreover, HN inhibited BimEL-induced release of SMAC and cytochrome c from mitochondria isolated from bax-/-cells, indicating that HN can suppress BimEL independently of its effect on Bax. Finally, we demonstrate that HN prevents BimEL-induced oligomerization of Bak using isolated mitochondria. Taken together, our results indicate that the inhibition of BimEL may contribute to the antiapoptotic properties of the HN peptide.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Carrier Proteins; COS Cells; Cytochromes c; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Peptide Fragments; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2