| Research Abstract |
Humanin (HN) is a secretory peptide that inhibits neurotoxicity by various Alzheimer's disease-relevant insults. In this project, we focused on HN structure-function analysis. Followings are the findings derived from this, project.
1) HN structure in solution by CD and sedimentation analysis.
Both HN and HNG showed some secondary structure typical of anti-parallel beta-sheets in water and saline : HNG is a mutant of HN and has a 1000-fold higher activity. The structure is different between the two solvents and between HN and HNG. Sedimentation analysis showed that HN and HNG form oligomers, greater than trimers, in, Saline, but are mostly monomeric in water. The size of oligomers in saline decreases as the peptide concentration is decreased.
2) Amino acid requirement of HN essential for the function
We have identified that the substitution of Leu9 for Arg nullifies the extracellular secretion of HN. While Ala substitution of neither residue in HN sequence affected HN secretion, Arg substit
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ution revealed that the two structures -Leu9-Leu11 and Pro19-Va120 -were essential for the secretion of full-length HN. In the Leu9-Leu11 domain, the, Leu10 residue turned out to play a central role in this function, because the Asp substitution of Leu10, but not Leu9 or Leu11, nullified the secretion of HN. We also investigated a comprehensive structure-function relationship for the neuroprotective function of full-length HN, which revealed (i) that Pro3, Ser7, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19 were essential for this function and (ii) that Ser7 and Leu9 were essential for self-dimerization of HN. These findings indicate that HN has activity similar to a signal peptide, for which the Leu9-Leu11 region, particularly Leu10, functions as a core domains,and suggest that self-dimerization of HN is a process essential for its neuroprotective function.
3) Mechanism of HN activation
We found that HN with a D-Ser substitution at position 14 exerted a grater neuroprotective activity than HN by two to three orders of magnitude, whereas D-Ser substitution at position 7 did not affect the HN action. The results demonstrated that the neuroprotective function of HN is regulated by D-racemation of Ser14.
This study provides definite clues to the understanding of how HN protects neuronal cells from AD relevant insults Alzheimer's Disease (AD) Less
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