期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 286, 期 13, 页码 11555-11562出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M110.181313
关键词
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资金
- Ministry of Health, Labor and Welfare
- Ministry of Education, Culture, Sports, Science, and Technology
- New Energy and Industrial Technology Development Organization
- Alzheimer's Drug Discovery and Biomarkers
- Grants-in-Aid for Scientific Research [19058001] Funding Source: KAKEN
Nonfibrillar assemblies of amyloid beta-protein (A beta) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using A beta labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical A beta assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of similar to 330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are similar to 128 kDa (similar to 32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the A beta(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying A beta assembly steps at which inhibition may be beneficial.
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