期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 36, 页码 13515-13524出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.008000
关键词
amyloid; fibril; apolipoprotein; protein structure; protein aggregation; activation energy; apolipoprotein A-I; G26R variant; hereditary systemic amyloidosis; pyrene labeling
资金
- Japan Society for the Promotion of Science (JSPS) KAKENHI [JP17H03979]
The N-terminal (1-83) fragment of the major constituent of plasma high-density lipoprotein, apolipoprotein A-I (apoA-I), strongly tends to form amyloid fibrils, leading to systemic amyloidosis. Here, using a series of deletion variants, we examined the roles of two major amyloidogenic segments (residues 14-22 and 50-58) in the aggregation and fibril formation of an amyloidogenic G26R variant of the apoA-I 1-83 fragment (apoA-I 1-83/G26R). Thioflavin T fluorescence assays and atomic force microscopy revealed that elimination of residues 14-22 completely inhibits fibril formation of apoA-I 1-83/G26R, whereas Delta 32-40 and Delta 50-58 variants formed fibrils with markedly reduced nucleation and fibril growth rates. CD measurements revealed structural transitions from random coil to beta-sheet structures in all deletion variants except for the Delta 14-22 variant, indicating that residues 14-22 are critical for the beta-transition and fibril formation. Thermodynamic analysis of the kinetics of fibril formation by apoA-I 1-83/G26R indicated that both nucleation and fibril growth are enthalpically unfavorable, whereas entropically, nucleation is favorable, but fibril growth is unfavorable. Interestingly, the nucleation of the Delta 50-58 variant was entropically unfavorable, indicating that residues 50-58 entropically promote the nucleation step in fibril formation of apoA-I 1-83/G26R. Moreover, a residue-level structural investigation of apoA-I 1-83/G26R fibrils with site-specific pyrene labeling indicated that the two amyloidogenic segments are in close proximity to form an amyloid core structure, whereas the N- and C-terminal tail regions are excluded from the amyloid core. These results provide critical insights into the aggregation mechanism and fibril structure of the amyloidogenic N-terminal fragment of apoA-I.
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