title: Dual role of an N-terminal amyloidogenic mutation in apolipoprotein A-I: destabilization of helix bundle and enhancement of fibril formation.
abstract: A number of naturally occurring mutations of apolipoprotein (apo) A-I, the major protein of HDL, are known to be associated with hereditary amyloidosis and atherosclerosis. Here, we examined the effects of the G26R point mutation in apoA-I (apoA-I(Iowa)) on the structure, stability, and aggregation propensity to form amyloid fibril of full-length apoA-I and the N-terminal fragment of apoA-I. Circular dichroism and fluorescence measurements demonstrated that the G26R mutation destabilizes the N-terminal helix bundle domain of full-length protein, leading to increased hydrophobic surface exposure, whereas it has no effect on the initial structure of the N-terminal 1-83 fragment, which is predominantly a random coil structure. Upon incubation for extended periods at neutral pH, the N-terminal 1-83 variants undergo a conformational change to β-sheet-rich structure with a great increase in thioflavin T fluorescence, whereas no structural change is observed in full-length proteins. Comparison of fibril-forming propensity among substituted mutants at Gly-26 position of 1-83 fragments demonstrated that the G26R mutation enhances the nucleation step of fibril formation, whereas G26K and G26E mutations have small or inhibiting effects on the formation of fibrils. These fibrils of the 1-83 variants have long and straight morphology as revealed by atomic force microscopy and exhibited significant toxicity with HEK293 cells. Our results indicate dual critical roles of the arginine residue at position 26 in apoA-I(Iowa): destabilization of the N-terminal helix bundle structure in full-length protein and enhancement of amyloid fibril formation by the N-terminal 1-83 fragment.
authors.affiliation: Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78-1 Shomachi, Tokushima 770-8505, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Adachi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakajima
authors.forename: Chiharu
authors.initials: C
authors.lastname: Mizuguchi
authors.forename: Padmaja
authors.initials: P
authors.lastname: Dhanasekaran
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kawashima
authors.forename: Kohjiro
authors.initials: K
authors.lastname: Nagao
authors.forename: Kenichi
authors.initials: K
authors.lastname: Akaji
authors.forename: Sissel
authors.initials: S
authors.lastname: Lund-Katz
authors.forename: Michael C
authors.initials: MC
authors.lastname: Phillips
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Saito
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D028226:Amyloidosis, Familial / Q000235:genetics* / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000235:genetics*; D002942:Circular Dichroism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force; D009154:Mutation; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D000682:Amyloid; D016632:Apolipoprotein A-I