Abstract
The misfolding and self-assembly of proteins into amyloid fibrils that occurs in several debilitating and age-related diseases is affected by common components of amyloid deposits, notably lipids and lipid complexes. We have examined the effect of the short-chain phospholipids, dihexanoylphosphatidylcholine (DHPC) and dihexanoylphosphatidylserine (DHPS), on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Micellar DHPC and DHPS strongly inhibited apoC-II fibril formation, whereas submicellar levels of these lipids accelerated apoC-II fibril formation to a similar degree. These results indicate that the net negative charge on DHPS, compared with the neutrally charged DHPC, is not critical for either the inhibition or activation process. We also investigated the mechanism for the submicellar, lipid-induced activation of fibril formation. Emission data for fluorescently labeled apoC-II indicated that DHPC and DHPS stimulate the early formation and accumulation of oligomeric species. Sedimentation velocity and equilibrium experiments using a new fluorescence detection system identified a discrete lipid-induced tetramer formed at low apoC-II concentrations in the absence of significant fibril formation. Seeding experiments showed that this tetramer was on the fibril-forming pathway. Fluorescence resonance energy transfer experiments established that this tetramer forms rapidly and is stabilized by submicellar, but not micellar, concentrations of DHPC and DHPS. Several recent studies show that oligomeric intermediates in amyloid fibril formation are toxic. Our results indicate that lipids promote on-pathway intermediates of apoC-II fibril assembly and that the accumulation of a discrete tetrameric intermediate depends on the molecular state of the lipid.
Highlights
Native form of the protein, a fibrillar cross--morphology, and the ability to interact with the dyes thioflavin T and Congo Red [1]
To investigate the role of individual lipid Materials—DHPS and DHPC were obtained from Avanti molecules, we explored the effects of the model short- Polar Lipids, Inc. (Alabaster, AL), and Alexa488 C5 maleimide chain phospholipid dihexanoylphosphatidylcholine (DHPC) on and Alexa594 C5 maleimide were obtained from Invitrogen. apolipoprotein C-II (apoC-II) amyloid fibril formation [18, 19]
The Effect of DHPC and DHPS on ApoC-II Fibril Formation— The time course for apoC-II fibril formation in the presence of various concentrations of DHPC and DHPS was monitored by thioflavin T (ThT) fluorescence (Fig. 1)
Summary
Native form of the protein, a fibrillar cross--morphology, and the ability to interact with the dyes thioflavin T and Congo Red [1]. Lipid bilayers composed of negatively charged phosphatidylserine or other anionic phospholipids accelerate amyloid fibril formation by several proteins relative to control neutral lipids [6] Micellar lipids, such as short-chain oxidized phospholipids, are present in human plasma [7]. Immunohistochemical studies indicate the presence of apoC-II deposits in human coronary artery plaques co-localized with serum amyloid P, a widely used marker of in vivo amyloid [13]. Fibrils formed from both apoC-II and A initiate early events in heart disease, including the induction of several markers of the macrophage inflammatory response [14, 15]. A major difficulty in defining these species is the low steady-state concentrations of intermediates in amyloid fibril-forming pathways
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