Journal of Molecular Biology
Volume 406, Issue 3, 25 February 2011, Pages 416-429
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High-Affinity Amphipathic Modulators of Amyloid Fibril Nucleation and Elongation

https://doi.org/10.1016/j.jmb.2010.12.023Get rights and content

Abstract

The misfolding and aggregation of proteins to form amyloid fibrils are associated with a number of debilitating, age-related diseases. Many of the proteins that form amyloid in vivo are lipid-binding proteins, accounting for the significant impact of lipids on the rate of formation and morphology of amyloid fibrils. To systematically investigate the effect of lipid-like compounds, we screened a range of amphipathic lipids and detergents for their effect on amyloid fibril formation by human apolipoprotein (apo) C-II. The initial screen, conducted using a set of amphiphiles at half critical micelle concentration, identified several activators and inhibitors that were selected for further analysis. Sedimentation analysis and circular dichroism studies of apoC-II at low, non-fibril-forming concentrations (0.05 mg/ml) revealed that all of the inhibitors induced the formation of apoC-II dimers enriched in α-helical content while the activators promoted the formation of stable apoC-II tetramers with increased β-structure. Kinetic analysis identified modulators of apoC-II fibril formation that were effective at concentrations as low as 10 μM, corresponding to a modulator-to-apoC-II ratio of approximately 1:10. Delayed addition of the test compounds after fibril formation had commenced allowed the effects of selected amphiphiles on fibril elongation to be determined separately from their effects on fibril nucleation. The results indicated that specific amphiphiles induce structural changes in apoC-II that cause separate and independent effects on fibril nucleation and elongation. Low-molecular-weight amphipathic lipids and detergents may serve as useful, stage-specific modulators of protein self-assembly and fibril formation in disease-prevention strategies.

Research Highlights

► We screened amphiphiles for their effect on apo C-II amyloid fibril formation. ► Several activators and inhibitors acted at submicellar concentrations. ► Activators promoted tetrameric β-rich intermediates. ► Inhibitors induced apoC-II dimers with increased α-structure. ► Modulators exerted independent effects on fibril nucleation and elongation.

Introduction

Amyloid deposits are characteristic of a number of devastating, age-related diseases, including Alzheimer's and Parkinson's diseases, type 2 diabetes, and atherosclerosis. These deposits consist mostly of amyloid fibrils, defined by their common cross-β secondary structure, fibrillar morphology, and the ability to bind the dyes thioflavin T (ThT) and Congo red. While the fibrillar end products in amyloid deposits have been implicated in the disease process,1, 2, 3 it is widely considered that toxicity is mediated by small oligomeric intermediates in the assembly pathway.4, 5 Accordingly, the characterization of these pathways and the factors that enhance or inhibit the various steps has drawn considerable research attention. A high proportion of proteins that form disease-related amyloid fibrils in vivo are lipid-binding proteins, accounting for the common observation that lipids have a significant impact on the kinetics of formation, morphology, and toxicity of amyloid fibrils.6, 7, 8, 9 These studies provide the rationale for exploring the potential of lipids and lipid-like amphiphiles to modulate amyloid fibril formation and reduce the accumulation of toxic species.

Human apolipoprotein (apo) C-II is a member of the plasma apolipoprotein family and is one of several apolipoproteins that accumulate in atherosclerotic plaques, colocalized with serum amyloid P, a marker of in vivo amyloid deposits.10 ApoC-II is a 79-amino-acid protein cofactor of lipoprotein lipase that circulates in the bloodstream bound to very low density lipoproteins and chylomicrons. In the lipid-free state, apoC-II spontaneously aggregates to form homogeneous amyloid fibrils with all of the hallmarks of amyloid fibrils.11 Amyloid fibrils formed by apoC-II initiate early events in heart disease, including the induction of the macrophage inflammatory response.12 ApoC-II fibrils are relatively homogeneous in structure and size and form readily under quiescent conditions. Analysis of the concentration dependence of the rate of apoC-II fibril formation coupled with size distribution analysis allowed the development of a detailed kinetic model for apoC-II fibril formation involving reversible nucleation and fibril elongation as well as end-to-end joining and breaking.13

Phospholipids exert significant effects on apoC-II fibril formation. At concentrations below their critical micelle concentration (CMC), phospholipids accelerate fibril formation while phospholipid micelles and bilayers induce a new, straight, rod-like fibril morphology relative to the twisted-ribbon morphology formed in the absence of lipids.7 Mechanistic studies show that the activation of apoC-II fibril formation by submicellar dihexanoylphosphatidylcholine (DHPC) occurs via the lipid-induced acceleration of the formation of a tetrameric intermediate that slowly isomerizes to form a nucleus. This nucleus then acts as a template for fibril elongation and the formation of mature fibrils.14 Studies conducted by adding DHPC after the completion of the initial nucleation phase indicated that although DHPC activated fibril nucleation, there was no discernable effect on fibril elongation.15 These studies demonstrate that submicellar phospholipids and self-assembled phospholipid complexes can selectively target individual steps in the amyloid-fibril-forming pathway. To explore the structural specificity of lipids and “lipid-like” mimetics as activators and inhibitors of amyloid fibril formation, we screened a range of amphipathic lipids and detergent molecules for their ability to modulate amyloid fibril formation by apoC-II. Our aim was to identify compounds that alter protein folding, self-assembly, and fibril formation pathways with the potential to prevent the accumulation of misfolded proteins in disease deposits.

Section snippets

Screen of 96 amphipathic lipids and detergents

To identify new activators and inhibitors of apoC-II fibril formation, we screened a commercial set of physiological lipids and amphipathic lipid-like detergents designed specifically for membrane protein crystallization studies. The identity, screening concentration, and CMC of the compounds used in the screen are given in Table S1. Lipid- and detergent-induced effects on fibril formation were assessed by continuous monitoring of the rate of induction of ThT fluorescence or turbidity relative

Discussion

Amphipathic lipids are a common component of amyloid deposits19 with the potential to determine the rate of formation, stability, morphology, and toxicity of amyloid fibrils. The results in Fig. 2 demonstrate the broad range of amphipathic lipid-like compounds that either activate or inhibit apoC-II fibril formation. The compounds used in the screen were from a commercial set developed for X-ray and NMR studies of membrane proteins and designed to minimize or manipulate nonspecific hydrophobic

Materials

ApoC-II and the mutant derivative apoC-II (S61C) were expressed, purified,14, 52 and stored as stock solutions at approximately 40 mg/ml in 5 M guanidine hydrochloride. The cysteine residue of apoC-II (S61C) was modified with either Alexa 488 C5 maleimide or Alexa 594 C5 maleimide (Invitrogen–Molecular Probes, Eugene, OR).14 A 96-well detergent screen (product HR2-406) was obtained from Hampton Research. Hydrophobicity values for the test compounds were calculated as octanol/water partitioning

Acknowledgement

This research was supported by the Australian Research Council (DP0877800).

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