Journal of Molecular Biology
High-Affinity Amphipathic Modulators of Amyloid Fibril Nucleation and Elongation
Graphical Abstract
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).
References (54)
- et al.
Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic/necrotic pathways
J. Mol. Biol.
(2007) - et al.
The non-core regions of human lysozyme amyloid fibrils influence cytotoxicity
J. Mol. Biol.
(2010) - et al.
Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways
J. Mol. Biol.
(2008) - et al.
Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications
J. Lipid Res.
(2007) - et al.
Fibrillar amyloid protein present in atheroma activates CD36 signal transduction
J. Biol. Chem.
(2004) - et al.
Apolipoprotein C-II amyloid fibrils assemble via a reversible pathway that includes fibril breaking and rejoining
J. Mol. Biol.
(2008) - et al.
Fluorescence detection of a lipid-induced tetrameric intermediate in amyloid fibril formation by apolipoprotein C-II
J. Biol. Chem.
(2008) - et al.
Phospholipids enhance nucleation but not elongation of apolipoprotein C-II amyloid fibrils
J. Mol. Biol.
(2010) - et al.
Sub-micellar phospholipid accelerates amyloid formation by apolipoprotein C-II
FEBS Lett.
(2001) - et al.
Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set
Anal. Biochem.
(2000)
On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation
Anal. Biochem.
Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry
Biophys. J.
A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis
J. Mol. Biol.
Alpha-synuclein selectively binds to anionic phospholipids embedded in liquid-disordered domains
J. Mol. Biol.
Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I
J. Biol. Chem.
Two types of Alzheimer's beta-amyloid (1–40) peptide membrane interactions: aggregation preventing transmembrane anchoring versus accelerated surface fibril formation
J. Mol. Biol.
Phospholipid catalysis of diabetic amyloid assembly
J. Mol. Biol.
Negatively charged phospholipid membranes induce amyloid formation of medin via an alpha-helical intermediate
J. Mol. Biol.
Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human alpha-synuclein
J. Mol. Biol.
A phospholipid acts as a chaperone in assembly of a membrane transport protein
J. Biol. Chem.
Lipid-assisted protein folding
J. Biol. Chem.
The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II
J. Biol. Chem.
Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct
J. Biol. Chem.
Effects of grape seed-derived polyphenols on amyloid beta-protein self-assembly and cytotoxicity
J. Biol. Chem.
Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling
Biophys. J.
Sedimentation equilibrium analysis of protein interactions with global implicit mass conservation constraints and systematic noise decomposition
Anal. Biochem.
Self-propagating, molecular-level polymorphism in Alzheimer's beta-amyloid fibrils
Science
Cited by (30)
Sclerotiorin Stabilizes the Assembly of Nonfibrillar Abeta42 Oligomers with Low Toxicity, Seeding Activity, and Beta-sheet Content
2020, Journal of Molecular BiologyEffects of lipid composition on the structural properties of human serum amyloid A in reconstituted high-density lipoprotein particles
2019, Chemistry and Physics of LipidsCitation Excerpt :Lipid binding has been observed to greatly influence fibril formation of amyloidogenic proteins. However, the combination of protein and lipid species encountered upon lipid binding could determine whether facilitative or inhibitory effects ensue (Butterfield and Lashuel, 2010; Ryan et al., 2011). For example, the interaction of SAA with neutral phospholipids has been observed to prevent fibril formation by stabilizing an α-helical structure; however, SAA interaction with acidic phospholipids appeared to promote fibril formation (Tanaka et al., 2017).
Lipid-apolipoprotein interactions in amyloid fibril formation and relevance to atherosclerosis
2019, Biochimica et Biophysica Acta - Proteins and ProteomicsEffect of lipid environment on amyloid fibril formation of human serum amyloid A
2017, Chemistry and Physics of LipidsA Stable Mutant Predisposes Antibody Domains to Amyloid Formation through Specific Non-Native Interactions
2016, Journal of Molecular BiologyApolipoprotein C-II Adopts Distinct Structures in Complex with Micellar and Submicellar Forms of the Amyloid-Inhibiting Lipid-Mimetic Dodecylphosphocholine
2016, Biophysical JournalCitation Excerpt :ApoC-II in the absence of detergent has <5% protein remaining in the supernatant, whereas both of the apoC-II/DPC complex samples contain 95% of the starting protein. We have previously investigated apoC-II in the presence of submicellar DPC using circular dichroism and analytical ultracentrifugation, indicating the formation of a dimer with a high content of α-helical secondary structure (18). However, we have little understanding of the molecular arrangement of apoC-II in complex with submicellar DPC.