Shear flow induced changes in apolipoprotein C-II conformation and amyloid fibril formation

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold. Fibrils produced at shear rates of 150 and 300 s(-1) were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s(-1), tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 μg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.     

Sedimentation velocity analysis of flexible macromolecules: self-association and tangling of amyloid fibrils

A novel bead modeling technique has been developed for the analysis of the sedimentation velocity behavior of flexible fibrils. The method involves the generation of a family of bead models representing a sample of the conformations available to the molecule and the calculation of the sedimentation coefficients of these models by established techniques. This approach has been used to investigate the size distribution of amyloid fibrils formed by human apolipoprotein C-II (apoC-II). ApoC-II fibrils have a simple and homogeneous ribbon morphology with no evidence of amorphous aggregation. Freshly prepared apoC-II forms fibrils with systematically larger sedimentation coefficients upon increasing protein concentration (modes of 100, 300, and 800 for apoC-II concentrations of 0.3, 0.7, and 1.0 mg/mL, respectively). The sedimentation coefficient distributions are not affected by rotor speed, and are not significantly changed by dilution once the fibrils are formed. The kinetics of aggregation (1 mg/mL apoC-II) as assessed using thioflavin T and preparative pelleting assays reveal that monomeric apoC-II is depleted after approximately 12 h incubation at room temperature. In contrast, the sedimentation coefficient distribution of fibrils continues to grow larger over a period of 48 h to an average value of 800 S. Calculations using the bead modeling procedure suggest maximum sedimentation coefficients for individual apoC-II fibrils to be around 100 S. The larger experimentally observed sedimentation coefficients for apoC-II fibrils indicate an extensive and time-dependent tangling or association of the fibrils to form specific networks.     

NBD-labeled phospholipid accelerates apolipoprotein C-II amyloid fibril formation but is not incorporated into mature fibrils

Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by submicellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by submicellar lipids using the fluorescently labeled, short-chain phospholipid 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxyglycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately 2-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril-forming concentrations of apoC-II indicated NBD-lyso-12-PC binds rapidly, on the millisecond time scale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-lyso-12-PC binds to both apoC-II monomers and tetramers at approximately five sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-lyso-12-PC were devoid of lipid, indicating a purely catalytic role for submicellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules in controlling protein folding and fibril assembly pathways.     

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.     

Visualization of polymorphism in apolipoprotein C-II amyloid fibrils

The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.     

Methionine oxidation inhibits assembly and promotes disassembly of apolipoprotein C-II amyloid fibrils

Methionine residues are linked to the pathogenicity of several amyloid diseases; however, the mechanism of this relationship is largely unknown. These diseases are characterized, in vivo, by the accumulation of insoluble proteinaceous plaques, of which the major constituents are amyloid fibrils. In vitro, methionine oxidation has been shown to modulate fibril assembly in several well-characterized amyloid systems. Human apolipoprotein (apo) C-II contains two methionine residues (Met-9 and Met-60) and readily self-assembles in vitro to form homogeneous amyloid fibrils, thus providing a convenient system to examine the effect of methionine oxidation on amyloid fibril formation and stability. Upon oxidation of the methionine residues of apoC-II with hydrogen peroxide, fibril formation was inhibited. Oxidized apoC-II molecules did not inhibit native apoC-II assembly, indicating that the oxidized molecules had a reduced ability to interact with the growing fibrils. Single Met-Val substitutions were performed and showed that oxidation of Met-60 had a more significant inhibitory effect than oxidation of Met-9. In addition, Met-Gln substitutions designed to mimic the effect of oxidation on side chain hydrophilicity showed that a change in hydrophobicity at position 60 within the core region of the fibril had a potent inhibitory effect. The oxidation of preformed apoC-II fibrils caused their dissociation; however, mutants in which the Met-60 was substituted with a valine were protected from this peroxide-induced dissociation. This work highlights an important role for methionine in the formation of amyloid fibril structure and gives new insight into how oxidation affects the stability of mature fibrils.     

A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.     

Cross-linking and amyloid formation by N- and C-terminal cysteine derivatives of human apolipoprotein C-II

We have investigated the effect of disulfide cross-linking on amyloid formation by human apolipoprotein (apo) C-II. Three derivatives of apoC-II were generated by inserting a cysteine residue on either the N-terminus (C(N)-apoC-II), C-terminus (C(C)-apoC-II), or both termini (C(N)C(C)-apoC-II). Under reducing conditions, all derivatives formed amyloid with a fibrous ribbon morphology similar to that of wild-type apoC-II. Under oxidizing conditions, C(N)- and C(N)C(C)-apoC-II formed a highly tangled network of fibrils, suggesting that the addition of an N-terminal cysteine to apoC-II promotes interfibril disulfide cross-links. Fibrils formed by C(C)-apoC-II under oxidizing conditions were closely packed but less tangled than fibrils formed by the C(N) and C(N)C(C) derivatives. The frequency of closed ring structures was more than doubled for C(C)-apoC-II compared to wild-type apoC-II. The kinetics of fibril formation by all cysteine derivatives was markedly enhanced under oxidizing conditions, suggesting that disulfide cross-linking promotes amyloid formation. Substoichiometric levels of preformed C(N)- and C(C)-apoC-II dimers accelerate amyloid formation by wild-type apoC-II. These data suggest that the N- and C-termini of apoC-II are close together in the amyloid fibril such that covalent cross-linking of either the N or C end of apoC-II promotes nucleation and the "seeding" of fibril growth.     

Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.     

Serum amyloid P colocalizes with apolipoproteins in human atheroma: functional implications

Serum amyloid P (SAP) is a common component of human amyloid deposits and has been identified in atherosclerotic lesions. We investigated the extent of the colocalization of SAP with apolipoprotein A-I (apoA-I), apoB, apoC-II, and apoE in human coronary arteries and explored potential roles for SAP in these regions, specifically the effect of SAP on the rate of formation and macrophage recognition of amyloid fibrils composed of apoC-II. Analysis of 42 human arterial sections by immunohistochemistry and double label fluorescence microscopy demonstrated that SAP and apoA-I, apoB, apoC-II, and apoE were increased significantly in atherosclerotic lesions compared with nonatherosclerotic segments. SAP colocalized with all four apolipoproteins to a similar extent, whereas plaque macrophages were found to correlate most strongly with apoC-II and apoB. In vitro studies showed that SAP accelerated the formation of amyloid fibrils by purified apoC-II. Furthermore, SAP strongly inhibited the phagocytosis of apoC-II amyloid fibrils by primary macrophages and macrophage cell lines and blocked the resultant production of reactive oxygen species. The ability of SAP to accelerate apoC-II amyloid fibril formation and inhibit macrophage recognition of apoC-II fibrils suggests that SAP may modulate the inflammatory response to amyloid fibrils in atherosclerosis.     

Fluorescence detection of a lipid-induced tetrameric intermediate in amyloid fibril formation by apolipoprotein C-II

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.     

Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.     

High density lipoproteins bind Abeta and apolipoprotein C-II amyloid fibrils

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.     

High-affinity amphipathic modulators of amyloid fibril nucleation and elongation

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.     

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.     

A cyclic peptide inhibitor of apoC-II peptide fibril formation: mechanistic insight from NMR and molecular dynamics analysis

The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation.     

The circularization of amyloid fibrils formed by apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.     

Structural basis for the recognition and cross-linking of amyloid fibrils by human apolipoprotein E

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.     

A Structural Model for Apolipoprotein C-II Amyloid Fibrils: Experimental Characterization and Molecular Dynamics Simulations

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple ‘letter-G-like’ β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops.