"Janus" Cyclic Peptides: A New Approach to Amyloid Fibril Inhibition?

Cyclic peptides are increasingly being shown as powerful inhibitors of fibril formation, and have the potential to be therapeutic agents for combating many debilitating amyloid-related diseases. One such example is a cyclic peptide derivative from the human apolipoprotein C-II, which has the ability to inhibit fibril formation by the fibrillogenic peptide apoC-II(60–70). Using classical molecular dynamics and electronic structure calculations, we were able to provide insight into the interaction between the amyloidogenic peptide apoC-II(60–70) and its cyclic derivative, cyc(60–70). Our results showed that cyc(60–70) induced increased flexibility in apoC-II(60–70), suggesting that one mechanism by which cyc(60–70) inhibits fibrillisation is by destabilising apoC-II(60–70) structure, rendering it incapable of adopting fibril favouring conformations. In contrast, cyc(60–70) shows less flexibility upon binding to apoC-II(60–70), which is predominantly mediated by hydrophobic interactions between the aromatic rings of the peptides. This effectively creates a cap around the fibril-forming region of apoC-II(60–70) and generates an outer hydrophilic shell that discourages further apoC-II(60–70) peptide self-association. We showed that apoC-II(60–70) exhibited stronger binding affinity for the hydrophobic face of cyc(60–70) and weakest binding affinity for the hydrophilic side. This suggests that cyc(60–70) can be an effective fibril inhibitor due to its amphipathic character, like that of the "Janus"-type particles. This property can be exploited in the design of specific inhibitors of amyloid fibril formation.     

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.     

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.     

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.     

Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin.

The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Amyloid fibril formation by a synthetic peptide from a region of human acetylcholinesterase that is homologous to the Alzheimer's amyloid-beta peptide

A region near the C-terminus of human acetylcholinesterase (AChE) is weakly homologous with the N-terminus of the Alzheimer's disease amyloid-beta peptide. We report that a 14-amino acid synthetic polypeptide whose sequence corresponds to residues 586-599 of the human synaptic or T form of AChE assembles into amyloid fibrils under physiological conditions. The fibrils have all the classical characteristics of amyloid: they have a diameter of 6-7 nm and bind both Congo red and thioflavin-T. Furthermore, the kinetics of assembly indicate that fibril formation proceeds via a two-step nucleation-dependent polymerization pathway, and a transition in the peptide conformation from random coil to beta-sheet is observed during fibril formation using far-UV circular dichroism spectroscopy. We also show that the peptide in aggregated fibrillar form has a toxic effect upon PC-12 cells in vitro. AChE normally resides mainly on cholinergic neuronal membranes, but is abnormally localized to senile plaques in Alzheimer's disease. Recently, an in vitro interaction between AChE and A beta, the principal constituent of the amyloid fibrils in senile plaques, has been documented. The presence of a fibrillogenic region within AChE may be relevant to the interaction of AChE with amyloid fibrils formed by Abeta.     

Amyloid fibril formation and chaperone-like activity of peptides from alphaA-crystallin

AlphaA-crystallin (alphaAC), a major component of eye lens, exhibits chaperone-like activity and is responsible for maintaining eye lens transparency. Synthetic peptides which corresponded to the putative substrate-binding site of alphaAC have been reported to prevent aggregation of proteins [Sharma, K. K., et al. (2000) J. Biol. Chem. 275, 3767-3771]. In this study, we found that these peptides, alphaAC(70-88), the peptide corresponding to amino acids 70-88 of alphaAC (KFVIFLDVKHFSPEDLTVK), and alphaAC(71-88), suppressed the amyloid fibril formation of amyloid beta protein (Abeta). On the other hand, while alphaAC(71-88) exhibited chaperone-like activity toward insulin, alphaAC(70-88) and alphaAC(70-88)K70D promoted rapid growth of aggregates consisting of insulin and these peptides in their solution mixtures. Interestingly, we found that alphaAC(71-88) itself can also form amyloid fibrils. It is possible that the chaperone-like activity of the alphaAC peptides is potentially related to their propensity for amyloid fibril formation. Analysis of variants of the alphaAC peptides suggested that F71 is important for amyloid formation, and interestingly, this same residue has previously been found to be essential for chaperone-like activity. Amyloid fibril formation was also observed with the shorter peptide, alphaAC(70-76)K70D, showing that the ability to form amyloid fibrils is maintained even with significant deletion of the C-terminal sequence. The formation of amyloid fibril was suppressed in alphaAC(70-88), suggesting that the K70 in the substrate binding site may play a role in suppressing the amyloid fibril formation of alphaAC, which agreed with recent proposals about the presence of an aggregation suppressor in the region flanking aggregation-prone hydrophobic sequences.     

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.     

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.     

Lipid-apolipoprotein interactions in amyloid fibril formation and relevance to atherosclerosis

The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-β structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma.     

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.     

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.     

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP)

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer's disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.     

Elucidating the Role of Disulfide Bond on Amyloid Formation and Fibril Reversibility of Somatostatin-14: RELEVANCE TO ITS STORAGE AND SECRETION*?

The storage of protein/peptide hormones within subcellular compartments and subsequent release are crucial for their native function, and hence these processes are intricately regulated in mammalian systems. Several peptide hormones were recently suggested to be stored as amyloids within endocrine secretory granules. This leads to an apparent paradox where storage requires formation of aggregates, and their function requires a supply of non-aggregated peptides on demand. The precise mechanism behind amyloid formation by these hormones and their subsequent release remain an open question. To address this, we examined aggregation and fibril reversibility of a cyclic peptide hormone somatostatin (SST)-14 using various techniques. After proving that SST gets stored as amyloid in vivo, we investigated the role of native structure in modulating its conformational dynamics and self-association by disrupting the disulfide bridge (Cys³–Cys¹⁴) in SST. Using two-dimensional NMR, we resolved the initial structure of somatostatin-14 leading to aggregation and further probed its conformational dynamics in silico. The perturbation in native structure (S-S cleavage) led to a significant increase in conformational flexibility and resulted in rapid amyloid formation. The fibrils formed by disulfide-reduced noncyclic SST possess greater resistance to denaturing conditions with decreased monomer releasing potency. MD simulations reveal marked differences in the intermolecular interactions in SST and noncyclic SST providing plausible explanation for differential aggregation and fibril reversibility observed experimentally in these structural variants. Our findings thus emphasize that subtle changes in the native structure of peptide hormone(s) could alter its conformational dynamics and amyloid formation, which might have significant implications on their reversible storage and secretion.     

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.     

Amyloid fibril formation by pentapeptide and tetrapeptide fragments of human calcitonin

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.     

Laminin affects polymerization,depolymerization and neurotoxicity of Abeta peptide

Amyloid deposition in Alzheimer fibrils forms neurotoxic senile plaques in a process that may be modulated by associated proteins. In this work we demonstrate the ability of laminin-1 and laminin-2 to inhibit fibril formation and toxicity on cultured rat hippocampal neurons. We confirm that the laminin-1-derived peptide YFQRYLI inhibits efficiently both fibril formation and neurotoxicity and show that the IKVAV peptide inhibits amyloid neurotoxicity despite its slight inhibition of fibril formation. On other hand, laminin-1 induces disaggregation of preformed fibrils in vitro, characterized as a progressive disassembly of fibrils into protofibrils and further clearance of these latter species, leading to a continual inhibition of amyloid neurotoxicity.     

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.     

All or none fibrillogenesis of a prion peptide.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.