Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates

Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.     

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

Mutation-dependent Polymorphism of Cu,Zn-Superoxide Dismutase Aggregates in the Familial Form of Amyotrophic Lateral Sclerosis

More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.     

Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its beta sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.     

Characterization of cytoplasmic alpha-synuclein aggregates. Fibril formation is tightly linked to the inclusion-forming process in cells

The alpha-synuclein fibrillation process has been associated with the pathogenesis of several neurodegenerative diseases. Here, we have characterized the cytoplasmic alpha-synuclein aggregates using a fractionation procedure with which different aggregate species can be separated. Overexpression of alpha-synuclein in cells produce two distinct types of aggregates: large juxtanuclear inclusion bodies and small punctate aggregates scattered throughout the cytoplasm. Biochemical fractionation results in an inclusion-enriched fraction and two small aggregate fractions. Electron microscopy and thioflavin S reactivity of the fractions show that the juxtanuclear inclusion bodies are filled with amyloid-like alpha-synuclein fibrils, whereas both the small aggregate fractions contain non-fibrillar spherical aggregates with distinct size distributions. These aggregates appear sequentially, with the smallest population appearing the earliest and the fibrillar inclusions the latest. Based on the structural and kinetic properties, we suggest that the small spherical aggregates are the cellular equivalents of the protofibrils. The proteins that co-exist in the Lewy bodies, such as proteasome subunit, ubiquitin, and hsp70 chaperone, are present in the fibrillar inclusions but absent in the protofibrils, suggesting that these proteins may not be directly involved in the early aggregation stage. As predicted in the aggresome model, disruption of microtubules with nocodazole reduced the number of inclusions and increased the size of the protofibrils. Despite the increased size, the protofibrils remained non-fibrillar, suggesting that the deposition of the protofibrils in the juxtanuclear region is important in fibril formation. This study provides evidence that the cellular fibrillation also involves non-fibrillar intermediate species, and the microtubule-dependent inclusion-forming process is required for the protofibril-to-fibril conversion in cells.     

Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Cytotoxicity of insulin within its self-assembly and amyloidogenic pathways

Solvational perturbations were employed to selectively tune the aggregational preferences of insulin at 60 degrees C in vitro in purely aqueous acidic solution and in the presence of the model co-solvent ethanol (EtOH) (at 40%(w/w)). Dynamic light scattering (DLS), thioflavin T (ThT)-fluorescence, Fourier transform infrared (FTIR) and atomic force microscopy (AFM) techniques were employed to characterize these pathways biophysically with respect to the pre-aggregational assembly of the protein, the aggregation kinetics, and finally the aggregate secondary structure and morphology. Using cell viability assays, the results were subsequently correlated with the cytotoxicity of the insulin species that form in the two distinct aggregation pathways. In the cosolvent-free solution, predominantly dimeric insulin self-assembles via the well-known amyloidogenic pathway, yielding exclusively fibrillar aggregates, whereas in the solution containing EtOH, the aggregation of predominantly monomeric insulin proceeds via a pathway that leads to exclusively non-fibrillar, amorphous aggregates. Initially present native insulin assemblies as well as partially unfolded monomeric species and low molecular mass oligomeric aggregates could be ruled out as direct and major cytotoxic species. Apart from the slower overall aggregation kinetics under amorphous aggregate promoting conditions, which is due to the chaotropic nature of high EtOH concentrations, however, both pathways were unexpectedly found to evoke insulin aggregates that were cytotoxic to cultured rat insulinoma cells. The observed kinetics of the decrease of cell viabilities correlated well with the results of the DLS, ThT, FTIR and AFM studies, revealing that the formation of cytotoxic species correlated well with the formation of large-sized, beta-sheet-rich assemblies (>500 nm) of both fibrillar and amorphous nature. These results suggest that large-sized, beta-sheet-rich insulin assemblies of both fibrillar and amorphous nature are toxic to pancreatic beta-cells. In the light of the ongoing discussion about putative cytotoxic effects of prefibrillar and fibrillar amyloid aggregates, our results support the hypothesis that, in the case of insulin, factors other than the specific secondary or quarternary structural features of the various different aggregates may define their cytotoxic properties. Two such factors might be the aggregate size and the aggregate propensity to expose hydrophobic surfaces to a polar environment.     

Immunomodulation of the human prion peptide 106-126 aggregation

Site-directed monoclonal antibodies (mAbs) may interact with their antigens, leading to stabilization, refolding, and suppression of aggregation. In the following study, we show that mAbs raised against the peptide 106-126 of human prion protein (PrP 106-126) modulate the conformational changes occurring in the peptide exposed to aggregation conditions. MAbs 3-11 and 2-40 prevent PrP 106-126's fibrillar aggregation, disaggregates already formed aggregates, and inhibits the peptide's neurotoxic effect on the PC12 cells system, while mAb 3F4 has no protective effect. We suggest that there are key positions within the PrP 106-126 molecule where unfolding is initiated and their locking with specific antibodies may maintain the prion peptide native structure, reverse the aggregated peptide conformation, and lead to rearrangements involved in the essential feature of prion diseases.     

Concentration dependent Cu2+ induced aggregation and dityrosine formation of the Alzheimer's disease amyloid-beta peptide

The Amyloid beta peptide (Abeta) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu2+ ions can induce the de novo aggregation of the Abeta peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu2+ mediates the change from amyloid material toward Cu2+ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Abeta1-42 at neutral pH is governed by the Cu2+:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu2+ switching effect centered at equimolar Cu2+:peptide ratios. At sub-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms both small spherical oligomers approximately 10-20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu2+ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu2+ induced aggregates are toxic when compared to Abeta1-42 aged in the absence of Cu2+. Importantly, the formation of dityrosine crosslinked Abeta, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu2+ induced aggregates. These results define the role Cu2+ plays in modulating the aggregation state and toxicity of Abeta1-42.     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.     

Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic lateral sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. SOD1 becomes stabilized and enzymatically active after copper and zinc binding and intramolecular disulfide formation, but it remains unknown which step(s) in the SOD1 maturation process is important in the pathological aggregation. In this study we have shown that apoSOD1 without disulfide is the most facile state for formation of amyloid-like fibrillar aggregates. fALS mutations impair either zinc binding, disulfide formation, or both, leading to accumulation of the aggregation-prone, apo, and disulfide-reduced SOD1. Moreover, we have found that the copper chaperone for SOD1 (CCS) facilitates maturation of SOD1 and that CCS overexpression ameliorates intracellular aggregation of mutant SOD1 in vivo. Based on our in vivo and in vitro results, we propose that facilitation of post-translational modifications is a promising strategy to reduce SOD1 aggregation in the cell.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Assessing mutant huntingtin fragment and polyglutamine aggregation by atomic force microscopy

Huntington disease (HD), a neurodegenerative disorder, is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats located near the N-terminus of the huntingtin (htt) protein. The expansion of the polyQ domain results in the ordered assembly of htt fragments into fibrillar aggregates that are the main constituents of inclusion bodies, which are a hallmark of the disease. This paper describes protocols for studying the aggregation of mutant htt fragments and synthetic polyQ peptides with atomic force microscopy (AFM). Ex situ AFM is used to characterize aggregate formation in protein incubation as a function of time. Methods to quickly and unambiguously distinguish specific aggregate species from complex, heterogeneous aggregation reactions based on simple morphological features are presented. Finally, the application of time lapse atomic force microscopy in solution is presented for studying synthetic model polyQ peptides, which allows for tracking the formation and fate of individual aggregates on surfaces over time. This ability allows for dynamic studies of the aggregation process and direct observation of the interplay between different types of aggregates.     

Cu(II) mediates kinetically distinct, non-amyloidogenic aggregation of amyloid-beta peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ(1-40) using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ(1-42) were compatible with the conclusions for Aβ(1-40) even though the low solubility of Aβ(1-42) precluded examination under the same conditions as for the Aβ(1-40). Experiments with Aβ(1-16) and Aβ(1-28) showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ(1-40) depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.     

Alternate aggregation pathways of the Alzheimer beta-amyloid peptide: Abeta association kinetics at endosomal pH

The deposition of beta-amyloid peptide (Abeta) fibrils around neurons is an invariable feature of Alzheimer's disease and there is increasing evidence that fibrillar deposits and/or prefibrillar intermediates play a central role in the observed neurodegeneration. One site of Abeta generation is the endosomes, and we have investigated the kinetics of Abeta association at endosomal pH over physiologically relevant time frames. We have identified three distinct Abeta association phases that occur at rates comparable to endosomal transit times. Rapid formation of burst phase aggregates, larger than 200nm, was observed within 15 seconds. Two slower association phases were detected by fluorescence resonance energy transfer and termed phase 1 and phase 2 aggregation reactions. At 20 microM Abeta, pH 6, the half lives of the phase 1 and phase 2 aggregation phases were 3.15 minutes and 17.66 minutes, respectively. Atomic force microscopy and dynamic light scattering studies indicate that the burst phase aggregate is large and amorphous, while phase 1 and 2 aggregates are spherical with hydrodynamic radii around 30 nm. There is an apparent equilibrium, potentially mediated through a soluble Abeta intermediate, between the large burst phase aggregates and phase 1 and 2 spherical particles. The large burst phase aggregates form quickly, however, they disappear as the equilibrium shifts toward the spherical aggregates. These aggregated species do not contain alpha-helical or beta-structure as determined by circular dichroism spectroscopy. However, after two weeks beta-structure is observed and is attributable to the insoluble portion of the sample. After two months, mature amyloid fibrils appear and the spherical aggregates are significantly diminished.     

Morphohistological study of the different constituents of a banana (Musa AAA, cv. Grande naine) embryogenic cell suspension

Five types of cellular aggregates have been characterised in embryogenic cell suspensions of banana (Musa AAA Grande naine cv.). Type I corresponded to isolated cells or to small cell aggregates. Type II were composed of embryogenic cells. Type III can be distinguished from type II due to the presence of peripheral proliferation zones with embryonic cells. Type IV were composed of protodermic masses histologically comparable to proembryos. Type V were nodules composed of a central zone of meristematic cells and of an external zone of starchy cells. Each culture flask of a cell line contained a majority of one of the above-mentioned aggregate types. Histological studies of somatic embryo developement on semi-solid regeneration medium showed that there were close similarities between the initial steps of ontogenesis of the embryos and the different cell aggregates in liquid multiplication medium. It appeared that aggregates II–IV of the suspension belong to the same development continuum which reproduces the initial phases of somatic embryo ontogenesis on semi-solid medium. Type V resulted from the development of type IV, for which ontogenesis is hindered by direct contact with 2,4-dichlorophenoxyacetic acid and the shaken liquid multiplication medium. Type I aggregates probably do not belong to the development continuum but rather correspond to the degeneration of the other types of aggregates in the suspension. The presence of intermediate types in the liquid medium reinforces the hypothesis of a relationship between the aggregates. The aggregates tended to develop through time from a majority of type II or III at the beginning of their culture to types IV–V for older suspensions. Received: 10 August 1999 / Revision received: 8 November 1999 / Accepted: 9 November 1999     

Point mutations in Aβ result in the formation of distinct polymorphic aggregates in the presence of lipid bilayers

A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.     

Amyloid fibril formation can proceed from different conformations of a partially unfolded protein

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high beta-sheet content from partially unfolded states with very different structural features. Moreover, the presence of alpha-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.     

Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23-98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23-98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23-231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.