Glycosaminoglycans (GAGs) suppress the toxicity of HypF-N prefibrillar aggregates

A group of diverse human pathologies is associated with proteins unable to retain their native state and convert into prefibrillar and fibrillar amyloid aggregates that are then deposited in the extracellular space. Glycosaminoglycans (GAGs) have been found to physically associate with these deposits and also to promote their formation in vitro. However, the effect of GAGs on the toxicity of these aggregates has been investigated in only one protein system, the amyloid β peptide associated with Alzheimer's disease. In this study, we investigate whether GAGs affect the toxicity of the N-terminal domain of Escherichia coli HypF (HypF-N) oligomers on Chinese hamster ovarian cells and the mechanism by which such suppression is mediated. The results show that heparin and other GAGs inhibit the toxicity observed by HypF-N oligomers in a dose-dependent manner. GAGs were not found to bind preformed HypF-N oligomers, change their morphological and structural characteristics or disaggregate them. Nevertheless, they were found to bind to the cells' surface and prevent the interaction of the oligomers with the cells. Overall, the results indicate that GAGs have a generic ability to inhibit the toxicity of aberrant protein oligomers and that such toxicity suppression can occur through different mechanisms, such as through binding to the oligomers with consequent loss of interaction of the oligomers to the GAGs present on the cell surface, as proposed previously for amyloid β aggregates, or through mechanisms independent of direct GAG-oligomer binding, as shown here for HypF-N aggregates.     

Searching for conditions to form stable protein oligomers with amyloid-like characteristics: The unexplored basic pH

Conversion of peptides and proteins from their native states into amyloid fibrillar aggregates is the hallmark of a number of pathological conditions, including Alzheimer's disease and amyloidosis. Evidence is accumulating that soluble oligomers, as opposed to mature fibrils, mediate cellular dysfunction, ultimately leading to disease onset. In this study, we have explored the ability of alkaline pH solutions, which have remained relatively unexplored so far, to form a partially folded state of the N-terminal domain of the Escherichia coli protein HypF (HypF-N), which subsequently assembles to form stable soluble oligomers. Results showed that HypF-N unfolds at high pH via a two-state process. Characterization of the resulting alkaline-unfolded state by near- and far-UV circular dichroism, intrinsic and ANS-derived fluorescence and DLS indicated characteristics of a monomeric, premolten globule state. Interestingly, alkaline-unfolded HypF-N aggregates, at high concentration in the presence of low concentrations of TFE, into stable oligomers. These are able to bind amyloid-specific dyes, such as Congo red, ThT, and ANS, contain extensive β-sheet structure, as detected with far-UV circular dichroism, and have a height of 2.0–3.9 nm when analysed using atomic force microscopy. This study, which complements our previous one in which morphologically, structurally, and tinctorially similar oligomers were formed at low and nearly neutral pH values by the same protein, offers opportunities to explore the fine differences existing in the mechanism of formation of these species under different conditions, in their precise molecular structure and in their ability to cause cellular dysfunction.     

A comparison of the biochemical modifications caused by toxic and non-toxic protein oligomers in cells

Peptides and proteins can convert from their soluble forms into highly ordered fibrillar aggregates, giving rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. It is increasingly recognized that protein oligomers forming early in the process of fibril aggregation represent the pathogenic species in protein deposition diseases. The N-terminal domain of the HypF protein from Escherichia coli (HypF-N) has previously been shown to form, under distinct conditions, two types of HypF-N oligomers with indistinguishable morphologies but distinct structural features at the molecular level. Only the oligomer type exposing hydrophobic surfaces and possessing sufficient structural plasticity is toxic (type A), whereas the other type is benign to cultured cells (type B). Here we show that only type A oligomers are able to induce a Ca(2+) influx from the cell medium to the cytosol, to penetrate the plasma membrane, to increase intracellular reactive oxygen species production, lipid peroxidation and release of intracellular calcein, resulting in the activation of the apoptotic pathway. Remarkably, these oligomers can also induce a loss of cholinergic neurons when injected into rat brains. By contrast, markers of cellular stress and viability were unaffected in cultured and rat neuronal cells exposed to type B oligomers. The analysis of the time scales of such effects indicates that the difference of toxicity between the two oligomer types involve the early events of the toxicity cascade, shedding new light on the mechanism of action of protein oligomers and on the molecular targets for the therapeutic intervention against protein deposition diseases.     

Amyloid formation from HypF-N under conditions in which the protein is initially in its native state

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.     

Natively folded HypF-N and its early amyloid aggregates interact with phospholipid monolayers and destabilize supported phospholipid bilayers

Recent data depict membranes as the main sites where proteins/peptides are recruited and concentrated, misfold, and nucleate amyloids; at the same time, membranes are considered key triggers of amyloid toxicity. The N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) in 30% trifluoroethanol undergoes a complex path of fibrillation starting with initial 2-3-nm oligomers and culminating with the appearance of mature fibrils. Oligomers are highly cytotoxic and permeabilize lipid membranes, both biological and synthetic. In this article, we report an in-depth study aimed at providing information on the surface activity of HypF-N and its interaction with synthetic membranes of different lipid composition, either in the native conformation or as amyloid oligomers or fibrils. Like other amyloidogenic peptides, the natively folded HypF-N forms stable films at the air/water interface and inserts into synthetic phospholipid bilayers with efficiencies depending on the type of phospholipid. In addition, HypF-N prefibrillar aggregates interact with, insert into, and disassemble supported phospholipid bilayers similarly to other amyloidogenic peptides. These results support the idea that, at least in most cases, early amyloid aggregates of different peptides and proteins produce similar effects on the integrity of membrane assembly and hence on cell viability.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Transthyretin suppresses the toxicity of oligomers formed by misfolded proteins in vitro

Although human transthyretin (TTR) is associated with systemic amyloidoses, an anti-amyloidogenic effect that prevents Aβ fibril formation in vitro and in animal models has been observed. Here we studied the ability of three different types of TTR, namely human tetramers (hTTR), mouse tetramers (muTTR) and an engineered monomer of the human protein (M-TTR), to suppress the toxicity of oligomers formed by two different amyloidogenic peptides/proteins (HypF-N and Aβ₄₂). muTTR is the most stable homotetramer, hTTR can dissociate into partially unfolded monomers, whereas M-TTR maintains a monomeric state. Preformed toxic HypF-N and Aβ₄₂ oligomers were incubated in the presence of each TTR then added to cell culture media. hTTR, and to a greater extent M-TTR, were found to protect human neuroblastoma cells and rat primary neurons against oligomer-induced toxicity, whereas muTTR had no protective effect. The thioflavin T assay and site-directed labeling experiments using pyrene ruled out disaggregation and structural reorganization within the discrete oligomers following incubation with TTRs, while confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and hTTR, particularly M-TTR. Moreover, atomic force microscopy (AFM), light scattering and turbidimetry analyses indicated that larger assemblies of oligomers are formed in the presence of M-TTR and, to a lesser extent, with hTTR. Overall, the data suggest a generic capacity of TTR to efficiently neutralize the toxicity of oligomers formed by misfolded proteins and reveal that such neutralization occurs through a mechanism of TTR-mediated assembly of protein oligomers into larger species, with an efficiency that correlates inversely with TTR tetramer stability.     

Effect of chemical chaperones in improving the solubility of recombinant proteins in Escherichia coli

The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.     

Comparison of the folding processes of distantly related proteins. Importance of hydrophobic content in folding

The N-terminal domain of HypF from Escherichia coli (HypF-N) is a 91 residue protein module sharing the same folding topology and a significant sequence identity with two extensively studied human proteins, muscle and common-type acylphosphatases (mAcP and ctAcP). With the aim of learning fundamental aspects of protein folding from the close comparison of so similar proteins, the folding process of HypF-N has been studied using stopped-flow fluorescence. While mAcP and ctAcP fold in a two-state fashion, HypF-N was found to collapse into a partially folded intermediate before reaching the fully folded conformation. Formation of a burst-phase intermediate is indicated by the roll over in the Chevron plot at low urea concentrations and by the large jump of intrinsic and 8-anilino-1-naphtalenesulphonic acid-derived fluorescence immediately after removal of denaturant. Furthermore, HypF-N was found to fold rapidly with a rate constant that is approximately two and three orders of magnitudes faster than ctAcP and mAcP, respectively. Differences between the bacterial protein and the two human counterparts were also found as to the involvement of proline isomerism in their respective folding processes. The results clearly indicate that features that are often thought to be relevant in protein folding are not highly conserved in the evolution of the acylphosphatase superfamily. The large difference in folding rate between mAcP and HypF-N cannot be entirely accounted for by the difference in relative contact order or related topological metrics. The analysis shows that the higher folding rate of HypF-N is in part due to the relatively high hydrophobic content of this protein. This conclusion, which is also supported by the highly significant correlation found between folding rate and hydrophobic content within a group of proteins displaying the topology of HypF-N and AcPs, suggests that the average hydrophobicity of a protein sequence is an important determinant of its folding rate.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

Differing molecular mechanisms appear to underlie early toxicity of prefibrillar HypF-N aggregates to different cell types

Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF-N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF-N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl-2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro-apoptotic stimuli, which are counteracted by upregulation of Bcl-2.     

Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Prefibrillar amyloid protein aggregates share common features of cytotoxicity

The intracellular free Ca(2+) concentration and redox status of murine fibroblasts exposed to prefibrillar aggregates of the HypF N-terminal domain have been investigated in vitro and in vivo using a range of fluorescent probes. Aggregate entrance into the cytoplasm is followed by an early rise of reactive oxygen species and free Ca(2+) levels and eventually by cell death. Such changes correlate directly with the viability of the cells and are not observed when cell are cultured in the presence of reducing agents or in Ca(2+)-free media. In addition, moderate cell stress following exposure to the aggregates was found to be fully reversible. The results show that the cytotoxicity of prefibrillar aggregates of HypF-N, a protein not associated with clinical disease, has the same fundamental origin as that produced by similar types of aggregates of proteins linked with specific amyloidoses. These findings suggest that misfolded proteinaceous aggregates stimulate generic cellular responses as a result of the exposure of regions of the structure (such as hydrophobic residues and the polypeptide main chain) that are buried in the normally folded proteins. They also support the idea that a higher number of degenerative pathologies than previously known might be considered as protein deposition diseases.     

Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.     

The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless of their aggregation state

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.     

Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with alpha-synuclein at the single molecule level

ABSTRACT: BACKGROUND: Fibrillar amyloid-like deposits and co-deposits of tau and alpha-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and alpha-synuclein are not well understood. RESULTS: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with alpha-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3beta exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of alpha-synuclein in pre-formed tau oligomers. CONCLUSIONS: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.     

Conformational properties of the aggregation precursor state of HypF-N

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.     

Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways

In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Molecular defects in variant forms of mannose-binding protein associated with immunodeficiency.

Distinct molecular mechanisms underlying immunodeficiency caused by three different naturally occurring point mutations within the collagen-like domain of human mannose-binding protein (MBP; also known as mannose-binding lectin) have been revealed by introduction of analogous mutations into rat serum MBP. The change Arg23-->Cys results in a lower proportion of the large oligomers most efficient at activating the complement cascade. The presence of cysteine at position 23, which forms aberrant interchain disulfide bonds, causes disruption of the normal oligomeric state. The deficiency in MBPs containing Gly25-->Asp and Gly28-->Glu substitutions also results in part from reduced formation of higher oligomers. However, decreased ability to interact with downstream components of the complement cascade due to changes in both the N-terminal disulfide-bonding arrangement and the local structure of the collagenous domain make more important contributions to the loss of activity in these mutants.