Benedetta Bolognesi

ANS binding reveals common features of cytotoxic amyloid species

Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimers disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β 1−40 peptide

In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a newly-discovered risk factor in Alzheimers disease. We have examined the interactions between human clusterin and the Alzheimers disease–associated amyloid-β1−40 peptide (Aβ1−40), which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single-molecule fluorescence methods, we have found that Aβ1−40 forms a heterogeneous distribution of small oligomers (from dimers to 50-mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence both the aggregation and disaggregation of Aβ1−40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and Alzheimers disease.

Detergent-like Interaction of Congo Red with the Amyloid β Peptide

Accumulating evidence links prefibrillar oligomeric species of the amyloid beta peptide (Abeta) to cellular toxicity in Alzheimers disease, potentially via disruption of biological membranes. Congo red (CR) affects protein aggregation. It is known to self-associate into micelle-like assemblies but still reduces the toxicity of Abeta aggregates in cell cultures and model organisms. We show here that CR interacts with Abeta(1-40) in a manner similar to that of anionic detergents. Although CR promotes beta sheet formation and peptide aggregation, it may also solubilize toxic protein species, making them less harmful to critical cellular components and thereby reducing amyloid toxicity.

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 Alzheimers disease-related amyloid beta peptide (Abeta) 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 Abeta40 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 Abeta42 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 Abeta peptide.

Disulfide Bonds Reduce the Toxicity of the Amyloid Fibrils Formed by an Extracellular Protein

The misfolding of proteins into amyloid fibrils constitutes the hallmark of many diseases.[1] Although relatively few physicochemical properties of protein sequences—charge, hydrophobicity, patterns of polar and nonpolar residues, and tendency to form secondary structures—are sufficient to rationalize in general terms their relative propensities to form amyloid fibrils,[2, 3] other properties can also be important. One example is intramolecular disulfide bonds, which limit the ways in which a polypeptide can be arranged in a fibril through the topological restraints that they impose. Although disulfide bonds are present in 15 % of the human proteome, in 65 % of secreted proteins, and in more than 50 % of those involved in amyloidosis, our understanding of how they influence the properties of amyloid fibrils is limited.[4–6] We have examined the formation of fibrils by human lysozyme[7, 8] in the presence and absence (Figure 1 a,b) of its native disulfide bonds, and found that they profoundly influence the fibrillar morphology and cytotoxicity. Figure 1 a) Structure (pdb code 1Lz1) of wild-type lysozyme (Lys) with the disulfide bonds shown in red. These were reduced as shown in (b) to obtain LysRA. c, d) Amyloid formation by Lys (c) and LysRA (d) monitored by light scattering (LS) at 500 nm and different ... As disulfide bonds stabilize folded proteins, they determine the conditions under which wild-type (Lys) and reduced and alkylated lysozyme (LysRA) are amyloidogenic. In agreement with previous reports, we found that it is necessary to incubate Lys under destabilizing conditions, such as low pH (pH 2.0) and high temperature (≥50 °C), to form amyloid fibrils within 24 h (Figure 1 c and Figure S1 in the Supporting Information).[8–10] By contrast, LysRA is amyloidogenic under milder conditions; at pH 2.0, for example, it forms fibrils at 20 °C (Figure 1 d and Figure S1 in the Supporting Information). We analyzed the conformational properties of Lys and LysRA by NMR spectroscopy and far-UV circular dichroism (CD) as a function of temperature. We found that Lys is folded at 20 °C (Figure 1 e,g) and experiences a well-defined unfolding transition at about 55 °C (Figure 1 g and Figure S2 in the Supporting Information).[10] By contrast, LysRA is unfolded at all temperatures (Figure 1 f,h). Our results, therefore, indicate that the presence of intact disulfide bonds decreases the rate at which lysozyme forms fibrils (Figure 1 c,d) by stabilizing the cooperatively folded native protein.[11] Disulfide bonds also determine the morphology of the fibrils. After 24 h of incubation under the mildest conditions that lead to aggregation (Figure 1 c,d), both Lys and LysRA had converted into fibrils as shown by transmission electron microscopy (TEM; Figure 2 a,b insets) and by thioflavin T (ThT) and Congo red (CR) binding (Figure 2 c,d and e,f, respectively). We analyzed the samples by far-UV CD and found that in both cases the spectra evolved from those corresponding to largely disordered proteins (Figure 2 a,b, blue) to those of species rich in β-sheet structure, with a minimum in the ellipticity at approximately 217 nm typical of amyloid fibrils (Figure 2 a,b, red). We also analyzed the amide I region (1580–1720 cm−1) of the infrared spectra of the fibrils by using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy (Figure 3 a,b and Table S1 in the Supporting Information),[9] and found that LysRA fibrils are less rich in β-sheet structure than those formed by Lys (51.5 versus 72.5 %). Figure 2 Aggregation kinetics of a) Lys at pH 2.0 and 60 °C and b) LysRA at pH 2 and 20 °C monitored by far UV-CD. Samples of fibrils formed after 24 h and isolated by ultracentrifugation have a fibrillar morphology (insets in (a) and (b)) and ... Figure 3 a, b) ATR-FTIR spectra of LysRA (a) and Lys fibrils (b), shown in black, with the contributions obtained by curve fitting colored as follows: red: β sheet, green: random/α helix, blue: turns and loops, gray: side chains. c) SDS-PAGE of ... To analyze the nature of the fibrillar core of the fibrils, we studied both types of fibrils by limited proteolysis (Figure 3 c,d). It was found that the fibrils formed by Lys are inert to proteolysis under the conditions used here but that those formed by LysRA are readily cleaved (Figure 3 c,d and Figure S3 in the Supporting Information) and have their diameter reduced from (5.1±0.6) to (3.6±0.6) nm (Figure 2 b and Figure S4 in the Supporting Information). The protease-resistant segment of the molecule, attributable to the core, is composed of about 80 residues, from residue 29 to 108. As susceptibility to proteases requires 10 to 12 unfolded residues,[12] our result is consistent with the number of residues in the β-sheet secondary structure determined by FTIR analysis (51.5 %, that is, 67 residues). We also probed the nature of the non-core regions by an 8-anilinonaphthalene-1-sulfonate (ANS) binding assay, in which interactions of this dye with solvent-exposed hydrophobic patches cause a blue shift in the maximum emission wavelength and an increase in emission intensity.[13] We found that the fluorescence intensity of ANS is higher in the presence of LysRA fibrils than in that of fibrils formed from Lys (Figure S5 in the Supporting Information), hence indicating a greater number of solvent-exposed hydrophobic residues. Since hydrogen-bonding interactions in the cross-β core stabilize amyloid fibrils,[14] we investigated whether differences in core size are reflected in their resistance to disaggregation. We measured the concentration of protein in equilibrium with fibrils at increasing concentrations of guanidine hydrochloride (GdnHCl)[15] and found that the fibrils formed by LysRA disaggregate at lower concentrations of GdnHCl than those formed by Lys (Figure 3 e). Our results indicate that the fibrillar core formed in the presence of disulfide bonds is larger than in their absence, thereby reducing the susceptibility of the fibrils to proteolysis and increasing their stability. Current evidence suggests that the most toxic forms of amyloid aggregates are not the mature fibrils but their less organized precursors.[16] In addition, recent studies have shown that partially structured fibrils can also give rise to toxicity as a result of their larger accessible hydrophobic area or by their greater tendency to generate toxic oligomeric species by fragmentation.[9] To investigate whether or not disulfide bonds alter the cytotoxicity of the fibrils, samples corresponding to protein concentrations of 5 to 20 μm were added to cultures of SH-SY5Y human neuroblastoma cells and the resulting changes in cell viability were measured using a calcein acetoxymethyl (AM) assay (Figure 3 f). The results, supported by an MTT assay (MTT=3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Figure S6 in the Supporting Information), show that the fibrils formed by LysRA have a significantly higher cytotoxic effect than those formed by Lys (p<0.0001; Figure 3 f), in agreement with the finding that ANS binding in amyloid species (Figure S5 in the Supporting Information) correlates with cytotoxicity.[17] This result shows that disulfide bonds can decrease toxicity by favoring the formation of highly structured amyloid fibrils, which suggests that disulfide bonds in extracellular proteins could be the result of evolutionary pressures[18] to minimize toxic aggregation in an environment where the redox potential favors disulfide bond formation. To investigate this hypothesis further, we analyzed the aggregation propensity of the human proteome using the well-established Zyggregator predictor.[3] We found that the sequences of extracellular proteins have higher intrinsic aggregation propensity than intracellular ones, an observation that has been related to the dilution that occurs upon secretion.[19, 20] We also found that disulfide bonds are associated with sequences of high aggregation propensity (Figure 3 g), which suggests that disulfide bonds have co-evolved with protein sequences[21] to minimize their propensity to form potentially toxic amyloid aggregates. This analysis would explain the high prevalence of disulfide bonds in extracellular proteins, where additional protective mechanisms that reduce misfolding and its consequences are likely to play a less significant role than inside the cell.[20, 22] The disulfide bonds of lysozyme inhibit the aggregation of this protein into amyloid fibrils by stabilizing the folded state—a fact that can be attributed to the reduction in the entropy of the unfolded state.[11] A partially unfolded state can nevertheless be populated as a result of changes in conditions, as in this work, or by mutations, as in patients with nonneuropathic systemic amyloidosis.[8] We have shown that, when this situation occurs, disulfide bonds allow the formation of fibrils with a large proportion of their sequence in the cross-β conformation. This result is at first sight unexpected, as one might have anticipated that the conformational constraints resulting from cross-linking would reduce the ability of the chain to fold into the complex β-sheet amyloid structure. It is, however, clear that lysozyme and other disulfide-linked proteins are able to form fibrils that contain a high fraction of sequence in the cross-β structure.[23] We conclude that intramolecular disulfide bonds can stabilize amyloid fibrils, as they do for the folded state, by decreasing the entropic penalty associated with the formation of this ordered form of protein structure. This can be concomitant with significant decreases in the toxicity of the resulting fibrils, which suggests that disulfide bonds have co-evolved with protein sequences to reduce toxic aggregation.[21]

Neurodegenerative diseases: Quantitative predictions of protein–RNA interactions

Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimers, and Parkinsons diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

Hydrophobicity and Conformational Change as Mechanistic Determinants for Nonspecific Modulators of Amyloid β Self-Assembly

The link between many neurodegenerative disorders, including Alzheimers and Parkinsons diseases, and the aberrant folding and aggregation of proteins has prompted a comprehensive search for small organic molecules that have the potential to inhibit such processes. Although many compounds have been reported to affect the formation of amyloid fibrils and/or other types of protein aggregates, the mechanisms by which they act are not well understood. A large number of compounds appear to act in a nonspecific way affecting several different amyloidogenic proteins. We describe here a detailed study of the mechanism of action of one representative compound, lacmoid, in the context of the inhibition of the aggregation of the amyloid β-peptide (Aβ) associated with Alzheimers disease. We show that lacmoid binds Aβ(1-40) in a surfactant-like manner and counteracts the formation of all types of Aβ(1-40) and Aβ(1-42) aggregates. On the basis of these and previous findings, we are able to rationalize the molecular mechanisms of action of nonspecific modulators of protein self-assembly in terms of hydrophobic attraction and the conformational preferences of the polypeptide.

The H50Q mutation induces a 10-fold decrease in the solubility of α-synuclein

Background: The basis of the pathogenicity of the H50Q variant α-synuclein is unknown. Results: The critical concentration of α-synuclein is decreased by 10-fold by the H50Q mutation, and its aggregation is modulated by the wild-type isoform. Conclusion: Key effects of the H50Q mutation on the aggregation of α-synuclein can be quantified. Significance: Our data provide insights into the mechanism of Lewy body formation in vivo. The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-β aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol−1, thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.

Principles of self-organization in biological pathways: a hypothesis on the autogenous association of alpha-synuclein

Previous evidence indicates that a number of proteins are able to interact with cognate mRNAs. These autogenous associations represent important regulatory mechanisms that control gene expression at the translational level. Using the catRAPID approach to predict the propensity of proteins to bind to RNA, we investigated the occurrence of autogenous associations in the human proteome. Our algorithm correctly identified binding sites in well-known cases such as thymidylate synthase, tumor suppressor P53, synaptotagmin-1, serine/ariginine-rich splicing factor 2, heat shock 70 kDa, ribonucleic particle-specific U1A and ribosomal protein S13. In addition, we found that several other proteins are able to bind to their own mRNAs. A large-scale analysis of biological pathways revealed that aggregation-prone and structurally disordered proteins have the highest propensity to interact with cognate RNAs. These findings are substantiated by experimental evidence on amyloidogenic proteins such as TAR DNA-binding protein 43 and fragile X mental retardation protein. Among the amyloidogenic proteins, we predicted that Parkinson’s disease-related α-synuclein is highly prone to interact with cognate transcripts, which suggests the existence of RNA-dependent factors in its function and dysfunction. Indeed, as aggregation is intrinsically concentration dependent, it is possible that autogenous interactions play a crucial role in controlling protein homeostasis.

X-inactivation: quantitative predictions of protein interactions in the Xist network

The transcriptional silencing of one of the female X-chromosomes is a finely regulated process that requires accumulation in cis of the long non-coding RNA X-inactive-specific transcript (Xist) followed by a series of epigenetic modifications. Little is known about the molecular machinery regulating initiation and maintenance of chromosomal silencing. Here, we introduce a new version of our algorithm catRAPID to investigate Xist associations with a number of proteins involved in epigenetic regulation, nuclear scaffolding, transcription and splicing processes. Our method correctly identifies binding regions and affinities of protein interactions, providing a powerful theoretical framework for the study of X-chromosome inactivation and other events mediated by ribonucleoprotein associations.