Methylene blue inhibits nucleation and elongation of SOD1 amyloid fibrils.
ABSTRACT: Protein aggregation into highly-structured amyloid fibrils is linked to several neurodegenerative diseases. Such fibril formation by superoxide dismutase I (SOD1) is considered to be related to amyotrophic lateral sclerosis, a late-onset and fatal disorder. Despite much effort and the discovery of numerous anti-amyloid compounds, no effective cure or treatment is currently available. Methylene blue (MB), a phenothiazine dye, has been shown to modulate the aggregation of multiple amyloidogenic proteins. In this work we show its ability to inhibit both the spontaneous amyloid aggregation of SOD1 as well as elongation of preformed fibrils.
Project description:The aggregation cascade of disease-related amyloidogenic proteins, terminating in insoluble amyloid fibrils, involves intermediate oligomeric states. The structural and biochemical details of these oligomers have been largely unknown. Here we report crystal structures of variants of the cytotoxic oligomer-forming segment residues 28-38 of the ALS-linked protein, SOD1. The crystal structures reveal three different architectures: corkscrew oligomeric structure, nontwisting curved sheet structure and a steric zipper proto-filament structure. Our work highlights the polymorphism of the segment 28-38 of SOD1 and identifies the molecular features of amyloidogenic entities.
Project description:Amyloid fibrils are mechanically robust and partly resistant to proteolytic degradation, making them potential candidates for scaffold materials in cell culture, tissue engineering, drug delivery and other applications. Such applications of amyloids would benefit from the possibility to functionalize the fibrils, for example by adding growth factors or cell attachment sites. The BRICHOS domain is found in a family of human proteins that harbor particularly amyloid-prone regions and can reduce aggregation as well as toxicity of several different amyloidogenic peptides. Recombinant human (rh) BRICHOS domains have been shown to bind to the surface of amyloid-? (A?) fibrils by immune electron microscopy. Here we produce fusion proteins between mCherry and rh Bri2 BRICHOS and show that they can bind to different amyloid fibrils with retained fluorescence of mCherry in vitro as well as in cultured cells. This suggests a "generic" ability of the BRICHOS domain to bind fibrillar surfaces that can be used to synthesize amyloid decorated with different protein functionalities.
Project description:Small-molecule inhibitors of abnormal protein self-assembly are promising candidates for developing therapy against proteinopathies. Such compounds have been examined primarily as inhibitors of amyloid β-protein (Aβ), whereas testing of inhibitors of other amyloidogenic proteins has lagged behind. An important issue with screening compound libraries is that although an inhibitor suitable for therapy must be both effective and nontoxic, typical screening focuses on efficacy, whereas safety typically is tested at a later stage using cells and/or animals. In addition, typical thioflavin T (ThT)-fluorescence-based screens use the final fluorescence value as a readout, potentially missing important kinetic information. Here, we examined potential inhibitors of superoxide dismutase 1 (SOD1) using ThT-fluorescence including the different phases of fluorescence change and added a parallel screen of SOD1 activity as a potential proxy for compound toxicity. Some compounds previously reported to inhibit other amyloidogenic proteins also inhibited SOD1 aggregation at low micromolar concentrations, whereas others were ineffective. Analysis of the lag phase and exponential slope added important information that could help exclude false-positive or false-negative results. SOD1 was highly resistant to inhibition of its activity, and therefore, did not have the necessary sensitivity to serve as a proxy for examining potential toxicity.
Project description:Protein aggregation into amyloid fibrils is linked to multiple neurodegenerative disorders, such as Alzheimer's, Parkinson's or Creutzfeldt-Jakob disease. A better understanding of the way these aggregates form is vital for the development of drugs. A large detriment to amyloid research is the ability of amyloidogenic proteins to spontaneously aggregate into multiple structurally distinct fibrils (strains) with different stability and seeding properties. In this work we show that prion proteins are capable of forming more than one type of fibril under the exact same conditions by assessing their Thioflavin T (ThT) binding ability, morphology, secondary structure, stability and seeding potential.
Project description:Protein misfolding and/or aggregation has been implicated as the cause of several human diseases, such as Alzheimer's and Parkinson's diseases and familial amyloid polyneuropathy. These maladies are referred to as amyloid diseases, named after the cross-?-sheet amyloid fibril aggregates or deposits common to these disorders. Epigallocatechin-3-gallate (EGCG), the principal polyphenol present in green tea, has been shown to be effective at preventing aggregation and is able to remodel amyloid fibrils comprising different amyloidogenic proteins, although the mechanistic underpinnings are unclear. Herein, we work toward an understanding of the molecular mechanism(s) by which EGCG remodels mature amyloid fibrils made up of A?(1-40), IAPP(8-24), or Sup35NM(7-16). We show that EGCG amyloid remodeling activity in vitro is dependent on auto-oxidation of the EGCG. Oxidized and unoxidized EGCG binds to amyloid fibrils, preventing the binding of thioflavin T. This engagement of the hydrophobic binding sites in A?(1-40), IAPP(8-24), or Sup35NM(Ac7-16) Y?F amyloid fibrils seems to be sufficient to explain the majority of the amyloid remodeling observed by EGCG treatment, although how EGCG oxidation drives remodeling remains unclear. Oxidized EGCG molecules react with free amines within the amyloid fibril through the formation of Schiff bases, cross-linking the fibrils, which may prevent dissociation and toxicity, but these aberrant post-translational modifications do not appear to be the major driving force for amyloid remodeling by EGCG treatment. These insights into the molecular mechanism of action of EGCG provide boundary conditions for exploring amyloid remodeling in more detail.
Project description:The acylation of lysine residues in superoxide dismutase-1 (SOD1) has been previously shown to decrease its rate of nucleation and elongation into amyloid-like fibrils linked to amyotrophic lateral sclerosis. The chemical mechanism underlying this effect is unclear, i.e. hydrophobic/steric effects versus electrostatic effects. Moreover, the degree to which the acylation might alter the prion-like seeding of SOD1 in vivo has not been addressed. Here, we acylated a fraction of lysine residues in SOD1 with groups of variable hydrophobicity, charge, and conformational entropy. The effect of each acyl group on the rate of SOD1 fibril nucleation and elongation were quantified in vitro with thioflavin-T (ThT) fluorescence, and we performed 594 iterate aggregation assays to obtain statistically significant rates. The effect of the lysine acylation on the prion-like seeding of SOD1 was assayed in spinal cord extracts of transgenic mice expressing a G85R SOD1-yellow fluorescent protein construct. Acyl groups with >2 carboxylic acids diminished self-assembly into ThT-positive fibrils and instead promoted the self-assembly of ThT-negative fibrils and amorphous complexes. The addition of ThT-negative, acylated SOD1 fibrils to organotypic spinal cord failed to produce the SOD1 inclusion pathology that typically results from the addition of ThT-positive SOD1 fibrils. These results suggest that chemically increasing the net negative surface charge of SOD1 via acylation can block the prion-like propagation of oligomeric SOD1 in spinal cord.
Project description:Inhibiting the cytotoxicity of amyloid aggregation by endogenous proteins is a promising strategy against degenerative amyloid diseases due to their intrinsically high biocompatibility and low immunogenicity. In this study, we investigated the inhibition mechanism of the structured core region of αB-crystallin (αBC) against Aβ fibrillization using discrete molecular dynamics simulations. Our computational results recapitulated the experimentally observed Aβ binding sites in αBC and suggested that αBC could bind to various Aβ aggregate species during the aggregation process-including monomers, dimers, and likely other high molecular weight oligomers, protofibrils, and fibrils-by capping the exposed β-sheet elongation surfaces. Thus, the nucleation of Aβ oligomers into fibrils and the fibril growth could be inhibited. Mechanistic insights obtained from our systematic computational studies may aid in the development of novel therapeutic strategies to modulate the aggregation of pathological, amyloidogenic protein in degenerative diseases.
Project description:The amyloid-forming proteins tau, ?B crystallin, and amyloid P protein are all found in lesions of multiple sclerosis (MS). Our previous work established that amyloidogenic peptides from the small heat shock protein ?B crystallin (HspB5) and from amyloid ? fibrils, characteristic of Alzheimer's disease, were therapeutic in experimental autoimmune encephalomyelitis (EAE), reflecting aspects of the pathology of MS. To understand the molecular basis for the therapeutic effect, we showed a set of amyloidogenic peptides composed of six amino acids, including those from tau, amyloid ? A4, major prion protein (PrP), HspB5, amylin, serum amyloid P, and insulin B chain, to be anti-inflammatory and capable of reducing serological levels of interleukin-6 and attenuating paralysis in EAE. The chaperone function of the fibrils correlates with the therapeutic outcome. Fibrils composed of tau 623-628 precipitated 49 plasma proteins, including apolipoprotein B-100, clusterin, transthyretin, and complement C3, supporting the hypothesis that the fibrils are active biological agents. Amyloid fibrils thus may provide benefit in MS and other neuroinflammatory disorders.
Project description:Typically, elongation of an amyloid fibril entails passing conformational details of the mother seed to daughter generations of fibrils with high fidelity. There are, however, several factors that can potentially prevent such transgenerational structural imprinting from perpetuating, for example heterogeneity of mother seeds or so-called conformational switching. Here, we examine phenotypic persistence of bovine insulin amyloid ([BI]) upon multiple rounds of self-seeding under quiescent conditions. According to infrared spectroscopy, with the following passages of homologous seeding, daughter fibrils gradually depart from the mother seed's spectral characteristics. We note that this transgenerational structural drift in [BI] amyloid leads toward fibrils with infrared, chiroptical, and morphological traits similar to those of the superstructural variant of fibrils which normally forms upon strong agitation of insulin solutions. However, in contrast to agitation-induced insulin amyloid, the superstructural assemblies of daughter fibrils isolated through self-seeding are sonication-resistant. Our results suggest that formation of single amyloid fibrils is not a dead-end of the amyloidogenic self-assembly. Instead, the process appears to continue toward the self-assembly of higher-order structures although on longer time-scales. From this perspective, the fast agitation-induced aggregation of insulin appears to be a shortcut to amyloid superstructures whose formation under quiescent conditions is slow.
Project description:Amyloid fibrils formed from prion protein (PrP) are associated with prion diseases. In this review we discuss a number of extrinsic and intrinsic experimental factors related to the formation of PrP amyloid fibrils in vitro. We first examined the effects of ultrasonic power on the induction of amyloid fibrillation from PrP. The most important conclusion drawn from the results is that an applied ultrasonic power of approximately 2 W enhanced the nucleation of amyloid fibrils efficiently but that more powerful ultrasonication led to retardation of growth. We also reviewed evidence on the amyloidogenic regions of PrP based on peptide screening throughout the polypeptide sequence. These results showed that helix 2 (H2) peptides of PrP were capable of both the fibrillation and propagation of straight, long fibrils. Moreover, the conformation of preformed H2 fibrils changed reversibly depending on the pH of the solution, implying that interactions between side-chains modulated the conformation of amyloid fibrils. The evidence discussed in this review relates specifically to PrP but may be relevant to other amyloidogenic proteins.