Controlling amyloid fibril formation by partial stirring

Many proteins undergoe self‐assembly into fibrillar structures known as amyloid fibrils. During the self‐assembly process, related structures known as spherulites can be formed. Herein we report a facile method where the balance between amyloid fibrils and spherulites can be controlled by stirring of the reaction mixture during the initial stages of the self‐assembly process. Moreover, we report how this methodology can be used to prepare non‐covalently functionalized amyloid fibrils. By stirring the reaction mixture continuously or for a limited time during the lag phase, the fibril length, and hence the propensity to form liquid crystalline phases, can be influenced. This phenomena is utilized in order to prepare films consisting of aligned protein fibrils incorporating the laser dye Nile red. The resulting films display polarized Nile red fluorescence. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 249–259, 2016.     

Modification of replicon operation in HeLa cells by 2,4-dinitrophenol

Cycloheximide causes inhibition of semiconservative DNA replication in HeLa cells by reducing the average rate of DNA chain elongation. 2,4-Dinitrophenol inhibits semiconservative DNA replication (50 to 80% inhibitions at 10^?3 to 5 × 10^?3 M-2,4-dinitrophenol) without affecting the average rate of DNA chain elongation. Therefore, at any given time the number of replicating sections of DNA per DNA-synthesizing (S-phase) cell appears to be reduced in the presence of 2,4-dinitrophenol.Radioactivity profiles of pulse-labeled DNA in alkaline sucrose gradients suggest that 2,4-dinitrophenol modifies initiation and termination patterns of replicating sections, most of which are found to be 10 to 80 μm (mode: 15 to 30 μm) under control conditions. DNA synthesized in the presence of 2,4-dinitrophenol has the density of control DNA, is metabolically stable, and after mitosis, functions normally as a template in the next round of replication.     

Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

The role of protein stability, solubility, and net charge in amyloid fibril formation

Ribonuclease Sa and two charge-reversal variants can be converted into amyloid in vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.     

Prevention of amyloid fibril formation of amyloidogenic chicken cystatin by site-specific glycosylation in yeast

To address the role of glycosylation on fibrillogenicity of amyloidogenic chicken cystatin, the consensus sequence for N-linked glycosylation (Asn106-Ile108 --> Asn106-Thr108) was introduced by site-directed mutagenesis into the wild-type and amyloidogenic chicken cystatins to construct the glycosylated form of chicken cystatins. Both the glycosylated and unglycosylated forms of wild-type and amyloidogenic mutant I66Q cystatin were expressed and secreted in a culture medium of yeast Pichia pastoris transformants. Comparison of the amount of insoluble aggregate, the secondary structure, and fibrillogenicity has shown that the N-linked glycosylation could prevent amyloid fibril formation of amyloidogenic chicken cystatin secreted in yeast cells without affecting its inhibitory activities. Further study showed this glycosylation could inhibit the formation of cystatin dimers. Therefore, our data strongly suggested that the mechanism causing the prevention of amyloidogenic cystation fibril formation may be realized through suppression of the formation of three-dimensional domain-swapped dimers and oligomers of amyloidogenic cystatin by the glycosylated chains at position 106.     

Comparative MD analysis of the stability of transthyretin providing insight into the fibrillation mechanism

Proteins can misfold and aggregate, which is believed to be the cause of a variety of diseases, affecting very diverse organs in the body. Many questions about the nature of aggregation and the proteins that are involved in these events are still left unanswered. One of the proteins that is known to form amyloids is transthyretin (TTR), the secondary transporter of thyroxine, and transporter of retinol-binding protein. Several experimental results have helped to explain this aberrant behavior of TTR; however, structural insights of the amyloidgenic process are still lacking. Therefore, we have used all-atom MD simulation and free energy calculations to study the initial phase of this process. We have calculated the free energy changes of the initial tetramer dissociation under different conditions and in the presence of thyroxine. We show that tetramer formation is indeed only thermodynamically favorable in neutral pH conditions. We find that binding of two thyroxine molecules stabilizes the complex, and that this occurs with negative cooperativity. In addition to the energetic calculations, we have also investigated the dominant motions of the TTR and found that only the dimeric form of the protein could undergo the initial fibril formation.     

Anaerobic aquifer transformations of 2,4-dinitrophenol under different terminal electron accepting conditions

We evaluated the susceptibility of 2,4-dinitrophenol (2,4-DNP) and 2,4-diaminophenol to anaerobic biodegradation in aquifer slurries. Aquifer microorganisms depleted 2,4-DNP at rates of 25, 9 and 0.4 microM/day under methanogenic, sulfate-reducing and nitrate-reducing conditions, respectively. Rates of abiotic, 2,4-DNP loss in autoclaved control incubations were 7.2, 6.2 and 0.95 microM/day respectively. Abiotic, 2,4-DNP reduction was especially important as the first step in its transformation. 2-Amino-4-nitrophenol was produced by this process, but this compound was further metabolized in methanogenic and sulfate-reducing aquifer slurries. This partially reduced compound persisted in autoclaved controls and in the nitrate-reducing aquifer slurries. Aquifer slurries incubated with either 2,4-DNP or 2,4-diaminophenol produced methane when incubated with no other electron acceptor suggesting that mineralization had occurred under these conditions. In parallel experiments, aquifer slurries amended with 2,6-dinitrophenol or picric acid did not produce methane at levels above the substrate unamended controls.     

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Pathways and intermediates of amyloid fibril formation

The lack of understanding of amyloid fibril formation at the molecular level is a major obstacle in devising strategies to interfere with the pathologies linked to peptide or protein aggregation. In particular, little is known on the role of intermediates and fibril elongation pathways as well as their dependence on the intrinsic tendency of a polypeptide chain to self-assembly by β-sheet formation (β-aggregation propensity). Here, coarse-grained simulations of an amphipathic polypeptide show that a decrease in the β-aggregation propensity results in a larger heterogeneity of elongation pathways, despite the essentially identical structure of the final fibril. Protofibrillar intermediates that are thinner, shorter and less structured than the final fibril accumulate along some of these pathways. Moreover, the templated formation of an additional protofilament on the lateral surface of a protofibril is sometimes observed as a collective transition. Conversely, for a polypeptide model with a high β-aggregation propensity, elongation proceeds without protofibrillar intermediates. Therefore, changes in intrinsic β-aggregation propensity modulate the relative accessibility of parallel routes of aggregation.     

Inhibition of amyloid fibril formation and cytotoxicity by hydroxyindole derivatives

Gaining insight into the mechanism of amyloid fibril formation, the hallmark of multiple degenerative syndromes of unrelated origin, and exploring novel directions of inhibition are crucial for preventing disease development. Specific interactions between aromatic moieties were suggested to have a key role in the recognition and self-assembly processes leading to the formation of amyloid fibrils by several amyloidogenic polypeptides, including the beta-amyloid polypeptide associated with Alzheimer's disease. Our finding of the high-affinity molecular recognition and intense amyloidogenic potential of tryptophan-containing peptide fragments led to the hypothesis that screening for indole derivatives might lead to the identification of potential inhibitors of amyloid formation. Such inhibitors could mediate specific recognition processes without allowing further growth of the well-ordered amyloid chain. Using fluorescence spectroscopy, atomic force microscopy, and electron microscopy to screen 29 indole derivatives, we identified three potent inhibitors: indole-3-carbinol (I3C), 3-hydroxyindole (3HI), and 4-hydroxyindole (4HI). The latter, a simple low-molecular weight aromatic compound, was the most effective, completely abrogating not only the formation of aggregated structures by Abeta but also the cytotoxic activity of aggregated Abeta toward cultured cells. The results of this study provide further experimental support for the paradigm of amyloid inhibition by heteroaromatic interaction and point to indole derivatives as a simple molecular platform for the development of novel fibrillization inhibitors.     

Sequence dependence of amyloid fibril formation: insights from molecular dynamics simulations

The clarification of the physico-chemical determinants underlying amyloid deposition is critical for our understanding of misfolding diseases. With this purpose we have performed a systematic all-atom molecular dynamics (MD) study of a series of single point mutants of the de novo designed amyloidogenic peptide STVIIE. Sixteen different 50ns long simulations using explicit solvent have been carried out starting from four different conformations of a polymeric six-stranded beta-sheet. The simulations have provided evidence for the influence of a small number of site-specific hydrophobic interactions on the packing and stabilization of nascent aggregates, as well as the interplay between side-chain interactions and the net charge of the molecule on the strand arrangement of polymeric beta-sheets. This MD analysis has also shed light into the origin of the position dependence on mutation of beta-sheet polymerization that was found experimentally for this model system. Our results suggest that MD can be applied to detect critical positions for beta-sheet aggregation within a given amyloidogenic stretch. Studies similar to the one presented here can guide site-directed mutations or the design of drugs that specifically disrupt the key stabilizing interactions of beta-sheet aggregates.     

Unfolding and aggregation of transthyretin by the truncation of 50 N-terminal amino acids

Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wild-type transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C-terminal fragments starting near amino acid residue 50 as well as full-length transthyretin. Although a number of previous studies suggest the importance of the C-terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C-terminal fragments of transthyretin. To understand the role of C-terminal fragments in SSA, we examined the effects of the truncation of the N-terminal portions on the structure and aggregation properties of wild-type transthyretin. The deletion mutant lacking 50 N-terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self-assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N-terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self-assembly into aggregations in SSA.     

Chromium(III) ion and thyroxine cooperate to stabilize the transthyretin tetramer and suppress in vitro amyloid fibril formation

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T(4)), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T(4) for TTR binding is limited due to the preferential binding of T(4) to globulin, the major T(4) carrier in plasma. Here, we show that Cr(3+) increased the T(4)-binding capacity of wild-type (WT) and amyloidogenic V30M-TTR. Moreover, we demonstrate that Cr(3+) and T(4) cooperatively suppressed in vitro fibril formation due to the stabilization of WT-TTR and V30M-TTR.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Mutations in the B1 domain of protein G that delay the onset of amyloid fibril formation in vitro

We previously reported that under certain experimental conditions, many variants of the B1 domain of IgG-binding protein G from Streptococcus form fibrils reproducibly. The variant I6T53 was the focus of the present study because the lag phase in the kinetics of fibril formation by this variant is significantly longer than that of other variants. This lag phase is distinguished by changes in both intrinsic fluorescence intensity and in light scattering of the protein. NMR diffusion measurements suggest that the soluble protein during the lag phase is monomeric. The kinetic profiles of fibril formation are found to depend on experimental conditions. The first kinetic phase diminishes almost completely when the reaction is seeded with preformed amyloid fibrils.     

Glyceraldehyde-3-phosphate dehydrogenase tetramer dissociation and amyloid fibril formation induced by negatively charged membranes

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme related with Huntington’s, Parkinson’s and Alzheimer’s diseases. The ability of negatively charged membranes to induce a rapid formation of GAPDH amyloid fibrils has been demonstrated, but the mechanisms by which GAPDH reaches the fibrillar state remains unclear. In this report, we describe the structural changes undergone by GAPDH at physiological pH and temperature conditions right from its interaction with acidic membranes until the amyloid fibril is formed. According to our results, the GAPDH-membrane binding induces a β-structuring process along with a loss of quaternary structure in the enzyme. In this way, experimental evidences on the initial steps of GAPDH amyloid fibrils formation pathway are provided.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.