Β-sheet Oligomers Research Articles

E6*, the 50 Amino Acid Product of the Most Abundant Spliced Transcript of the E6 Oncoprotein in High-Risk Human Papillomavirus, Is a Promiscuous Folder and Binder

High-risk human papillomavirus E6 participates in tumorigenic progression, mainly by its ability to promote p53 degradation. HPV transcripts show a complex splicing pattern, where E6* is the most abundant transcript in high-risk HPV types, comprising the first 50 amino acids of E6. No structural or biochemical information of this polypeptide, which contains half of the first zinc binding motif of E6, is available, due to the difficulty to acquire a compact monomeric fold in such a small polypeptide. We show that HPV16-E6* can fold into either α-helix or β-sheet large oligomers at pH 7.5 and 5.0, respectively, in the absence of zinc. The β-sheet oligomers are highly stable and unaffected by the presence of zinc, while the α-helix oligomers tend to rapidly form aggregates, prevented by the presence of the metal. Two E6* molecules bind per atom of zinc, suggesting a tetrahedral, high-affinity arrangement (K(D) < 10(-12) M), which results in a zinc-mediated E6* dimer with significant secondary structure. Endogenous E6 oligomers were previously found in the cytosol of high-risk HPV transformed cell lines, and we propose that the oligomerization determinant resides within E6*. E6* effects were reported to counteract those of E6 in cells, and the ratio between these two species modulates p53 degradation and other apoptosis-dependent signaling cascades. A residue of an evolved splicing event related to regulation of oncogene expression in HPV or a splicing event resulting from the selection of a small deleterious viral polypeptide, the abundant existence of E6* with a "chameleon" nature correlates with target plasticity, and its fate is linked to a balance between protein levels, zinc availability, redox potential, and oligomerization. In addition, the results presented here have strong implications for zinc binding sites in nascent polypeptides. This evolved promiscuous folder speaks of effect rather than function of a viral product that, when highly increased, can directly or indirectly affect various cellular processes leading to cell deregulation and tumorigenesis.

Ion mobility–mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation

Amyloid cascades leading to peptide β-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with the cellular machinery. The location and cause of the transformation from natively unstructured assembly to the beta-sheet oligomers found in all fibrils is important in understanding disease onset and the development of therapeutic agents. Research on this early oligomeric region has largely been unsuccessful since all traditional techniques measure only ensemble average oligomer properties. Here, ion mobility methods are utilized to deduce the peptide self-assembly mechanism. We look at a series of amyloid forming peptides clipped from larger peptides or proteins associated with disease. We provide unambiguous evidence for structural transitions in each of these fibril forming peptide systems establishing the potential of this method for the development of therapeutic agents and drug evaluation.

Macromolecular crowding converts the human recombinant PrPcto the soluble neurotoxic β‐oligomers

Prion diseases are fatal neurodegenerative disorders and are linked with the conversion of the cellular isoform of the prion protein (PrP(C)) into the abnormal beta-sheet-rich isoform. It is widely accepted that the soluble oligomers of beta-PrP are neurotoxic and that they are more pathologically significant. To unravel the molecular mechanism under the conversion process, it is critical to identify the factors that can promote the conversion from PrP(C) to the beta-oligomers. By recording circular dichroism spectra and performing a size-exclusion HPLC assay, we found that the conformation of the recombinant human prion protein (rPrP(C)) was converted from an alpha-helical conformation into beta-sheet oligomers under a macromolecular crowding condition. The soluble beta-oligomers of rPrP were resistant to proteinase K digestion and could bind to the dyes thioflavin T and 8-anilino-1-naphthalene sulfonate. Furthermore, by the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, we showed that the soluble beta-oligomers were neurotoxic. These results suggest that macromolecular crowding, which has not been considered before, is a key intracellular factor in the formation of soluble neurotoxic beta-oligomers in prion diseases.

Suppression of IAPP fibrillation at anionic lipid membranes via IAPP-derived amyloid inhibitors and insulin

Aggregation of human islet amyloid polypeptide (hIAPP) into cytotoxic β-sheet oligomers and amyloid plaques is considered a key event in pancreatic β-cell degeneration in type 2 diabetes (T2D). hIAPP is synthesized in the pancreatic β-cells and it is stored, co-processed in the secretory granules, and co-secreted to the extracellular matrix together with insulin. In vivo, hIAPP aggregation may start and proceed at the water-cell membrane interface and anionic lipid membranes strongly enhance the process of hIAPP fibrillization which is causally linked to membrane disintegration and cell degeneration. In this study we explored the amyloidogenic propensity and conformational properties of hIAPP in the presence of negatively charged membrane (DOPC/DOPG phospholipid bilayers) surfaces upon addition of two recently designed potent hIAPP-derived inhibitors of hIAPP amyloidogenesis, the hexapeptide NF(N-Me)GA(N-Me)IL (NFGAIL-GI) and the 37-residue non-amyloidogenic hIAPP analog [(N-Me)G24, (N-Me)I26]-IAPP (IAPP-GI). For comparison, the effects of insulin, which is a natively occurring hIAPP aggregation inhibitor, rat IAPP (rIAPP), which is a natively non-amyloidogenic hIAPP analog, and the hIAPP amyloid core peptide hIAPP(22-27) or NFGAIL were also studied. The aim of our study was to test whether and how the above peptides which have been shown to completely block or suppress hIAPP amyloidogenesis in bulk solution in vitro would also affect these processes in the presence of lipid membranes. To this end, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was applied. We find that IAPP-GI, NFGAIL-GI, insulin, and rIAPP are potent inhibitors of hIAPP fibrillization. Importantly, our data also suggest that the hetero-complexes of IAPP-GI, rIAPP, and insulin with hIAPP although non-amyloidogenic per se are still able to adsorb at the lipid membrane. By contrast, in the presence of NFGAIL-GI, interaction of hIAPP with the lipid membrane is completely abolished, consistent with NFGAIL-GI mediated sequestration of hIAPP via hetero-complexation in the aqueous phase mainly accounting for the observed strong effect of NFGAIL-GI on hIAPP fibrillogenesis at the lipid membrane interface. Finally, our studies show that once hIAPP is fibrillized at the water-lipid membrane interface with fibrils being attached to the lipid membrane, it cannot be disaggregated by all above peptides.

Membrane charge dependent states of the β-amyloid fragment Aβ (16–35) with differently charged micelle aggregates

A β (16–35) is the hydrophobic central core of β-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, β-amyloid peptides undergo a conformational transition from random coil or α-helical monomers, to highly toxic β-sheet oligomers and aggregate fibrils. The behavior of β-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of A β (16–35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles “doped” with small amounts of SDS. To analyze the tendency of A β (16–35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of A β (16–35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, A β (16–35) undergoes a conformational transition from a soluble helical–kink–helical structure, to a U-turn shaped conformation that resembles protofibril models.

Association Thermodynamics and Conformational Stability of β-Sheet Amyloid β(17-42) Oligomers: Effects of E22Q (Dutch) Mutation and Charge Neutralization

Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type A β 17–42 oligomers with different protonation states of Glu 22, as well the E22Q (Dutch) mutants. The association free energy of small β-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the β-sheet oligomers. The charge reduction of the wild-type A β 17–42 oligomers upon protonation of the solvent-exposed Glu 22 at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type A β 17–42 oligomers at neutral pH and the Dutch mutants, on the one hand, and the A β 17–42 oligomers with protonated Glu 22, on the other, may be explained by destabilization of the inter- and intrapeptide salt bridges between Asp 23 and Lys 28. Peculiarities in the conformational stability and the association thermodynamics for the different models of the A β 17–42 oligomers are rationalized based on the analysis of the local physical interactions and the microscopic solvation structure.

Amyloidogenesis abolished by proline substitutions but enhanced by lipid binding.

The influence of lipid molecules on the aggregation of a highly amyloidogenic segment of human islet amyloid polypeptide, hIAPP20–29, and the corresponding sequence from rat has been studied by all-atom replica exchange molecular dynamics (REMD) simulations with explicit solvent model. hIAPP20–29 fragments aggregate into partially ordered β-sheet oligomers and then undergo large conformational reorganization and convert into parallel/antiparallel β-sheet oligomers in mixed in-register and out-of-register patterns. The hydrophobic interaction between lipid tails and residues at positions 23–25 is found to stabilize the ordered β-sheet structure, indicating a catalysis role of lipid molecules in hIAPP20–29 self-assembly. The rat IAPP variants with three proline residues maintain unstructured micelle-like oligomers, which is consistent with non-amyloidogenic behavior observed in experimental studies. Our study provides the atomic resolution descriptions of the catalytic function of lipid molecules on the aggregation of IAPP peptides.

A Molecular Dynamics Study of the Interaction of d-Peptide Amyloid Inhibitors with Their Target Sequence Reveals a Potential Inhibitory Pharmacophore Conformation

The self-assembly of soluble proteins and peptides into β-sheet-rich oligomeric structures and insoluble fibrils is a hallmark of a large number of human diseases known as amyloid diseases. Drugs that are able to interfere with these processes may be able to prevent and/or cure these diseases. Experimental difficulties in the characterization of the intermediates involved in the amyloid formation process have seriously hampered the application of rational drug design approaches to the inhibition of amyloid formation and growth. Recently, short model peptide systems have proved useful in understanding the relationship between amino acid sequence and amyloid formation using both experimental and theoretical approaches. Moreover, short d-peptide sequences have been shown to specifically interfere with those short amyloid stretches in proteins, blocking oligomer formation or disassembling mature fibrils. With the aim of rationalizing which interactions drive the binding of inhibitors to nascent β-sheet oligomers, in this study, we have carried out extensive molecular dynamics simulations of the interaction of selected d-peptide sequences with oligomers of the target model sequence STVIIE. Structural analysis of the simulations helped to identify the molecular determinants of an inhibitory core whose conformational and physicochemical properties are actually shared by nonpeptidic small-molecule inhibitors of amyloidogenesis. Selection of one of these small molecules and experimental validation against our model system proved that it was indeed an effective inhibitor of fibril formation by the STVIIE sequence, supporting theoretical predictions. We propose that the inhibitory determinants derived from this work be used as structural templates in the development of pharmacophore models for the identification of novel nonpeptidic inhibitors of aggregation.

Stabilization of Neurotoxic Soluble β-Sheet-Rich Conformations of the Alzheimer's Disease Amyloid-β Peptide

An emerging paradigm for degenerative diseases associated with protein misfolding, such as Alzheimer's disease, is the formation of a toxic species due to structural transitions accompanied by oligomerization. Increasingly, the focus in Alzheimer's disease is on soluble oligomeric forms of the amyloid-β peptide (Aβ) as the potential toxic species. Using a variety of methods, we have analyzed how sodium dodecyl sulphate (SDS) modulates the folding of Aβ40 and 42 and found that submicellar concentrations of SDS solubilize Aβ and induce structural transitions. Under these conditions, Aβ40 and 42 are interconverting oligomeric ensembles with a predominantly β-sheet structure. The Aβ42 soluble oligomers form β-sheet structures more readily and have increased stability compared with Aβ40 under identical conditions. The presence of added Cu2+ significantly promotes and stabilizes the formation of the soluble oligomeric β-sheet structures but these structures are nonamyloidogenic. In contrast, in the absence of added Cu2+, these β-sheet oligomers possess the hallmarks of amyloidogenic structures. These SDS-induced β-sheet forms of Aβ, both in the presence and absence of Cu2+, are toxic to neuronal cells.

Conversion of non-fibrillar β-sheet oligomers into amyloid fibrils in Alzheimer’s disease amyloid peptide aggregation

Aβ(1–40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer’s disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular β-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Aβ(1–40) fibril formation process. A unique sample containing 90 μM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar β-structures. The number of oligomers and the amount of non-fibrillar β-structures grows throughout the lag phase and during the elongation phase these non-fibrillar β-structures are transformed into fibrillar (amyloid) β-structures, formed by association of high molecular weight intermediates.

Amyloid Fibrils of Mammalian Prion Protein Are Highly Toxic to Cultured Cells and Primary Neurons

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.

Human amyloid-derived aßcontains tyrosine cross-linked oligomers

Amyloid fibrils are associated with many neurodegenerative diseases. It was found that amyloidogenic oligomers, not mature fibrils, are neurotoxic agents related to these diseases. Molecular mechanisms of infectivity, pathways of aggregation, and molecular structure of these oligomers remain elusive. Here, we use all-atom molecular dynamics, molecular mechanics combined with solvation analysis by statistical-mechanical, three-dimensional molecular theory of solvation (also known as 3D-RISM-KH) in a new MM-3D-RISM-KH method to study conformational stability, and association thermodynamics of small wild-type Aβ17–42 oligomers with different protonation states of Glu22, as well the E22Q (Dutch) mutants. The association free energy of small β-sheet oligomers shows near-linear trend with the dimers being thermodynamically more stable relative to the larger constructs. The linear (within statistical uncertainty) dependence of the association free energy on complex size is a consequence of the unilateral stacking of monomers in the β-sheet oligomers. The charge reduction of the wild-type Aβ17–42 oligomers upon protonation of the solvent-exposed Glu22 at acidic conditions results in lowering the association free energy compared to the wild-type oligomers at neutral pH and the E22Q mutants. The neutralization of the peptides because of the E22Q mutation only marginally affects the association free energy, with the reduction of the direct electrostatic interactions mostly compensated by the unfavorable electrostatic solvation effects. For the wild-type oligomers at acidic conditions such compensation is not complete, and the electrostatic interactions, along with the gas-phase nonpolar energetic and the overall entropic effects, contribute to the lowering of the association free energy. The differences in the association thermodynamics between the wild-type Aβ17–42 oligomers at neutral pH and the Dutch mutants, on the one hand, and the Aβ17–42 oligomers with protonated Glu22, on the other, may be explained by destabilization of the inter- and intrapeptide salt bridges between Asp23 and Lys28. Peculiarities in the conformational stability and the association thermodynamics for the different models of the Aβ17–42 oligomers are rationalized based on the analysis of the local physical interactions and the microscopic solvation structure.