Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool

Huntington disease is caused by expanded polyglutamine sequences in huntingtin, which procures its aggregation into intracellular inclusion bodies (IBs). Aggregate intermediates, such as soluble oligomers, are predicted to be toxic to cells, yet because of a lack of quantitative methods, the kinetics of aggregation in cells remains poorly understood. We used sedimentation velocity analysis to define and compare the heterogeneity and flux of purified huntingtin with huntingtin expressed in mammalian cells under non-denaturing conditions. Non-pathogenic huntingtin remained as hydrodynamically elongated monomers in vitro and in cells. Purified polyglutamine-expanded pathogenic huntingtin formed elongated monomers (2.4 S) that evolved into a heterogeneous aggregate population of increasing size over time (100–6,000 S). However, in cells, mutant huntingtin formed three major populations: monomers (2.3 S), oligomers (mode s_20,w = 140 S) and IBs (mode s_20,w = 320,000 S). Strikingly, the oligomers did not change in size heterogeneity or in their proportion of total huntingtin over 3 days despite continued monomer conversion to IBs, suggesting that oligomers are rate-limiting intermediates to IB formation. We also determined how a chaperone known to modulate huntingtin toxicity, Hsc70, influences in-cell huntingtin partitioning. Hsc70 decreased the pool of 140 S oligomers but increased the overall flux of monomers to IBs, suggesting that Hsc70 reduces toxicity by facilitating transfer of oligomers into IBs. Together, our data suggest that huntingtin aggregation is streamlined in cells and is consistent with the 140 S oligomers, which remain invariant over time, as a constant source of toxicity to cells irrespective of total load of insoluble aggregates.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

Two different binding modes of α-synuclein to lipid vesicles depending on its aggregation state

Aggregation of α-synuclein is involved in the pathogenesis of Parkinson's disease (PD). Studies of in vitro aggregation of α-synuclein are rendered complex because of the formation of a heterogeneous population of oligomers. With the use of confocal single-molecule fluorescence techniques, we demonstrate that small aggregates (oligomers) of α-synuclein formed from unbound monomeric species in the presence of organic solvent (DMSO) and iron (Fe³⁺) ions have a high affinity to bind to model membranes, regardless of the lipid-composition or membrane curvature. This binding mode contrasts with the well-established membrane binding of α-synuclein monomers, which is accompanied with α-helix formation and requires membranes with high curvature, defects in the lipid packing, and/or negatively charged lipids. Additionally, we demonstrate that membrane-bound α-synuclein monomers are protected from aggregation. Finally, we identified compounds that potently dissolved vesicle-bound α-synuclein oligomers into monomers, leaving the lipid vesicles intact. As it is commonly believed that formation of oligomers is related PD progression, such compounds may provide a promising strategy for the design of novel therapeutic drugs in Parkinson's disease.     

Elucidating the Aggregation Number of Dopamine-Induced α-Synuclein Oligomeric Assemblies

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson’s disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15–19 monomers and 34–38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.     

Pentameric ellagitannin oligomers in melastomataceous plants--chemotaxonomic significance

The pantropical plant family Melastomataceae produces characteristic hydrolyzable tannin oligomers. The latter in this family are distinguished from those in other plant families by the fact that the oligomers from dimers to tetramers are composed of two different alternating monomeric units: casuarictin and pterocaryanin C. These oligomers are metabolites that are produced by intermolecular C-O oxidative coupling between the monomers (or their desgalloyl-or des-hexahydroxydiphenoyl derivatives) forming a valoneoyl group as the link between monomers. The chemotaxonomically significant oligomerization pattern of melastomataceous plants provided helpful suggestions for determining the structures of new oligomers (nobotanins Q-T and melastoflorins A-D) isolated from Monochaetum multiflorum, which belongs to this family. Melastoflorins A-D were characterized as pentamers, which are the largest hydrolyzable tannins composed of different monomeric units.     

Probing the nucleus model for oligomer formation during insulin amyloid fibrillogenesis

We find evidence for a direct transition of insulin monomers into amyloid fibrils without measurable concentrations of oligomers or protofibrils, suggesting that fibrillogenesis may occur directly from assembly of denaturing insulin monomers rather than by successive transitions through protofibril nuclei. To support our finding, we obtain size distributions using electrospray differential mobility analysis (ES-DMA), which provides excellent resolution to clearly distinguish among small oligomers and rapidly generates statistically significant size distributions. The distributions detect an absence of significant peaks between 6 nm and 17 nm as the monomer reacts into fibers—exactly the size range observed by others for small-angle-neutron-scattering-measured intermediates and for circular supramolecular structures. They report concentrations in the nanomolar range, whereas our limit of detection remains three-orders-of-magnitude lower (<5 pmol/L). This finding, along with the lack of significant increases in the β-sheet content of monomers using circular dichroism, suggests monomers do not first structurally rearrange and accumulate in a β-rich state but react and reorganize at the growing fiber's tip. These results quantitatively inform reaction-based theories of amyloid fiber formation and have implications for neurodegenerative, protein conformation ailments including Alzheimer's disease and bovine spongiform encephalopathy.     

Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway

Although actin filaments can form by oligomer annealing in vitro, they are assumed to assemble exclusively from actin monomers in vivo. In this study, we show that a pool of actin resistant to the monomer-sequestering drug latrunculin A (lat A) contributes to filament assembly in vivo. Furthermore, we show that the cofilin accessory protein Aip1 is important for establishment of normal actin monomer concentration in cells and efficiently converts cofilin-generated actin filament disassembly products into monomers and short oligomers in vitro. Additionally, in aip1Δ mutant cells, lat A–insensitive actin assembly is significantly enhanced. We conclude that actin oligomer annealing is a physiologically relevant actin filament assembly pathway in vivo and identify Aip1 as a crucial factor for shifting the distribution of short actin oligomers toward monomers during disassembly.     

Early Aggregation Steps in α-Synuclein as Measured by FCS and FRET: Evidence for a Contagious Conformational Change

The kinetics of aggregation of α-synuclein are usually studied by turbidity or Thio-T fluorescence. Here we follow the disappearance of monomers and the formation of early oligomers using fluorescence correlation spectroscopy. Alexa488-labeled A140C-synuclein was used as a fluorescent probe in trace amounts in the presence of excess unlabeled α-synuclein. Repeated short measurements produce a distribution of diffusion coefficients. Initially, a sharp peak is obtained corresponding to monomers, followed by a distinct transient population and the gradual formation of broader-sized distributions of higher oligomers. The kinetics of aggregation can be followed by the decreasing number of fast-diffusing species. Both the disappearance of fast-diffusing species and the appearance of turbidity can be fitted to the Finke-Watzky equation, but the apparent rate constants obtained are different. This reflects the fact that the disappearance of fast species occurs largely during the lag phase of turbidity development, due to the limited sensitivity of turbidity to the early aggregation process. The nucleation of the early oligomers is concentration-dependent and accompanied by a conformational change that precedes β-structure formation, and can be visualized using fluorescence resonance energy transfer between the donor-labeled N-terminus and the acceptor-labeled cysteine in the mutant A140C.     

Cloning,Expression, and Biochemical Characterization of Two New Oligoalginate Lyases with Synergistic Degradation Capability

Alginate, the most abundant carbohydrate presents in brown macroalgae, has recently gained increasing attention as an alternative biomass for the production of biofuel. Oligoalginate lyases catalyze the degradation of alginate oligomers into monomers, a prerequisite for bioethanol production. In this study, two new oligoalginate lyase genes, oalC6 and oalC17, were cloned from Cellulophaga sp. SY116, and expressed them in Escherichia coli. The deduced oligoalginate lyases, OalC6 and OalC17, belonged to the polysaccharide lyase (PL) family 6 and 17, respectively. Both showed less than 50% amino acid identity with all of the characterized oligoalginate lyases. Moreover, OalC6 and OalC17 could degrade both alginate polymers and oligomers into monomers in an exolytic mode. Substrate specificity studies demonstrated that OalC6 preferred α-L-guluronate (polyG) blocks, while OalC17 preferred poly β-D-mannuronate (polyM) blocks. The combination of OalC6 and OalC17 showed synergistic degradation ability toward both alginate polymers and oligomers. Finally, an efficient process for the production of alginate monomers was established by combining the new-isolated exotype alginate lyases (i.e., OalC6 and OalC17) and the endotype alginate lyase AlySY08. Overall, our work provides new insights for the development of novel biotechnologies for biofuel production from seaweed.     

Fibrillar Oligomers Nucleate the Oligomerization of Monomeric Amyloid �� but Do Not Seed Fibril Formation

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid β (Aβ) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 × g, rich in β-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Aβ FOs do not seed the formation of thioflavin T-positive fibrils from Aβ monomers but instead seed the formation of FOs from Aβ monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.     

Enhancement of transfecting activity of bacteriophage P22 DNA upon exonucleolytic erosion

The transfecting efficiency of P22 DNA on “rough” strains of Salmonella typhimurium or non-restricting mutants of Escherichia coli K12 approaches 3 × 10^?8 plaques/genome equivalent. It increases 20-fold upon complete erosion of the terminally redundant regions of the DNA molecule with either λ exonuclease or exonuclease III. Eroded DNA molecules form circles and linear oligomers upon annealing. The circular monomers display transfecting activity about ten times higher than that of eroded linear monomers or hydrogen-bonded oligomers. recB recC sbcB strains of E. coli K12 are transfected with P22 DNA with an efficiency of 1.5 × 10^?6 plaques/genome equivalent. The activity of DNA molecules on these strains is not augmented by erosion. This suggests that the activation by erosion, seen in assays on rec⁺ genotypes, is due to the formation of hydrogen-bonded circular molecules, which more readily escape degradation by the recBC nuclease.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

Enzymic cross-linkage of monomeric extensin precursors in vitro

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.     

Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly

The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.     

Probing the efficacy of peptide-based inhibitors against acid- and zinc-promoted oligomerization of amyloid-β peptide via single-oligomer spectroscopy

One avenue for prevention and treatment of Alzheimer's disease involves inhibiting the aggregation of amyloid-β peptide (Aβ). Given the deleterious effects reported for Aβ dimers and trimers, it is important to investigate inhibition of the earliest association steps. We have employed quantized photobleaching of dye-labeled Aβ peptides to characterize four peptide-based inhibitors of fibrillogenesis and/or cytotoxicity, assessing their ability to inhibit association in the smallest oligomers (n = 2-5). Inhibitors were tested at acidic pH and in the presence of zinc, conditions that may promote oligomerization in vivo. Distributions of peptide species were constructed by examining dozens of surface-tethered monomers and oligomers, one at a time. Results show that all four inhibitors shift the distribution of Aβ species toward monomers; however, efficacies vary for each compound and sample environment. Collectively, these studies highlight promising design strategies for future oligomerization inhibitors, affording insight into oligomer structures and inhibition mechanisms in two physiologically significant environments.     

Oligomerization of Optineurin and Its Oxidative Stress- or E50K Mutation-Driven Covalent Cross-Linking: Possible Relationship with Glaucoma Pathology

The optineurin gene, OPTN, is one of the causative genes of primary open-angle glaucoma. Although oligomerization of optineurin in cultured cells was previously observed by gel filtration analysis and blue native gel electrophoresis (BNE), little is known about the characteristics of optineurin oligomers. Here, we aimed to analyze the oligomeric state of optineurin and factors affecting oligomerization, such as environmental stimuli or mutations in OPTN. Using BNE or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we demonstrated that both endogenous and transfected optineurin exist as oligomers, rather than monomers, in NIH3T3 cells. We also applied an in situ proximity ligation assay to visualize the self-interaction of optineurin in fixed HeLaS3 cells and found that the optineurin oligomers were localized diffusely in the cytoplasm. Optineurin oligomers were usually detected as a single band of a size equal to that of the optineurin monomer upon SDS-PAGE, while an additional protein band of a larger size was observed when cells were treated with H₂O₂. We showed that larger protein complex is optineurin oligomers by immunoprecipitation and termed it covalent optineurin oligomers. In cells expressing OPTN bearing the most common glaucoma-associated mutation, E50K, covalent oligomers were formed even without H₂O₂ stimulation. Antioxidants inhibited the formation of E50K-induced covalent oligomers to various degrees. A series of truncated constructs of OPTN was used to reveal that covalent oligomers may be optineurin trimers and that the ubiquitin-binding domain is essential for formation of these trimers. Our results indicated that optineurin trimers may be the basic unit of these oligomers. The oligomeric state can be affected by many factors that induce covalent bonds, such as H₂O₂ or E50K, as demonstrated here; this provides novel insights into the pathogenicity of E50K. Furthermore, regulation of the oligomeric state should be studied in the future.     

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.     

Protein N-terminal analysis using fast atom bombardment mass spectrometry

An immunoassay is described that discriminates between monomers and oligomers of human leukocyte interferon. The assay in principle can be used to distinguish between monomers and oligomers of any substance.     

γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the stablest one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.