Stabilization of nontoxic Ajβ-oligomers: Insights into the mechanism of action of hydroxyquinolines in alzheimer’s disease

©2015 the authors. The extracellular accumulation of amyloid β (A/β) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aβ, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from A/β:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aβ fibrillar polymerization and direct depolymerization of existing Aβ fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aβ and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aβ associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aβ with an affinity of 1-10 μam and suppress the formation of large (>30kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aβ toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aβ oligomer formation through stabilization of small (dimeric) nontoxic Aβ conformers

Structure of the endocytic adaptor complex reveals the basis for efficient membrane anchoring during clatharin-mediated endocytosis

During clathrin-mediated endocytosis, a complex and dynamic network of protein-membrane interactions cooperate to achieve membrane invagination. Throughout this process in yeast, endocytic coat adaptors, Sla2 and Ent1, must remain attached to the plasma membrane to transmit force from the actin cytoskeleton required for successful membrane invagination. Here, we present a cryo-EM structure of a 16-mer complex of the ANTH and ENTH membrane-binding domains from Sla2 and Ent1 bound to PIP2 that constitutes the anchor to the plasma membrane. Detailed in vitro and in vivo mutagenesis of the complex interfaces delineate the key interactions for complex formation and deficient cell growth phenotypes demonstrate its biological relevance. A hetero-tetrameric unit binds PIP2 molecules at the ANTH-ENTH interfaces and can form larger assemblies to contribute to membrane remodeling. Finally, a time-resolved small-angle X-ray scattering study of the interaction of these adaptor domains in vitro suggests that ANTH and ENTH domains have evolved to achieve a fast subsecond timescale assembly in the presence of PIP2 and do not require further proteins to form a stable complex. Together, these findings provide a molecular understanding of an essential piece in the molecular puzzle of clathrin-coated endocytic sites

Small angle X-ray scattering analysis of Cu2+-induced oligomers of the Alzheimer's amyloid beta peptide

Research into causes of Alzheimer's disease and its treatment has produced a tantalising array of hypotheses about the role of transition metal dyshomeostasis, many of them on the interaction of these metals with the neurotoxic amyloid-β peptide (Aβ). Here, we have used small angle X-ray scattering (SAXS) to study the effect of the molar ratio, Cu(2+)/Aβ, on the early three-dimensional structures of the Aβ1-40 and Cu(2+)/Aβ1-42 peptides in solution. We found that at molar ratios of 0.5 copper to peptide Aβ1-40 aggregated, while Aβ1-42 adopted a relatively monodisperse cylindrical shape, and at a ratio of 1.5 copper to peptide Aβ1-40 adopted a monodisperse cylindrical shape, while Aβ1-42 adopted the shape of an ellipsoid of rotation. We also found, via in-line rapid mixing SAXS analysis, that both peptides in the absence of copper were monodisperse at very short timeframes (<2 s). Kratky plots of these scattering profiles indicated that immediately after mixing both were intrinsically disordered. Ensemble optimisation modelling reflected this, indicating a wide range of structural conformers. These data reflect the ensembles from which the Cu(2+)-promoted oligomers were derived. Our results are discussed in the light of other studies that have shown that the Cu(2+)/Aβ has a marked effect on fibril and oligomer formation by this peptide, with a higher ratio favouring the formation of cytotoxic non-amyloid oligomers. Our results are relatively consistent with previous two-dimensional studies of the conformations of these Cu(2+)-induced entities, made on a much longer time-scale than SAXS, by transmission electron microscopy and atomic force microscopy, which showed that a range of oligomeric species are formed. We propose that SAXS carried out on a modern synchrotron beamline enables studies on initial events in disordered protein folding on physiologically-relevant time-scales, and will likely provide great insight into the initiating processes of the Aβ misfolding, oligomerisation and amyloid formation

Δεύτερη εθνική έκθεση για την εφαρμογή της Οδηγίας 92/43 ΕΟΚ στην Ελλάδα (περίοδος αναφοράς: 2001-2006): Χάρτης εξάπλωσης και εύρους εξάπλωσης του τύπου οικοτόπου 3150

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production

Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

The 140 residue intrinsically disordered protein α-synuclein (α-syn) self-associates to form fibrils that are the major constituent of the Lewy body intracellular protein inclusions, and neurotoxic oligomers. Both of these macromolecular structures are associated with a number of neurodegenerative diseases, including Parkinson's disease and dementia with Lewy bodies. Using ensemble optimisation modelling (EOM) and small angle X-ray scattering (SAXS) on a size-exclusion column equipped beamline, we studied how the distribution of structural conformers in α-syn may be influenced by the presence of the familial early-onset mutations A30P, E45K and A53T, by substituting the four methionine residues with alanines and by reaction with copper (Cu2+) or an anti-fibril organic platinum (Pt) complex. We found that the WT had two major conformer groups, representing ensembles of compact and extended structures. The population of the extended group was increased in the more rapidly fibril-forming E45K and A53T mutants, while the compact group was enlarged in the oligomer-forming A30P mutant. Addition of Cu2+ resulted in the formation of an ensemble of compact conformers, while the anti-fibril agent and alanine substitution substantially reduced the population of extended conformers. Since our observations with the mutants suggest that fibrils may be drawn from the extended conformer ensemble, we propose that the compact and extended ensembles represent the beginning of oligomer and fibril formation pathways respectively, both of which have been reported to lead to a toxic gain of function. Manipulating these pathways and monitoring the results by EOM and SAXS may be useful in the development of anti-Parkinson's disease therapies

Ammonium hydroxide treatment of A beta produces an aggregate free solution suitable for biophysical and cell culture characterization

Alzheimer's disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays

Characterisation of assembly and ubiquitylation by the RBCC motif of Trim5α

The post-entry restriction factor Trim5α blocks infection of retroviral pathogens shortly after the virus gains entry to the cell, preventing reverse transcription and integration into the host genome. Central to the mechanism of restriction is recognition of the lattice of capsid protein that forms the inner-shell of the retrovirus. To recognise this lattice, Trim5α has been shown to assemble into a large hexagonal array, complementary to the capsid lattice. Structures of the Trim5α coiled-coil region reveal an elongated anti-parallel dimer consistent with the edges of this array placing the Bbox domain at each end of the coiled-coil to facilitate assembly. To investigate the nature of this assembly we have designed and characterised a monomeric version of the TRIM RBCC motif with a truncated coiled-coil. Biophysical characterisation by SEC-MALLS, AUC, and SAXS demonstrate that this construct forms compact folded domain that assembles into a trimer that would support the formation of a hexagonal lattice. Furthermore, the RING domain and elements of the coiled-coil region are shown to contribute to assembly. Ubiquitylation assays demonstrate that this assembly increases ubiquitylation activity providing a link from recognition of the capsid lattice and assembly to the activation of innate immune signalling and restriction

Two-dimensional structure from random multiparticle X-ray scattering images using cross-correlations

Knowledge of the structure of biological macromolecules, especially in their native environment, is crucial because of the close structure–function relationship. X-ray small-angle scattering is used to determine the shape of particles in solution, but the achievable resolution is limited owing to averaging over particle orientations. In 1977, Kam proposed to obtain additional structural information from the cross-correlation of the scattering intensities. Here we develop the method in two dimensions, and give a procedure by which the single-particle diffraction pattern is extracted in a model-independent way from the correlations. We demonstrate its application to a large set of synchrotron X-ray scattering images on ensembles of identical, randomly oriented particles of 350 or 200 nm in size. The obtained 15 nm resolution in the reconstructed shape is independent of the number of scatterers. The results are discussed in view of proposed ‘snapshot’ scattering by molecules in the liquid phase at X-ray free-electron lasers