Solvent exposure of Tyr10 as a probe of structural differences between monomeric and aggregated forms of the amyloid-β peptide.

Aggregation of amyloid-β (Aβ) peptides is a characteristic pathological feature of Alzheimer's disease. We have exploited the relationship between solvent exposure and intrinsic fluorescence of a single tyrosine residue, Tyr10, in the Aβ sequence to probe structural features of the monomeric, oligomeric and fibrillar forms of the 42-residue Aβ1-42. By monitoring the quenching of Tyr10 fluorescence upon addition of water-soluble acrylamide, we show that in Aβ1-42 oligomers this residue is solvent-exposed to a similar extent to that found in the unfolded monomer. By contrast, Tyr10 is significantly shielded from acrylamide quenching in Aβ1-42 fibrils, consistent with its proximity to the fibrillar cross-β core. Furthermore, circular dichroism measurements reveal that Aβ1-42 oligomers have a considerably lower β-sheet content than the Aβ1-42 fibrils, indicative of a less ordered molecular arrangement in the former. Taken together these findings suggest significant differences in the structural assembly of oligomers and fibrils that are consistent with differences in their biological effects.This work was funded by grants to E.K.E from the Wenner-Gren Foundations, the Hasselblad Foundation, and the Swedish Innovation Agency (Vinnova) and to C.M.D from the Wellcome Trust. The TEM imaging was carried out in the Multi-Imaging Unit in the Department of Physiology, Development and Neuroscience, University of Cambridge, UK and quantitative amino acid analysis was carried out at the Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge, UK.This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.bbrc.2015.11.01

Exogenous misfolded protein oligomers can cross the intestinal barrier and cause a disease phenotype in C. elegans.

Misfolded protein oligomers are increasingly recognized as highly cytotoxic agents in a wide range of human disorders associated with protein aggregation. In this study, we assessed the possible uptake and resulting toxic effects of model protein oligomers administered to C. elegans through the culture medium. We used an automated machine-vision, high-throughput screening procedure to monitor the phenotypic changes in the worms, in combination with confocal microscopy to monitor the diffusion of the oligomers, and oxidative stress assays to detect their toxic effects. Our results suggest that the oligomers can diffuse from the intestinal lumen to other tissues, resulting in a disease phenotype. We also observed that pre-incubation of the oligomers with a molecular chaperone (αB-crystallin) or a small molecule inhibitor of protein aggregation (squalamine), reduced the oligomer absorption. These results indicate that exogenous misfolded protein oligomers can be taken up by the worms from their environment and spread across tissues, giving rise to pathological effects in regions distant from their place of absorbance

Structural Characterization of Covalently Stabilized Human Cystatin C Oligomers

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10−12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease

Rapid Structural, Kinetic, and Immunochemical Analysis of Alpha-Synuclein Oligomers in Solution.

Oligomers comprised of misfolded proteins are implicated as neurotoxins in the pathogenesis of protein misfolding conditions such as Parkinson's and Alzheimer's diseases. Structural, biophysical, and biochemical characterization of these nanoscale protein assemblies is key to understanding their pathology and the design of therapeutic interventions, yet it is challenging due to their heterogeneous, transient nature and low relative abundance in complex mixtures. Here, we demonstrate separation of heterogeneous populations of oligomeric α-synuclein, a protein central to the pathology of Parkinson's disease, in solution using microfluidic free-flow electrophoresis. We characterize nanoscale structural heterogeneity of transient oligomers on a time scale of seconds, at least 2 orders of magnitude faster than conventional techniques. Furthermore, we utilize our platform to analyze oligomer ζ-potential and probe the immunochemistry of wild-type α-synuclein oligomers. Our findings contribute to an improved characterization of α-synuclein oligomers and demonstrate the application of microchip electrophoresis for the free-solution analysis of biological nanoparticle analytes

Using Tetracysteine-Tagged TDP-43 with a Biarsenical Dye To Monitor Real-Time Trafficking in a Cell Model of Amyotrophic Lateral Sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol

Engineering mono- and multi-valent inhibitors on a modular scaffold.

Here we exploit the simple, ultra-stable, modular architecture of consensus-designed tetratricopeptide repeat proteins (CTPRs) to create a platform capable of displaying both single as well as multiple functions and with diverse programmable geometrical arrangements by grafting non-helical short linear binding motifs (SLiMs) onto the loops between adjacent repeats. As proof of concept, we built synthetic CTPRs to bind and inhibit the human tankyrase proteins (hTNKS), which play a key role in Wnt signaling and are upregulated in cancer. A series of mono-valent and multi-valent hTNKS binders was assembled. To fully exploit the modular scaffold and to further diversify the multi-valent geometry, we engineered the binding modules with two different formats, one monomeric and the other trimeric. We show that the designed proteins are stable, correctly folded and capable of binding to and inhibiting the cellular activity of hTNKS leading to downregulation of the Wnt pathway. Multivalency in both the CTPR protein arrays and the hTNKS target results in the formation of large macromolecular assemblies, which can be visualized both in vitro and in the cell. When delivered into the cell by nanoparticle encapsulation, the multivalent CTPR proteins displayed exceptional activity. They are able to inhibit Wnt signaling where small molecule inhibitors have failed to date. Our results point to the tremendous potential of the CTPR platform to exploit a range of SLiMs and assemble synthetic binding molecules with built-in multivalent capabilities and precise, pre-programmed geometries.BBSRC Doctoral Training Programme (DTP) scholarship Oliver Gatty Studentship AstraZeneca PhD studentship. UK Medical Research Foundation. CRUK Pioneer Award (C17838/A22676) CRUK BTERP Award (C17838/A27225) Leverhulme Trust (RPG-2014-089) Cambridge Newton Trust BBSRC project grant (BB/T002697/1)

Evidence for Direct CP Violation in B±→ηh±B^\pm \to \eta h^\pm and Observation of B0→ηK0B^0 \to \eta K^0

We report measurements of the branching fractions and CP asymmetries for B^+- -> eta h^+- (h = K or pi) and the observation of the decay B^0 -> eta K^0 from the final data sample of 772x10^6 BBbar pairs collected with the Belle detector at the KEKB asymmetric-energy e^+e^- collider. The measured branching fractions are Br(B^+- -> eta K^+-) = (2.12 +- 0.23 +- 0.11)x10^-6}, Br(B^+- -> eta pi^+-) = (4.07 +- 0.26 +- 0.21)x10^{-6} and Br(B^0 -> eta K^0) = (1.27^{+0.33}_{-0.29} +- 0.08)x10^-6, where the last decay is observed for the first time with a significance of 5.4 standard deviations (\sigma). We also find evidence for CP violation in the charged B modes, A_{CP}(B^+- -> eta K^+-) = -0.38 +- 0.11 +- 0.01 and A_{CP}(B^+- -> eta pi^+- = -0.19 +- 0.06 +- 0.01 with significances of 3.8 sigma and 3.0 sigma, respectively. For all measurements, the first and second uncertainties are statistical and systematic, respectively.Comment: 6 pages, 3 figures, 1 tabl

Measurement of CP asymmetries in B0→K0π0B^0 \to K^0\pi^0 decays

We report measurements of CP violation parameters in $B^0 \to K^0 \pi^0$ decays based on a data sample of $657 \times 10^6 B\bar{B}$ pairs collected with the Belle detector at the KEKB $e^+ e^-$ asymmetric-energy collider. We use $B^0 \to K^0_S \pi^0$ decays for both mixing-induced and direct $CP$ violating asymmetry measurements and $B^0 \to K^0_L \pi^0$ decays for the direct CP violation measurement. The CP violation parameters obtained are $\sin 2 \phi_1^{\rm eff} = +0.67 \pm 0.31{(stat)} \pm 0.08 {(syst)}$ and $\mathcal{A}_{K^0 \pi^0} = +0.14 \pm 0.13{(stat)} \pm 0.06 {(syst)}$. The branching fraction of $B^0 \to K^0 \pi^0$ decay is measured to be $\mathcal{B}(B^0 \to K^0 \pi^0) = (8.7\pm0.5 (\rm{stat.})\pm0.6 (\rm{syst.}))\times 10^{-6}$. The observed $\mathcal{A}_{K^0 \pi^0}$ value differs by 1.9 standard deviations from the value expected from an isospin sum rule.Comment: 6 pages, 3 figure

Search for CP Violation in D Meson Decays to phi pi+

We search for CP violation in Cabibbo-suppressed charged D meson decays by measuring the difference between the CP violating asymmetries for the Cabibbo-suppressed decays D+ -> K+K-pi+ and the Cabibbo-favored decays Ds -> K+K-pi+ in the K+K- mass region of the phi resonance. Using 955/fb of data collected with the Belle detector we obtain A_CP(D+ -> phi pi+) = (+0.51 +- 0.28 +- 0.05)%. The measurement improves the sensitivity of previous searches by more than a factor of five. We find no evidence for direct CP violation.Comment: submitted to PR

Invariant-mass and fractional-energy dependence of inclusive production of di-hadrons in e+e−e^+e^- annihilation at s=\sqrt{s}= 10.58 GeV

The inclusive cross sections for di-hadrons of charged pions and kaons ($e^+e^- \rightarrow hhX$) in electron-positron annihilation are reported. They are obtained as a function of the total fractional energy and invariant mass for any di-hadron combination in the same hemisphere as defined by the thrust event-shape variable and its axis. Since same-hemisphere di-hadrons can be assumed to originate predominantly from the same initial parton, di-hadron fragmentation functions are probed. These di-hadron fragmentation functions are needed as an unpolarized baseline in order to quantitatively understand related spin-dependent measurements in other processes and to apply them to the extraction of quark transversity distribution functions in the nucleon. The di-hadron cross sections are obtained from a $655\,{\rm fb}^{-1}$ data sample collected at or near the $\Upsilon(4S)$ resonance with the Belle detector at the KEKB asymmetric-energy $e^+ e^-$ collider.Comment: 21 pages, 18 figures plus 25 figures in supplemental material, submitted to PR