| 1. |
- Bahri, Salima, et al.
(författare)
-
1H detection and dynamic nuclear polarization–enhanced NMR of Aβ1-42 fibrils
- 2022
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 119:1
-
Tidskriftsartikel (refereegranskat)abstract
- Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aβ1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aβ1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.
|
|
| 2. |
- Buell, Alexander K., et al.
(författare)
-
Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation
- 2014
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:21, s. 7671-7676
-
Tidskriftsartikel (refereegranskat)abstract
- The formation of amyloid fibrils by the intrinsically disordered protein alpha-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that alpha-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of alpha-synuclein aggregation and aggregate spreading in the context of Parkinson disease.
|
|
| 3. |
- Cedervall, Tommy, et al.
(författare)
-
Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles
- 2007
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 104:7, s. 2050-2055
-
Tidskriftsartikel (refereegranskat)abstract
- Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.
|
|
| 4. |
- Chia, Sean, et al.
(författare)
-
Monomeric and fibrillar α-synuclein exert opposite effects on the catalytic cycle that promotes the proliferation of Aβ42 aggregates
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 114:30, s. 8005-8010
-
Tidskriftsartikel (refereegranskat)abstract
- The coaggregation of the amyloid-β peptide (Aβ) and α-synuclein is commonly observed in a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. The complex interplay between Aβ and α-synuclein has led to seemingly contradictory results on whether α-synuclein promotes or inhibits Aβ aggregation. Here, we show how these conflicts can be rationalized and resolved by demonstrating that different structural forms of α-synuclein exert different effects on Aβ aggregation. Our results demonstrate that whereas monomeric α-synuclein blocks the autocatalytic proliferation of Aβ42 (the 42-residue form of Aβ) fibrils, fibrillar α-synuclein catalyses the heterogeneous nucleation of Aβ42 aggregates. It is thus the specific balance between the concentrations of monomeric and fibrillar α-synuclein that determines the outcome of the Aβ42 aggregation reaction.
|
|
| 5. |
- Chia, Sean, et al.
(författare)
-
SAR by kinetics for drug discovery in protein misfolding diseases
- 2018
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 115:41, s. 10245-10250
-
Tidskriftsartikel (refereegranskat)abstract
- To develop effective therapeutic strategies for protein misfolding diseases, a promising route is to identify compounds that inhibit the formation of protein oligomers. To achieve this goal, we report a structure.activity relationship (SAR) approach based on chemical kinetics to estimate quantitatively how small molecules modify the reactive flux toward oligomers. We use this estimate to derive chemical rules in the case of the amyloid beta peptide (Aβ), which we then exploit to optimize starting compounds to curtail Aâ oligomer formation. We demonstrate this approach by converting an inactive rhodanine compound into an effective inhibitor of Aβ oligomer formation by generating chemical derivatives in a systematic manner. These results provide an initial demonstration of the potential of drug discovery strategies based on targeting directly the production of protein oligomers.
|
|
| 6. |
- Cohen, Samuel I. A., et al.
(författare)
-
Proliferation of amyloid-beta 42 aggregates occurs through a secondary nucleation mechanism
- 2013
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 110:24, s. 9758-9763
-
Tidskriftsartikel (refereegranskat)abstract
- The generation of toxic oligomers during the aggregation of the amyloid-beta (A beta) peptide A beta 42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer's disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer's disease. These results reveal that the aggregation of A beta 42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the A beta aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic A beta 42 oligomers.
|
|
| 7. |
- Dear, Alexander J., et al.
(författare)
-
Kinetic diversity of amyloid oligomers
- 2020
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 117:22
-
Tidskriftsartikel (refereegranskat)abstract
- The spontaneous assembly of proteins into amyloid fibrils is a phenomenon central to many increasingly common and currently incurable human disorders, including Alzheimer's and Parkinson's diseases. Oligomeric species form transiently during this process and not only act as essential intermediates in the assembly of new filaments but also represent major pathogenic agents in these diseases. While amyloid fibrils possess a common, defining set of physicochemical features, oligomers, by contrast, appear much more diverse, and their commonalities and differences have hitherto remained largely unexplored. Here, we use the framework of chemical kinetics to investigate their dynamical properties. By fitting experimental data for several unrelated amyloidogenic systems to newly derived mechanistic models, we find that oligomers present with a remarkably wide range of kinetic and thermodynamic stabilities but that they possess two properties that are generic: they are overwhelmingly nonfibrillar, and they predominantly dissociate back to monomers rather than maturing into fibrillar species. These discoveries change our understanding of the relationship between amyloid oligomers and amyloid fibrils and have important implications for the nature of their cellular toxicity.
|
|
| 8. |
- Habchi, Johnny, et al.
(författare)
-
Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer's disease
- 2017
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 114:2, s. 200-208
-
Tidskriftsartikel (refereegranskat)abstract
- The aggregation of the 42-residue form of the amyloid-β peptide (Aβ42) is a pivotal event in Alzheimer's disease (AD). The use of chemical kinetics has recently enabled highly accurate quantifications of the effects of small molecules on specific microscopic steps in Aβ42 aggregation. Here, we exploit this approach to develop a rational drug discovery strategy against Aβ42 aggregation that uses as a readout the changes in the nucleation and elongation rate constants caused by candidate small molecules. We thus identify a pool of compounds that target specific microscopic steps in Aβ42 aggregation. We then test further these small molecules in human cerebrospinal fluid and in a Caenorhabditis elegans model of AD. Our results show that this strategy represents a powerful approach to identify systematically small molecule lead compounds, thus offering an appealing opportunity to reduce the attrition problem in drug discovery.
|
|
| 9. |
- Lattanzi, Veronica, et al.
(författare)
-
Amyloid β 42 fibril structure based on small-angle scattering
- 2021
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 118:48
-
Tidskriftsartikel (refereegranskat)abstract
- Amyloid fibrils are associated with a number of neurodegenerative diseases, including fibrils of amyloid β42 peptide (Aβ42) in Alzheimer's disease. These fibrils are a source of toxicity to neuronal cells through surface-catalyzed generation of toxic oligomers. Detailed knowledge of the fibril structure may thus facilitate therapeutic development. We use small-angle scattering to provide information on the fibril cross-section dimension and shape for Aβ42 fibrils prepared in aqueous phosphate buffer at pH = 7.4 and pH 8.0 under quiescent conditions at 37°C from pure recombinant Aβ42 peptide. Fitting the data using a continuum model reveals an elliptical cross-section and a peptide mass-per-unit length compatible with two filaments of two monomers, four monomers per plane. To provide a more detailed atomistic model, the data were fitted using as a starting state a high-resolution structure of the two-monomer arrangement in filaments from solid-state NMR (Protein Data Bank ID 5kk3). First, a twofold symmetric model including residues 11 to 42 of two monomers in the filament was optimized in terms of twist angle and local packing using Rosetta. A two-filament model was then built and optimized through fitting to the scattering data allowing the two N-termini in each filament to take different conformations, with the same conformation in each of the two filaments. This provides an atomistic model of the fibril with twofold rotation symmetry around the fibril axis. Intriguingly, no polydispersity as regards the number of filaments was observed in our system over separate samples, suggesting that the two-filament arrangement represents a free energy minimum for the Aβ42 fibril.
|
|
| 10. |
- Lindman, Stina, et al.
(författare)
-
In vivo protein stabilization based on fragment complementation and a split GFP system.
- 2010
-
Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 107:46, s. 19826-19831
-
Tidskriftsartikel (refereegranskat)abstract
- Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.
|
|
