| 1. |
- Kakkar, Vaishali, et al.
(författare)
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The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model
- 2016
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Ingår i: Molecular Cell. - : Elsevier BV. - 1097-2765. ; 62:2, s. 272-283
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Tidskriftsartikel (refereegranskat)abstract
- Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases. Kakkar et al. show that DNAJB6 is a chaperone that inhibits early steps in the formation of polyQ amyloid fibrils. An S/T-rich region in DNAJB6 is crucial for this function. In a polyQ mouse model, the inhibitory effects of DNAJB6 delay disease onset and increase lifespan.
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| 2. |
- Arosio, Paolo, et al.
(författare)
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Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.
- 2016
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Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 10:1, s. 333-341
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.
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| 3. |
- Habchi, Johnny, et al.
(författare)
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Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes
- 2018
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Ingår i: Nature Chemistry. - : Springer Science and Business Media LLC. - 1755-4330 .- 1755-4349. ; 10:6, s. 673-683
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer’s disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrate that lipid membranes containing cholesterol promote Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further show that this process occurs as a result of cooperativity in the interaction of multiple cholesterol molecules with Aβ42. These results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer’s disease and the impairment of cholesterol homeostasis.
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| 4. |
- Herling, Therese W., et al.
(författare)
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A Microfluidic Platform for Real-Time Detection and Quantification of Protein-Ligand Interactions
- 2016
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 110:9, s. 1957-1966
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Tidskriftsartikel (refereegranskat)abstract
- The key steps in cellular signaling and regulatory pathways rely on reversible noncovalent protein-ligand binding, yet the equilibrium parameters for such events remain challenging to characterize and quantify in solution. Here, we demonstrate a microfluidic platform for the detection of protein-ligand interactions with an assay time on the second timescale and without the requirement for immobilization or the presence of a highly viscous matrix. Using this approach, we obtain absolute values for the electrophoretic mobilities characterizing solvated proteins and demonstrate quantitative comparison of results obtained under different solution conditions. We apply this strategy to characterize the interaction between calmodulin and creatine kinase, which we identify as a novel calmodulin target. Moreover, we explore the differential calcium ion dependence of calmodulin ligand-binding affinities, a system at the focal point of calcium-mediated cellular signaling pathways. We further explore the effect of calmodulin on creatine kinase activity and show that it is increased by the interaction between the two proteins. These findings demonstrate the potential of quantitative microfluidic techniques to characterize binding equilibria between biomolecules under native solution conditions.
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| 5. |
- Saar, Kadi L., et al.
(författare)
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On-chip label-free protein analysis with downstream electrodes for direct removal of electrolysis products
- 2017
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 18:1, s. 162-170
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Tidskriftsartikel (refereegranskat)abstract
- The ability to apply highly controlled electric fields within microfluidic devices is valuable as a basis for preparative and analytical processes. A challenge encountered in the context of such approaches in conductive media, including aqueous buffers, is the generation of electrolysis products at the electrode/liquid interface which can lead to contamination, perturb fluid flows and generally interfere with the measurement process. Here, we address this challenge by designing a single layer microfluidic device architecture where the electric potential is applied outside and downstream of the microfluidic device while the field is propagated back to the chip via the use of a co-flowing highly conductive electrolyte solution that forms a stable interface at the separation region of the device. The co-flowing electrolyte ensures that all the generated electrolysis products, including Joule heat and gaseous products, are flowed away from the chip without coming into contact with the analytes while the single layer fabrication process where all the structures are defined lithographically allows producing the devices in a simple yet highly reproducible manner. We demonstrate that by allowing stable and effective application of electric fields in excess of 100 V cm-1, the described platform provides the basis for rapid separation of heterogeneous mixtures of proteins and protein complexes directly in their native buffers as well as for the simultaneous quantification of their charge states. We illustrate this by probing the interactions in a mixture of an amyloid forming protein, amyloid-β, and a molecular chaperone, Brichos, known to inhibit the process of amyloid formation. The availability of a platform for applying stable electric fields and its compatibility with single-layer soft-lithography processes opens up the possibility of separating and analysing a wide range of molecules on chip, including those with similar electrophoretic mobilities.
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| 6. |
- Weiffert, Tanja, et al.
(författare)
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Increased Secondary Nucleation Underlies Accelerated Aggregation of the Four-Residue N-Terminally Truncated Aβ42 Species Aβ5-42
- 2019
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193.
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Tidskriftsartikel (refereegranskat)abstract
- Aggregation of the amyloid-β (Aβ) peptide into plaques is believed to play a crucial role in Alzheimer's disease. Amyloid plaques consist of fibrils of full length Aβ peptides as well as N-terminally truncated species. β-Site amyloid precursor protein-cleaving enzyme (BACE1) cleaves amyloid precursor protein in the first step in Aβ peptide production and is an attractive therapeutic target to limit Aβ generation. Inhibition of BACE1, however, induces a unique pattern of Aβ peptides with increased levels of N-terminally truncated Aβ peptides starting at position 5 (Aβ5-X), indicating that these peptides are generated through a BACE1-independent pathway. Here we elucidate the aggregation mechanism of Aβ5-42 and its influence on full-length Aβ42. We find that, compared to Aβ42, Aβ5-42 is more aggregation prone and displays enhanced nucleation rates. Aβ5-42 oligomers cause nonspecific membrane disruption to similar extent as Aβ42 but appear at earlier time points in the aggregation reaction. Noteworthy, this implies similar toxicity of Aβ42 and Aβ5-42 and the toxic species are generated faster by Aβ5-42. The increased rate of secondary nucleation on the surface of existing fibrils originates from a higher affinity of Aβ5-42 monomers for fibrils, as compared to Aβ42: an effect that may be related to the reduced net charge of Aβ5-42. Moreover, Aβ5-42 and Aβ42 peptides coaggregate into heteromolecular fibrils and either species can elongate existing Aβ42 or Aβ5-42 fibrils but Aβ42 fibrils are more catalytic than Aβ5-42 fibrils. Our findings highlight the importance of the N-terminus for surface-catalyzed nucleation and thus the production of toxic oligomers.
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| 7. |
- Yates, Emma V., et al.
(författare)
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Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity
- 2015
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Ingår i: Nature Chemistry. - 1755-4330. ; 7:10, s. 802-809
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Tidskriftsartikel (refereegranskat)abstract
- The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a 'latent' analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.
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| 8. |
- Aprile, Francesco A., et al.
(författare)
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Selective targeting of primary and secondary nucleation pathways in Ab42 aggregation using a rational antibody scanning method
- 2017
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 3:6
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies targeting Ab42 are under intense scrutiny because of their therapeutic potential for Alzheimer’s disease. To enable systematic searches, we present an “antibody scanning” strategy for the generation of a panel of antibodies against Ab42. Each antibody in the panel is rationally designed to target a specific linear epitope, with the selected epitopes scanning the Ab42 sequence. By screening in vitro the panel to identify the specific microscopic steps in the Ab42 aggregation process influenced by each antibody, we identify two antibodies that target specifically the primary and the secondary nucleation steps, which are key for the production of Ab42 oligomers. These two antibodies act, respectively, to delay the onset of aggregation and to block the proliferation of aggregates, and correspondingly reduce the toxicity in a Caenorhabditis elegans model over-expressing Ab42. These results illustrate how the antibody scanning method described here can be used to readily obtain very small antibody libraries with extensive coverage of the sequences of target proteins.
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| 9. |
- Arosio, Paolo, et al.
(författare)
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Analysis of the length distribution of amyloid fibrils by centrifugal sedimentation
- 2016
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697. ; 504, s. 7-13
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of normally soluble peptides and proteins into amyloid fibrils is a process associated with a wide range of pathological conditions, including Alzheimer's and Parkinson's diseases. It has become apparent that aggregates of different sizes possess markedly different biological effects, with aggregates of lower relative molecular weight being associated with stronger neurotoxicity. Yet, although many approaches exist to measure the total mass concentration of aggregates, the ability to probe the length distribution of growing aggregates in solution has remained more elusive. In this work, we applied a differential centrifugation technique to measure the sedimentation coefficients of amyloid fibrils produced during the aggregation process of the amyloid β (M1-42) peptide (Aβ42). The centrifugal method has the advantage of providing structural information on the fibril distribution directly in solution and affording a short analysis time with respect to alternative imaging and analytical centrifugation approaches. We show that under quiescent conditions interactions between Aβ42 fibrils lead to lateral association and to the formation of entangled clusters. By contrast, aggregation under shaking generates a population of filaments characterized by shorter lengths. The results, which have been validated by cryogenic transmission electron microscopy (cryo-TEM) analysis, highlight the important role that fibril-fibril assembly can play in the deposition of aggregation-prone peptides.
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| 10. |
- Arosio, Paolo, et al.
(författare)
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Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.
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