| 1. |
- Speliotes, Elizabeth K., et al.
(författare)
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Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index
- 2010
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 42:11, s. 937-948
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Tidskriftsartikel (refereegranskat)abstract
- Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ~2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10−8), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.
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| 2. |
- Heid, Iris M, et al.
(författare)
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Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution
- 2010
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Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718. ; 42:11, s. 949-960
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Tidskriftsartikel (refereegranskat)abstract
- Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10⁻⁹ to P = 1.8 × 10⁻⁴⁰) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10⁻³ to P = 1.2 × 10⁻¹³). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.
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| 3. |
- Lango Allen, Hana, et al.
(författare)
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Hundreds of variants clustered in genomic loci and biological pathways affect human height.
- 2010
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 467:7317, s. 832-8
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Tidskriftsartikel (refereegranskat)abstract
- Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P<0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.
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| 4. |
- 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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| 5. |
- Eves, Ben J., et al.
(författare)
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Elongation rate and average length of amyloid fibrils in solution using isotope-labelled small-angle neutron scattering
- 2021
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Ingår i: RSC Chemical Biology. - : Royal Society of Chemistry (RSC). - 2633-0679. ; 2:4, s. 1232-1238
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a solution method that allows both elongation rate and average fibril length of assembling amyloid fibrils to be estimated. The approach involves acquisition of real-time neutron scattering data during the initial stages of seeded growth, using contrast matched buffer to make the seeds effectively invisible to neutrons. As deuterated monomers add on to the seeds, the labelled growing ends give rise to scattering patterns that we model as cylinders whose increase in length with time gives an elongation rate. In addition, the absolute intensity of the signal can be used to determine the number of growing ends per unit volume, which in turn provides an estimate of seed length. The number of ends did not change significantly during elongation, demonstrating that any spontaneous or secondary nucleation was not significant compared with growth on the ends of pre-existing fibrils, and in addition providing a method of internal validation for the technique. Our experiments on initial growth of alpha synuclein fibrils using 1.2 mg ml-1 seeds in 2.5 mg ml-1 deuterated monomer at room temperature gave an elongation rate of 6.3 ± 0.5 Å min-1, and an average seed length estimate of 4.2 ± 1.3 μm. This journal is
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| 6. |
- Simatos, Dimitrios, et al.
(författare)
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Effects of Processing-Induced Contamination on Organic Electronic Devices
- 2023
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Ingår i: Small Methods. - : Wiley. - 2366-9608. ; 7:11
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Tidskriftsartikel (refereegranskat)abstract
- Organic semiconductors are a family of pi-conjugated compounds used in many applications, such as displays, bioelectronics, and thermoelectrics. However, their susceptibility to processing-induced contamination is not well understood. Here, it is shown that many organic electronic devices reported so far may have been unintentionally contaminated, thus affecting their performance, water uptake, and thin film properties. Nuclear magnetic resonance spectroscopy is used to detect and quantify contaminants originating from the glovebox atmosphere and common laboratory consumables used during device fabrication. Importantly, this in-depth understanding of the sources of contamination allows the establishment of clean fabrication protocols, and the fabrication of organic field effect transistors (OFETs) with improved performance and stability. This study highlights the role of unintentional contaminants in organic electronic devices, and demonstrates that certain stringent processing conditions need to be met to avoid scientific misinterpretation, ensure device reproducibility, and facilitate performance stability. The experimental procedures and conditions used herein are typical of those used by many groups in the field of solution-processed organic semiconductors. Therefore, the insights gained into the effects of contamination are likely to be broadly applicable to studies, not just of OFETs, but also of other devices based on these materials.
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| 7. |
- 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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| 8. |
- Axell, Emil, et al.
(författare)
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The role of shear forces in primary and secondary nucleation of amyloid fibrils
- 2024
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490. ; 121:25, s. 2322572121-2322572121
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Tidskriftsartikel (refereegranskat)abstract
- Shear forces affect self-assembly processes ranging from crystallization to fiber formation. Here, the effect of mild agitation on amyloid fibril formation was explored for four peptides and investigated in detail for Aβ42, which is associated with Alzheimer's disease. To gain mechanistic insights into the effect of mild agitation, nonseeded and seeded aggregation reactions were set up at various peptide concentrations with and without an inhibitor. First, an effect on fibril fragmentation was excluded by comparing the monomer-concentration dependence of aggregation kinetics under idle and agitated conditions. Second, using a secondary nucleation inhibitor, Brichos, the agitation effect on primary nucleation was decoupled from secondary nucleation. Third, an effect on secondary nucleation was established in the absence of inhibitor. Fourth, an effect on elongation was excluded by comparing the seeding potency of fibrils formed under idle or agitated conditions. We find that both primary and secondary nucleation steps are accelerated by gentle agitation. The increased shear forces facilitate both the detachment of newly formed aggregates from catalytic surfaces and the rate at which molecules are transported in the bulk solution to encounter nucleation sites on the fibril and other surfaces. Ultrastructural evidence obtained with cryogenic transmission electron microscopy and free-flow electrophoresis in microfluidics devices imply that agitation speeds up the detachment of nucleated species from the fibril surface. Our findings shed light on the aggregation mechanism and the role of detachment for efficient secondary nucleation. The results inform on how to modulate the relative importance of different microscopic steps in drug discovery and investigations.
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| 9. |
- Braun, Gabriel A., et al.
(författare)
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On the Mechanism of Self-Assembly by a Hydrogel-Forming Peptide
- 2020
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 21:12, s. 4781-4794
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Tidskriftsartikel (refereegranskat)abstract
- Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.
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| 10. |
- Dear, Alexander J., et al.
(författare)
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Aβ Oligomer Dissociation Is Catalyzed by Fibril Surfaces
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Ingår i: ACS Chemical Neuroscience. - 1948-7193.
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric assemblies consisting of only a few protein subunits are key species in the cytotoxicity of neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Their lifetime in solution and abundance, governed by the balance of their sources and sinks, are thus important determinants of disease. While significant advances have been made in elucidating the processes that govern oligomer production, the mechanisms behind their dissociation are still poorly understood. Here, we use chemical kinetic modeling to determine the fate of oligomers formed in vitro and discuss the implications for their abundance in vivo. We discover that oligomeric species formed predominantly on fibril surfaces, a broad class which includes the bulk of oligomers formed by the key Alzheimer's disease-associated Aβ peptides, also dissociate overwhelmingly on fibril surfaces, not in solution as had previously been assumed. We monitor this "secondary nucleation in reverse" by measuring the dissociation of Aβ42 oligomers in the presence and absence of fibrils via two distinct experimental methods. Our findings imply that drugs that bind fibril surfaces to inhibit oligomer formation may also inhibit their dissociation, with important implications for rational design of therapeutic strategies for Alzheimer's and other amyloid diseases.
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| 11. |
- Dear, Alexander J., et al.
(författare)
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Identification of on- And off-pathway oligomers in amyloid fibril formation
- 2020
-
Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 11:24, s. 6236-6247
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Tidskriftsartikel (refereegranskat)abstract
- The misfolding and aberrant aggregation of proteins into fibrillar structures is a key factor in some of the most prevalent human diseases, including diabetes and dementia. Low molecular weight oligomers are thought to be a central factor in the pathology of these diseases, as well as critical intermediates in the fibril formation process, and as such have received much recent attention. Moreover, on-pathway oligomeric intermediates are potential targets for therapeutic strategies aimed at interrupting the fibril formation process. However, a consistent framework for distinguishing on-pathway from off-pathway oligomers has hitherto been lacking and, in particular, no consensus definition of on- and off-pathway oligomers is available. In this paper, we argue that a non-binary definition of oligomers' contribution to fibril-forming pathways may be more informative and we suggest a quantitative framework, in which each oligomeric species is assigned a value between 0 and 1 describing its relative contribution to the formation of fibrils. First, we clarify the distinction between oligomers and fibrils, and then we use the formalism of reaction networks to develop a general definition for on-pathway oligomers, that yields meaningful classifications in the context of amyloid formation. By applying these concepts to Monte Carlo simulations of a minimal aggregating system, and by revisiting several previous studies of amyloid oligomers in light of our new framework, we demonstrate how to perform these classifications in practice. For each oligomeric species we obtain the degree to which it is on-pathway, highlighting the most effective pharmaceutical targets for the inhibition of amyloid fibril formation. This journal is
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| 12. |
- Dear, Alexander J., et al.
(författare)
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Kinetic diversity of amyloid oligomers
- 2020
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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
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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.
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| 13. |
- Dear, Alexander J., et al.
(författare)
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The catalytic nature of protein aggregation
- 2020
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Ingår i: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 152:4
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Tidskriftsartikel (refereegranskat)abstract
- The formation of amyloid fibrils from soluble peptide is a hallmark of many neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Characterization of the microscopic reaction processes that underlie these phenomena have yielded insights into the progression of such diseases and may inform rational approaches for the design of drugs to halt them. Experimental evidence suggests that most of these reaction processes are intrinsically catalytic in nature and may display enzymelike saturation effects under conditions typical of biological systems, yet a unified modeling framework accounting for these saturation effects is still lacking. In this paper, we therefore present a universal kinetic model for biofilament formation in which every fundamental process in the reaction network can be catalytic. The single closed-form expression derived is capable of describing with high accuracy a wide range of mechanisms of biofilament formation and providing the first integrated rate law of a system in which multiple reaction processes are saturated. Moreover, its unprecedented mathematical simplicity permits us to very clearly interpret the effects of increasing saturation on the overall kinetics. The effectiveness of the model is illustrated by fitting it to the data of in vitro Aβ40 aggregation. Remarkably, we find that primary nucleation becomes saturated, demonstrating that it must be heterogeneous, occurring at interfaces and not in solution.
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| 14. |
- Flagmeier, Patrick, et al.
(författare)
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Direct measurement of lipid membrane disruption connects kinetics and toxicity of Aβ42 aggregation
- 2020
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Ingår i: Nature Structural and Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 27:10, s. 886-891
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Tidskriftsartikel (refereegranskat)abstract
- The formation of amyloid deposits in human tissues is a defining feature of more than 50 medical disorders, including Alzheimer’s disease. Strong genetic and histological evidence links these conditions to the process of protein aggregation, yet it has remained challenging to identify a definitive connection between aggregation and pathogenicity. Using time-resolved fluorescence microscopy of individual synthetic vesicles, we show for the Aβ42 peptide implicated in Alzheimer’s disease that the disruption of lipid bilayers correlates linearly with the time course of the levels of transient oligomers generated through secondary nucleation. These findings indicate a specific role of oligomers generated through the catalytic action of fibrillar species during the protein aggregation process in driving deleterious biological function and establish a direct causative connection between amyloid formation and its pathological effects.
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| 15. |
- 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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| 16. |
- 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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| 17. |
- Michaels, Thomas C.T., et al.
(författare)
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Dynamics of oligomer populations formed during the aggregation of Alzheimer’s Aβ42 peptide
- 2020
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Ingår i: Nature Chemistry. - : Springer Science and Business Media LLC. - 1755-4330 .- 1755-4349. ; 12:5, s. 445-451
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Tidskriftsartikel (refereegranskat)abstract
- Oligomeric species populated during the aggregation of the Aβ42 peptide have been identified as potent cytotoxins linked to Alzheimer’s disease, but the fundamental molecular pathways that control their dynamics have yet to be elucidated. By developing a general approach that combines theory, experiment and simulation, we reveal, in molecular detail, the mechanisms of Aβ42 oligomer dynamics during amyloid fibril formation. Even though all mature amyloid fibrils must originate as oligomers, we found that most Aβ42 oligomers dissociate into their monomeric precursors without forming new fibrils. Only a minority of oligomers converts into fibrillar structures. Moreover, the heterogeneous ensemble of oligomeric species interconverts on timescales comparable to those of aggregation. Our results identify fundamentally new steps that could be targeted by therapeutic interventions designed to combat protein misfolding diseases. [Figure not available: see fulltext.].
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| 18. |
- 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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| 19. |
- 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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| 20. |
- 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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| 21. |
- 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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| 22. |
- 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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| 23. |
- 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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| 24. |
- Arosio, Paolo, et al.
(författare)
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On the lag phase in amyloid fibril formation
- 2015
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Ingår i: Physical Chemistry Chemical Physics. - : Royal Society of Chemistry (RSC). - 1463-9084 .- 1463-9076. ; 17:12, s. 7606-7618
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Tidskriftsartikel (refereegranskat)abstract
- The formation of nanoscale amyloid fibrils from normally soluble peptides and proteins is a common form of self-assembly phenomenon that has fundamental connections with biological functions and human diseases. The kinetics of this process has been widely studied and exhibits on a macroscopic level three characteristic stages: a lag phase, a growth phase and a final plateau regime. The question of which molecular events take place during each one of these phases has been a central element in the quest for a mechanism of amyloid formation. In this review, we discuss the nature and molecular origin of the lag-phase in amyloid formation by making use of tools and concepts from physical chemistry, in particular from chemical reaction kinetics. We discuss how, in macroscopic samples, it has become apparent that the lag-phase is not a waiting time for nuclei to form. Rather, multiple parallel processes exist and typically millions of primary nuclei form during the lag phase from monomers in solution. Thus, the lag-time represents a time that is required for the nuclei that are formed early on in the reaction to grow and proliferate in order to reach an aggregate concentration that is readily detected in bulk assays. In many cases, this proliferation takes place through secondary nucleation, where fibrils may present a catalytic surface for the formation of new aggregates. Fibrils may also break (fragmentation) and thereby provide new ends for elongation. Thus, at least two -primary nucleation and elongation - and in many systems at least four - primary nucleation, elongation, secondary nucleation and fragmentation - microscopic processes occur during the lag phase. Moreover, these same processes occur during all three phases of the macroscopic aggregation process, albeit at different rates as governed by rate constants and by the concentration of reacting species at each point in time.
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| 25. |
- Arosio, Paolo, et al.
(författare)
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Quantification of the Concentration of A beta 42 Propagons during the Lag Phase by an Amyloid Chain Reaction Assay
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 136:1, s. 219-225
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Tidskriftsartikel (refereegranskat)abstract
- The aggregation of the amyloid beta peptide, A beta 42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar A beta 42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.
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