24 research outputs found
Heparin acts as a structural component of β-endorphin amyloid fibrils rather than a simple aggregation promoter.
The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide β-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging β-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on β-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.This work was funded by grants from the Wellcome Trust, the Medical Research Council UK, the Alzheimer Research UK Trust, the Engineering and Physical Sciences Research Council UK, and the Biotechnology and Biological Sciences Research Council. NN was supported through Early PostDoc.Mobility personal fellowship from Swiss National Science Foundation
Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles
Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn(6)) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ~23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of approximately 8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles
C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies
Recommended from our members
Peptide hormones and lipopeptides: from self-assembly to therapeutic applications
This review describes the properties and activities of lipopeptides and peptide hormones and how the lipidation of peptide hormones could potentially produce therapeutic agents combating some of the most prevalent diseases and conditions. The self-assembly of these types of molecules is outlined, and how this can impact on bioactivity. Peptide hormones specific to the uptake of food and produced in the gastrointestinal tract are discussed in detail. The advantages of lipidated peptide hormones over natural peptide hormones are summarised, in terms of stability and renal clearance, with potential application as therapeutic agent
Recommended from our members
Different structural conformers of monomeric alpha-synuclein identified after lyophilising and freezing
Understanding the mechanisms behind amyloid protein aggregation in diseases such as Parkinson’s and Alzheimer’s disease is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein alpha-synuclein (aSyn), a protein involved in Parkinson’s disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods including, size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterise the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterise the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilising the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.G.S.K.S. and C.F.K. acknowledge funding from the Wellcome Trust, the UK Medical Research Council (MRC), Alzheimer Research UK (ARUK), and Infinitus China Ltd. C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (EPSRC). M.Z. acknowledges funding from the Eugenides Foundation, Newnham College (Cambridge), and George and Marie Vergottis Foundation (Cambridge Trust). A.D.S. and M.Z. acknowledge the European Biophysical Societies’ Association (EBSA), Alzheimer Research UK (ARUK), Newnham College (Cambridge) and British Mass Spectrometry Society (BMSS) for travel grants
Heparin acts as a structural component of β-endorphin amyloid fibrils rather than a simple aggregation promoter
A New, Modular Mass Calibrant for High-Mass MALDI-MS
The
application of matrix-assisted laser desorption/ionization
mass spectrometry (MALDI-MS) for the analysis of high-mass proteins
requires suitable calibration standards at high <i>m</i>/<i>z</i> ratios. Several possible candidates were investigated,
and concatenated polyproteins based on recombinantly expressed maltodextrin-binding
protein (MBP) are shown here to be well-suited for this purpose. Introduction
of two specific recognition sites into the primary sequence of the
polyprotein allows for the selective cleavage of MBP<sub>3</sub> into
MBP and MBP<sub>2</sub>. Moreover, these MBP<sub>2</sub> and MBP<sub>3</sub> oligomers can be dimerized specifically, such that generation
of MPB<sub>4</sub> and MBP<sub>6</sub> is possible as well. With the
set of calibrants presented here, the <i>m</i>/<i>z</i> range of 40–400 kDa is covered. Since all calibrants consist
of the same species and differ only in mass, the ionization efficiency
is expected to be similar. However, equimolar mixtures of these proteins
did not yield equal signal intensities on a detector specifically
designed for detecting high-mass molecules
Dynamic Assembly and Disassembly of Functional β‑Endorphin Amyloid Fibrils
Neuropeptides
and peptide hormones are stored in the amyloid state
in dense-core vesicles of secretory cells. Secreted peptides experience
dramatic environmental changes in the secretory pathway, from the
endoplasmic reticulum via secretory vesicles to release into the interstitial
space or blood. The molecular mechanisms of amyloid formation during
packing of peptides into secretory vesicles and amyloid dissociation
upon release remain unknown. In the present work, we applied thioflavin
T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy
measurements, and solid-state NMR spectroscopy to study the influence
of physiologically relevant environmental factors on the assembly
and disassembly of β-endorphin amyloids in vitro. We found that
β-endorphin aggregation and dissociation occur in vitro on relatively
short time scales, comparable to times required for protein synthesis
and the rise of peptide concentration in the blood, respectively.
Both assembly and disassembly of amyloids strongly depend on the presence
of salts of polyprotic acids (such as phosphate and sulfate), while
salts of monoprotic acids are not effective in promoting aggregation.
A steep increase of the peptide aggregation rate constant upon increase
of solution pH from 5.0 to 6.0 toward the isoelectric point as well
as more rapid dissociation of β-endorphin amyloid fibrils at
lower pH indicate the contribution of ion-specific effects into dynamics
of the amyloid. Several low-molecular-weight carbohydrates exhibit
the same effect on β-endorphin aggregation as phosphate. Moreover,
no structural difference was detected between the phosphate- and carbohydrate-induced
fibrils by solid-state NMR. In contrast, β-endorphin amyloid
fibrils obtained in the presence of heparin demonstrated distinctly
different behavior, which we attributed to a dramatic change of the
amyloid structure. Overall, the presented results support the hypothesis
that packing of peptide hormones/neuropeptides in dense-core vesicles
do not necessarily require a specialized cellular machinery