Distinct higher-order alpha-synuclein oligomers induce intracellular aggretation

Misfolding and aggregation of alpha-synuclein (α-syn) into Lewy bodies (LB) is associated with a range of neurological disorders, including Parkinson's disease (PD). The cell to cell transmission of α-syn pathology has been linked to soluble amyloid oligomer populations that preceded LB formation. Oligomers produced in vitro under certain conditions have been demonstrated to induce intracellular aggregation in cell culture models. Here we characterize, by electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS), a specific population of α-syn oligomers. These mass spectrometry compatible oligomers were compared with oligomers with known seeding and pore forming capabilities and were shown to have the ability to induce intracellular aggregation. Each oligomer type was shown to have distinct epitope profiles that correlated with their toxic gain of function. Structurally the mass spectrometry compatible oligomers populated a range of species from dimers through to hexamers. Lower order oligomers were structurally diverse and consistent with unstructured assemblies. Higher order oligomers were shown to be compact with ring-like structures. The observation of this compact state may explain how this natively disordered protein is able to transfer pathology from cell to cell and avoid degradation by cellular proteases

The N-Terminal residues 43 to 60 form the interface for dopamine mediated α-synuclein dimerisation

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson's disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43-140) and C-terminally (1-95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA: α-syn oligomers, albeit 1-95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43-140 protein, we analysed the structural characteristics of the DA:α-syn 43-140 dimer and α-syn 43-140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers

Conformations and Assembly of Amyloid Oligomers by Electrospray Ionisation - Ion Mobility Spectrometry - Mass Spectrometry

Amyloid structures accumulate and propagate through self-assembly of partially folded proteins and peptides, resulting in a range of disease states. Key to understanding amyloid disease is the characterisation of the often toxic oligomeric species formed during the early stages of fibril assembly. Electrospray ionisation- ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) has emerged as a powerful tool to investigate amyloid oligomer assembly and protein conformation change. In this review we focus on the role of ESI-IMS-MS in understanding and probing conformational changes and the early stages of protein aggregation

Mass Spectrometry Detection and Imaging of a Non-Covalent Protein-Drug Complex in Tissue from Orally Dosed Rats

Here, we demonstrate detection by mass spectrometry of an intact protein–drug complex directly from liver tissue from rats that had been orally dosed with the drug. The protein–drug complex comprised fatty acid binding protein 1, FABP1, non‐covalently bound to the small molecule therapeutic bezafibrate. Moreover, we demonstrate spatial mapping of the [FABP1+bezafibrate] complex across a thin section of liver by targeted mass spectrometry imaging. This work is the first demonstration of in situ mass spectrometry analysis of a non‐covalent protein–drug complex formed in vivo and has implications for early stage drug discovery by providing a route to target‐drug characterization directly from the physiological environment

Quantitative Characterization of Three Carbonic Anhydrase Inhibitors by LESA Mass Spectrometry

[Image: see text] Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein–ligand interactions through calculation of equilibrium dissociation constants (K(d) values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the K(d) values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, K(d) values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein–ligand interactions when the dried sample comprises both protein and ligand

Oligomerisation of α-syn on treatment with DA.

<p><b>A</b>. Schematic of the different α-syn constructs used in this study. The black bars represent the imperfect repeats (residues 10–16, 21–27, 32–37, 43–49, 57–63, 80–86). NAC region (stippled bar, residues 60–95). <b>B</b>. Silver stain SDS-PAGE gel of truncated α-syn incubated in the presence or absence of DA. Lane 1: α-syn 1–140. Lane 2: α-syn 1–140 + DA. Lane 3: α-syn 1–95. Lane 4: α-syn 1–95 + DA. Lane 5: α-syn 43–140. Lane 6: α-syn 43–140 + DA. The α-syn to DA ratio was 1:10. <b>C</b>. Size exclusion chromatography of DA:α-syn 43–140 and DA:α-syn 1–95 oligomers. 200 μM α-syn was incubated with 2 mM DA for 7 days. The reaction was centrifuged at 100,000 rpm, 1 hour, 4°C and then analysed on a Superdex 200 10/300GL column using 10 mM sodium phosphate pH 7.5 buffer with a flow rate of 0.5 mL/min. Proteins were detected at A280nm.</p

Stability of DA:α-syn 43–140 dimer exposed to denaturants.

<p>Silver stain SDS-PAGE gel of DA:α-syn 43–140 dimer incubated with different amounts of denaturants HFIP or TFE. The dimers did not dissociate under any of the conditions tested, suggesting a covalent cross-link. <b>Lane 1:</b> DA:α-syn 43–140 dimer + 1% HFIP. <b>Lane 2:</b> DA:α-syn 43–140 dimer + 2% HFIP. <b>Lane 3:</b> DA:α-syn 43–140 dimer + 4% HFIP. <b>Lane 4:</b> DA:α-syn 43–140 dimer + 5% HFIP. <b>Lane 5:</b> DA:α-syn 43–140 dimer + 4% TFE. <b>Lane 6:</b> DA:α-syn 43–140 dimer + 8% TFE. <b>Lane 7:</b> DA:α-syn 43–140 dimer + 10% TFE. <b>Lane 8:</b> DA:α-syn 43–140 dimer + 15% TFE. Arrow indicates the DA:α-syn 43–140 dimer band.</p