How important is the N-terminal acetylation of alpha-synuclein for its function and aggregation into amyloids?

N-α-acetylation is a frequently occurring post-translational modification in eukaryotic proteins. It has manifold physiological consequences on the regulation and function of several proteins, with emerging studies suggesting that it is a global regulator of stress responses. For decades, biochemical investigations into the precise role of the intrinsically disordered protein alpha-synuclein (αS) in the etiology of Parkinson's disease (PD) were performed using non-acetylated αS.

Intracellular Protein-Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy.

Protein-membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied , but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells.

Lipid-Conjugated Rigidochromic Probe Discloses Membrane Alteration in Model Cells of Krabbe Disease.

The plasma membrane of cells has a complex architecture based on the bidimensional liquid-crystalline bilayer arrangement of phospho- and sphingolipids, which in turn embeds several proteins and is connected to the cytoskeleton. Several studies highlight the spatial membrane organization into more ordered (L or lipid raft) and more disordered (L) domains. We here report on a fluorescent analog of the green fluorescent protein chromophore that, when conjugated to a phospholipid, enables the quantification of the L and L domains in living cells on account of its large fluorescence lifetime variation in the two phases.

Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils.

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data.

Hydrophobic-Interaction-Induced Stiffening of α-Synuclein Fibril Networks.

In water, networks of semiflexible fibrils of the protein α-synuclein stiffen significantly with increasing temperature. We make plausible that this reversible stiffening is a result of hydrophobic contacts between the fibrils that become more prominent with increasing temperature. The good agreement of our experimentally observed temperature dependence of the storage modulus of the network with a scaling theory linking network elasticity with reversible cross-linking enables us to quantify the endothermic binding enthalpy and estimate the effective size of hydrophobic patches on the fibril surface.

C-Terminal Truncated α-Synuclein Fibrils Contain Strongly Twisted β-Sheets.

C-terminal truncations of monomeric wild-type alpha-synuclein (henceforth WT-αS) have been shown to enhance the formation of amyloid aggregates both in vivo and in vitro and have been associated with accelerated progression of Parkinson's disease (PD). The correlation with PD may not solely be a result of faster aggregation, but also of which fibril polymorphs are preferentially formed when the C-terminal residues are deleted. Considering that different polymorphs are known to result in distinct pathologies, it is important to understand how these truncations affect the organization of αS into fibrils.

Membrane Binding of Parkinson's Protein α-Synuclein: Effect of Phosphorylation at Positions 87 and 129 by the S to D Mutation Approach.

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction.

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags.

We report oriented immobilization of proteins using the standard hexahistidine (His)-Ni:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His-tag on the TagRFP proteins. All TagRFP variants with His-tags located on only one side of the barrel-shaped protein yielded a 1.

Non-uniform self-assembly: On the anisotropic architecture of α-synuclein supra-fibrillar aggregates.

Although the function of biopolymer hydrogels in nature depends on structural anisotropy at mesoscopic length scales, the self-assembly of such anisotropic structures in vitro is challenging. Here we show that fibrils of the protein α-synuclein spontaneously self-assemble into structurally anisotropic hydrogel particles. While the fibrils in the interior of these supra-fibrillar aggregates (SFAs) are randomly oriented, the fibrils in the periphery prefer to cross neighboring fibrils at high angles.

Room-temperature in-cell EPR spectroscopy: alpha-Synuclein disease variants remain intrinsically disordered in the cell.

Human alpha-Synuclein (aS), implicated in Parkinson's disease, adopts a rich variety of different conformations depending on the macromolecular context. In order to unravel its pathophysiological role, monitoring its intracellular conformational state and identifying differences for the disease variants is crucial. Here, we present an intracellular spectroscopy approach based on a systematic spin-labeling site-scan in combination with intracellular electron paramagnetic resonance spectroscopy determining conformations on a molecular scale.

Direct Visualization of Model Membrane Remodeling by α-Synuclein Fibrillization.

The interaction of α-synuclein (αS) with membranes is thought to be critical in the etiology of Parkinson's disease. Besides oligomeric αS aggregates that possibly form membrane pores, the aggregation of αS into amyloid fibrils has been reported to disrupt membranes. The mechanism by which aggregation affects the integrity of membranes is, however, unknown.

Alpha-Synuclein Disease Mutations Are Structurally Defective and Locally Affect Membrane Binding.

The intrinsically disordered human protein alpha-Synuclein (αS) has a prominent role in Parkinson's disease (PD) pathology. Several familial variants of αS are correlated with inherited PD. Disease mutations have been shown to have an impact on lipid membrane binding.

Distinct Mechanisms Determine α-Synuclein Fibril Morphology during Growth and Maturation.

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism.

Evidence for Intramolecular Antiparallel Beta-Sheet Structure in Alpha-Synuclein Fibrils from a Combination of Two-Dimensional Infrared Spectroscopy and Atomic Force Microscopy.

The aggregation of the intrinsically disordered protein alpha-synuclein (αS) into amyloid fibrils is thought to play a central role in the pathology of Parkinson's disease. Using a combination of techniques (AFM, UV-CD, XRD, and amide-I 1D- and 2D-IR spectroscopy) we show that the structure of αS fibrils varies as a function of ionic strength: fibrils aggregated in low ionic-strength buffers ([NaCl] ≤ 25 mM) have a significantly different structure than fibrils grown in higher ionic-strength buffers. The observations for fibrils aggregated in low-salt buffers are consistent with an extended conformation of αS molecules, forming hydrogen-bonded intermolecular β-sheets that are loosely packed in a parallel fashion.

Membrane-Bound Alpha Synuclein Clusters Induce Impaired Lipid Diffusion and Increased Lipid Packing.

The aggregation of membrane-bound α-synuclein (αS) into oligomers and/or amyloid fibrils has been suggested to cause membrane damage in in vitro model phospholipid membrane systems and in vivo. In this study, we investigate how αS interactions that precede the formation of well-defined aggregates influence physical membrane properties. Using three truncated variants of αS with different aggregation propensities and comparable phospholipid membrane binding affinities we show, using fluorescence recovery after photobleaching (FRAP) and fluorescence anisotropy measurements, that formation of αS clusters on supported lipid bilayers (SLBs) impairs lateral lipid diffusion and increases lipid packing beneath the αS clusters.

p53 Specifically Binds Triplex DNA In Vitro and in Cells.

Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein.

Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers.

Phagocytosis is an important process of the immune system by which pathogens are internalized and eliminated by phagocytic cells. Upon internalization, the phagosome matures and acidifies while being transported in a centripetal fashion. In this chapter, we describe protocols for simultaneous imaging of phagosomal acidification as well as their spatial manipulation by magnetic tweezers.

Solubilization of lipids and lipid phases by the styrene-maleic acid copolymer.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction.

α-Synuclein Oligomers Stabilize Pre-Existing Defects in Supported Bilayers and Propagate Membrane Damage in a Fractal-Like Pattern.

Phospholipid vesicles are commonly used to get insights into the mechanism by which oligomers of amyloidogenic proteins damage membranes. Oligomers of the protein α-synuclein (αS) are thought to create pores in phospholipid vesicles containing a high amount of anionic phospholipids but fail to damage vesicle membranes at low surface charge densities. The current understanding of how αS oligomers damage the membranes is thus incomplete.

The Impact of N-terminal Acetylation of α-Synuclein on Phospholipid Membrane Binding and Fibril Structure.

Human α-synuclein (αS) has been shown to be N terminally acetylated in its physiological state. This modification is proposed to modulate the function and aggregation of αS into amyloid fibrils. Using bacterially expressed acetylated-αS (NTAc-αS) and endogenous αS (Endo-αS) from human erythrocytes, we show that N-terminal acetylation has little impact on αS binding to anionic membranes and thus likely not relevant for regulating membrane affinity.

Chip based common-path optical coherence tomography system with an on-chip microlens and multi-reference suppression algorithm.

We demonstrate an integrated optical probe including an on-chip microlens for a common-path swept-source optical coherence tomography system. This common-path design uses the end facet of the silicon oxynitride waveguide as the reference plane, thus eliminating the need of a space-consuming and dispersive on-chip loop reference arm, thereby obviating the need for dispersion compensation. The on-chip micro-ball lens eliminates the need of external optical elements for coupling the light between the chip and the sample.

Size-selective analyte detection with a Young interferometer sensor using multiple wavelengths.

We present a method to discriminate between analytes based on their size using multiple wavelengths in a Young interferometer. We measured the response of two wavelengths when adding 85 nm beads (representing specific binding), protein A (representing non-specific binding) and D-glucose (inducing a bulk change) to our sensor. Next, the measurements are analysed using a approach based on theoretical analysis, and a ratio-based analysis approach to discriminate between bulk changes and the binding of the different sized substances.

Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.

Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells.

Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein.

Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all proteins are known to cross-seed aggregation.