Α-syn Membrane Binding Research Articles

Influence of Cholesterol on the Membrane Binding and Conformation of α-Synuclein.

The α-Synuclein (α-Syn) plays an important role in the pathology of Parkinson's disease (PD), and its oligomers and fibrils are toxic to the nervous system. As organisms age, the cholesterol content in biological membranes increases, which is a potential cause of PD. Cholesterol may affect the membrane binding of α-Syn and its abnormal aggregation, but the mechanism remains unclear. Here, we present our molecular dynamics simulation studies on the interaction between α-Syn and lipid membranes, with or without cholesterol. It is demonstrated that cholesterol provides additional hydrogen bond interaction with α-Syn; however, the coulomb interaction and hydrophobic interaction between α-Syn and lipid membranes could be weakened by cholesterol. In addition, cholesterol leads to the shrinking of lipid packing defects and the decrease of lipid fluidity, thereby shortening the membrane binding region of α-Syn. Under these multifaceted effects of cholesterol, membrane-bound α-Syn shows signs of forming a β-sheet structure, which may further induce the formation of abnormal α-Syn fibrils. These results provide important information for the understanding of membrane binding of α-Syn, and they are expected to promote the bridging between cholesterol and the pathological aggregation of α-Syn.

Protein kinase R dependent phosphorylation of α-synuclein regulates its membrane binding and aggregation.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

Restricting α-synuclein transport into mitochondria by inhibition of α-synuclein-VDAC complexation as a potential therapeutic target for Parkinson's disease treatment.

Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outermembrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Membrane Interactions of α-Synuclein Probed by Neutrons and Photons.

α-Synuclein (α-syn) is a key protein in the etiology of Parkinson's disease. In a disease state, α-syn accumulates as insoluble amyloid fibrils enriched in β-sheet structure. However, in its functional state, α-syn adopts an amphipathic helix upon membrane association and plays a role in synaptic vesicle docking, fusion, and clustering. In this Account, we describe our contributions made in the past decade toward developing a molecular understanding of α-syn membrane interactions, which are crucial for function and have pathological implications. Three topics are covered: α-syn membrane binding probed by neutron reflectometry (NR), the effects of membrane on α-syn amyloid formation, and interactions of α-syn with cellular membranes.NR offers a unique perspective by providing direct measurements of protein penetration depth. By the use of segmentally deuterated α-syn generated through native chemical ligation, the spatial resolution of specific membrane-bound polypeptide regions was determined by NR. Additionally, we used NR to characterize the membrane-bound complex of α-syn and glucocerebrosidase, a lysosomal hydrolase whose mutations are a common genetic risk factor for Parkinson's disease. Although phosphatidylcholine (PC) is the most abundant lipid species in mammalian cells, interactions of PC with α-syn have been largely ignored because they are substantially weaker compared with the electrostatically driven binding of negatively charged lipids. We discovered that α-syn tubulates zwitterionic PC membranes, which is likely related to its involvement in synaptic vesicle fusion by stabilization of membrane curvature. Interestingly, PC lipid tubules inhibit amyloid formation, in contrast to anionic phosphatidylglycerol lipid tubules, which stimulate protein aggregation. We also found that membrane fluidity influences the propensity of α-synuclein amyloid formation. Most recently, we obtained direct evidence of binding of α-syn to exocytic sites on intact cellular membranes using a method called cellular unroofing. This method provides direct access to the cytosolic plasma membrane. Importantly, measurements of fluorescence lifetime distributions revealed that α-syn is more conformationally dynamic at the membrane interface than previously appreciated. This exquisite responsiveness to specific lipid composition and membrane topology is important for both its physiological and pathological functions. Collectively, our work has provided insights into the effects of the chemical nature of phospholipid headgroups on the interplay among membrane remodeling, protein structure, and α-syn amyloid formation.

Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn-membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm ). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34-55 °C) and preserved in its homologs β- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16-20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.

Exploration of membrane binding of α‐synuclein through Site Based Spin Labeling

Neurodegenerative conditions, including Alzheimer's and Parkinson's disease, are becoming more prevalent in the aging population, and one of the main perpetrators behind these symptoms is the protein α‐synuclein (α‐syn). The cytosolic neural protein has a role in neurotransmitter transport within cells through membrane binding to intracellular vesicles. It is strongly linked to Parkinson's disease as a result of the A30P, E46K, and A53T point mutations, which are believed to cause α‐syn aggregation. These point mutations disrupt the formation of amphipathic α‐helices that directly bind to the vesicle surface. To gain further information on the membrane binding tendencies of α‐syn, each of these mutations, as well as a neutrally mutated C‐terminal tail (CTT), is being separately tested using site directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. The normally negatively charged CTT is the only region of the protein that does not directly contact the phospholipids during the membrane binding. Each of the mutant proteins' interactions with synthesized lipid vesicles will confirm the known malfunctions of the Parkinson's mutations, in addition to expanding of knowledge of the C‐terminal tail and its function in the membrane binding process. Results and analysis from the EPR spectroscopy are currently still being compiled. It is expected that each of the Parkinson's mutations and neutrally mutated CTT will decrease α‐syn membrane binding. These, or other conclusions from the analysis, can provide more information on the uncommonly studied α‐synuclein CTT and a basis on which to continue research for future undergraduate biochemistry students at Westminster College.Support or Funding InformationWestminster College Drinko Center Undergraduate Research GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Alpha-Synuclein Binding and Translocation by Alpha-Hemolysin Channel

Gauging the pathways of a natively unfolded Parkinson disease-related protein, α-synuclein (α-syn), between and within cells, is important for understanding pathogenesis. Here, we use a robust β-barrel ion channel, α-hemolysin (α-HL), as a model system to investigate the possibility of channel-assisted membrane transport of α-syn. The interaction of α-syn with the channel was observed as a transient, ∼95 % block of the channel current when (i) α-syn was applied from the membrane side where the shorter (stem) part of the α-HL heptamer is exposed and (ii) the applied potential is lower on the side of α-syn application. While the on-rate of α-syn binding to the channel exponentially increases with the applied field, the off-rate displays a crossover behavior. Statistical analysis of the blockage events indicates that at voltages >80 mV a significant portion of α-HL-bound α-syn undergoes subsequent translocation. The observed on-rate varies by more than 100 times, depending on bilayer lipid composition. To delineate the role of specific domains, N-terminal membrane-interacting and acidic C-terminal tail, we produced and examined 1) a C-terminal truncation variant lacking the last 25 amino acids (α-syn115) and 2) a synthetic peptide composed of the last 23 residues (C23). In the case of α-syn115, the absence of the highly negatively charged C-terminal tail leads to a significant decrease in the rates of both binding and translocation events. For C23, no time-resolvable blockades of the channel current could be detected, suggesting that membrane binding of α-syn plays an important role in the translocation process. Surprisingly, we found that α-syn can also effectively block mitochondrial channel VDAC, an endogenous β-barrel protein, suggesting that “large” membrane pores may serve as gateways for α-synuclein transport in vivo.

Effect of Ser-129 Phosphorylation on Interaction of α-Synuclein with Synaptic and Cellular Membranes

In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.

RETRACTED ARTICLE: Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors

BackgroundAlpha-Synuclein (α-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the α-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of α-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.ResultsIn the present study, we analysed the ability of cytosolic factors to regulate α-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant α-syn. To characterize cytosolic factor(s) that modulate α-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate α-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T α-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance α-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P α-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.ConclusionThese findings suggest that endogenous brain cytosolic factors regulate Wt and mutant α-syn membrane binding, and could represent potential targets to influence α-syn solubility in brain.