Interaction of α-synuclein with vesicles that mimic mitochondrial membranes

Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 12/2011; 1818(3):512-9. DOI: 10.1016/j.bbamem.2011.11.024
Source: PubMed


α-Synuclein, an intrinsically-disordered protein associated with Parkinson's disease, interacts with mitochondria, but the details of this interaction are unknown. We probed the interaction of α-synuclein and its A30P variant with lipid vesicles by using fluorescence anisotropy and (19)F nuclear magnetic resonance. Both proteins interact strongly with large unilamellar vesicles whose composition is similar to that of the inner mitochondrial membrane, which contains cardiolipin. However, the proteins have no affinity for vesicles mimicking the outer mitochondrial membrane, which lacks cardiolipin. The (19)F data show that the interaction involves α-synuclein's N-terminal region. These data indicate that the middle of the N-terminal region, which contains the KAKEGVVAAAE repeats, is involved in binding, probably via electrostatic interactions between the lysines and cardiolipin. We also found that the strength of α-synuclein binding depends on the nature of the cardiolipin acyl side chains. Eliminating one double bond increases affinity, while complete saturation dramatically decreases affinity. Increasing the temperature increases the binding of wild-type, but not the A30P variant. The data are interpreted in terms of the properties of the protein, cardiolipin demixing within the vesicles upon binding of α-synuclein, and packing density. The results advance our understanding of α-synuclein's interaction with mitochondrial membranes.

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    • "They hypothesized that the variations in mitochondria morphology are due to the direct interaction of αsyn oligomers with mitochondrial membranes. This idea is further supported by the evidence of αsyn interaction with mitochondria [36] and localization of αsyn at membranes mimicking mitochondrial membranes [42] or within mitochondria [61]. Moreover, it was proposed that αsyn can inhibit mitochondrial fusion processes, again because of the protein special interactions with mitochondrial membranes [43] or that mitochondrial fission is increased through an increased Drp1 translocation to mitochondria due to αsyn overexpression [62]. "
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    ABSTRACT: Background Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease. Methods We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging. Results Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria may be dysfunctional. Conclusions We showed that alpha-synuclein overexpression in SH-SY5Y causes the formation of alpha-synuclein oligomeric species, whose presence is associated with mitochondrial fragmentation and autophagic-lysosomal pathway activation in live cells. General significance The unique capability provided by the Number and Brightness analysis to study alpha-synuclein oligomers distribution and properties, and the study their association to intracellular components in single live cells is important to forward our understanding of the molecular mechanisms Parkinson’s disease and it may be of general significance when applied to the study of other aggregating proteins in cellular models.
    Biochimica et Biophysica Acta (BBA) - General Subjects 06/2014; 1840(6). DOI:10.1016/j.bbagen.2014.02.013 · 4.38 Impact Factor
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    • "It has been suggested to be a signaling molecule in apoptosis [1], [2], and changes in cardiolipin concentration have been related to a number of different diseases and pathological conditions, including Parkinson's disease, ageing and heart failure [3]–[5]. Many proteins have high affinity for cardiolipin-containing membranes, for example, α-synuclein, a amyloid protein related to Parkinson's disease [6], [7], and membrane proteins that are involved in the synthesis of ATP in the mitochondrion [8]–[11]. "
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    ABSTRACT: Cardiolipin is a phospholipid found in the inner mitochondrial membrane and in bacteria, and it is associated with many physiological functions. Cardiolipin has a dimeric structure consisting of two phosphatidyl residues connected by a glycerol bridge and four acyl chains, and therefore it can carry two negative charges. The pKa values of the phosphate groups have previously been reported to differ widely with pKa1 = 2.8 and pKa2 = 7.5-9.5. Still, there are several examples of experimental observations from cardiolipin-containing systems that do not fit with this dissociation behavior. Therefore, we have carried out pH-titration and titration calorimetric experiments on two synthetic cardiolipins, 1,1',2,2'-tetradecanoyl cardiolipin, CL (C14∶0), and 1,1',2,2'-tetraoctadecenoyl cardiolipin, CL (C18∶1). Our results show that both behave as strong dibasic acids with pKa1 about the same as the first pKa of phosphoric acid, 2.15, and pKa2 about one unit larger. The characterization of the acidic properties of cardiolipin is crucial for the understanding of the molecular organization in self-assembled systems that contain cardiolipin, and for their biological function.
    PLoS ONE 09/2013; 8(9):e73040. DOI:10.1371/journal.pone.0073040 · 3.23 Impact Factor
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    • "The α-synuclein gene encodes a 140 amino acid residue protein that is expressed ubiquitously in the brain and is enriched in presynaptic terminals [1], [2]. The α-synuclein protein exists mostly as an unfolded soluble monomer of ∼14 kDa, but it can assume an amphipathic, α-helical conformation when bound to acidic phospholipids in a variety of organelles including lysosomes, mitochondria, and endoplasmic reticulum/Golgi vesicles [3]–[8]. In keeping with a presynaptic function, both mutant and over-expressed forms of normal human α-synuclein interact with synaptic vesicles at presynaptic terminals and have been shown to negatively impact synaptic vesicle function, likely at a step prior to docking and upstream of the pool of vesicles poised for rapid neurotransmitter release [9]–[13]. "
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    ABSTRACT: While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.
    PLoS ONE 05/2013; 8(5):e63557. DOI:10.1371/journal.pone.0063557 · 3.23 Impact Factor
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