Abstract
alpha-Synuclein (alphaS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. Although unstructured in solution, alphaS forms alpha-helices in the presence of negatively charged lipid surfaces. Moreover, alphaS was shown to interact with FAs in a manner that promotes protein aggregation. Here, we investigate whether alphaS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of alphaS signal in both (1)H and (13)C spectra and ii) the appearance of a very broad FA (13)C-carboxyl signal. These data exclude high-affinity binding of FA molecules to specific alphaS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were easily disrupted to form smaller particles in the presence of alphaS, indicating a direct protein-lipid interaction. We conclude that alphaS is not likely to act as an intracellular FA carrier. Binding to negatively charged membranes, however, appears to be an intrinsic property of alphaS that is most likely related to its physiological role(s) in the cell.
Highlights
Abstract a-Synuclein is an amyloidogenic neuronal protein associated with several neurodegenerative disorders
Supplementary key words multidimensional nuclear magnetic resonance spectroscopy . electron microscopy . fatty acid binding . proteinlipid complexes . neurodegenerative disorder a-Synuclein, a 140 residue cytosolic protein that is water-soluble in its native state, represents in fibrillar form the most abundant protein component of the characteristic Lewy bodies, the pathological hallmark of Parkinson’s disease
With protocols designed to detect specific interactions between a particular protein and individual FA molecules by the use of NMR, we explored whether aS has high-affinity FA binding sites, similar to those revealed for (intestinal) fatty acid binding protein (FABP) and serum albumin via the same method [28, 31]
Summary
Abstract a-Synuclein (aS) is an amyloidogenic neuronal protein associated with several neurodegenerative disorders. We investigate whether aS has specific FA binding site(s) similar to fatty acid binding proteins (FABPs), such as the intracellular FABPs. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of aS signal in both 1H and 13C spectra and ii) the appearance of a very broad FA 13C-carboxyl signal. Our NMR experiments reveal that FA addition results in i) the simultaneous loss of aS signal in both 1H and 13C spectra and ii) the appearance of a very broad FA 13C-carboxyl signal These data exclude high-affinity binding of FA molecules to specific aS sites, as in FABPs. One possible mode of binding was revealed by electron microscopy studies of oleic acid bilayers at pH 7.8; these high-molecular-weight FA aggregates possess a net negative surface charge because they contain FA anions, and they were disrupted to form smaller particles in the presence of aS, indicating a direct proteinlipid interaction. In the example of Alzheimer’s disease, there is evidence that soluble Ab oligomers can interfere with synaptic efficacy in the absence of protofibrils or fibrils [15]
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