A combinatorial code for the interaction of alpha-synuclein with membranes.
A combinatorial code for the interaction of alpha-synuclein with membranes.
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发表时间：
2005
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通讯作者：
S. Kubo;V. Nemani;R. Chalkley;M. Anthony;N. Hattori;Y. Mizuno;R. Edwards;Doris L. Fortin
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作者：
S. Kubo;V. Nemani;R. Chalkley;M. Anthony;N. Hattori;Y. Mizuno;R. Edwards;Doris L. Fortin
大量的遗传学和病理学证据表明，小分子可溶性蛋白质α - 突触核蛋白与家族性和散发性帕金森病（PD）的发病机制有关。然而，α - 突触核蛋白在疾病过程中的确切作用及其正常功能仍知之甚少。我们最近发现，与脂筏的相互作用对α - 突触核蛋白在突触前的正常定位至关重要。为了了解α - 突触核蛋白如何与脂筏相互作用，我们现在开发了一种体外结合测定法，用于检测从天然膜纯化的脂筏。重组野生型α - 突触核蛋白（而非与PD相关的A30P突变型α - 突触核蛋白）与从培养细胞和纯化的突触小泡中分离出的脂筏结合，这重现了体内观察到的特异性。对脂筏进行蛋白水解消化不会破坏α - 突触核蛋白的结合，这表明它是与这些膜的脂质成分而非蛋白质成分相互作用。我们还发现，α - 突触核蛋白直接与脂质组成模拟脂筏的人工膜结合。α - 突触核蛋白与这些类脂筏脂质体的结合需要酸性磷脂，且更倾向于磷脂酰丝氨酸（PS）。有趣的是，各种具有特定酰基链的合成PS单独使用时并不支持结合。相反，α - 突触核蛋白的相互作用需要PS与油酸（18:1）和多不饱和（20:4或22:6）脂肪酰基链的组合，这表明膜内的相分离起作用。此外，α - 突触核蛋白对多不饱和脂肪酰基链上具有PS头部基团而非油酰侧链上具有PS头部基团的人工膜具有更高的亲和力，这表明α - 突触核蛋白与膜相互作用存在严格的组合规则。
Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.