The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal of the neuron. The SNARE complex is composed of four helices contributed by three proteins: one from syntaxin (localized at the plasma membrane), one from synaptobrevin (localized at the synaptic vesicle), and two from the intrinsically disordered and aggregation-prone SNAP-25, which is localized to the plasma membrane by virtue of palmitoylation of cysteine residues. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its J-protein co-chaperone CSPα. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25, disfavoring its aggregation and keeping it in a folding state competent for SNARE complex formation. To test this hypothesis, we employed a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. We showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25-derived peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, including native-PAGE, binding affinity by fluorescence anisotropy, ATPase-activity of Hsc70, and NMR. We have identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell to facilitate SNARE complex formation.

中文翻译：

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Hsc70 系统将突触 SNARE 蛋白 SNAP-25 维持在组装感受态并延迟其聚集。


突触小泡与质膜融合以释放神经递质的复杂机制是由神经元突触前末端 SNARE 复合物的形成启动的。SNARE 复合物由三种蛋白质贡献的四个螺旋组成：一个来自突触融合素（位于质膜），一个来自突触蛋白（位于突触小泡），两个来自固有无序且易聚集的 SNAP-25，它通过半胱氨酸残基的棕榈酰化定位于质膜。融合过程受到严格调节，需要组成型表达的 Hsp70 伴侣 （Hsc70） 及其 J 蛋白共伴侣 CSPα。我们假设 Hsc70 和 CSPα 合作陪伴 SNAP-25，不利于其聚集并保持其处于能够形成 SNARE 复合物的折叠状态。为了检验这一假设，我们采用了自下而上的方法，并在体外研究了 Hsc70 和 CSPα 与 SNAP-25 之间的相互作用。我们表明，在 Hsc70 和 CSPα 存在下，SNAP-25 的聚集会延迟。使用跨越 SNAP-25 序列的肽阵列，我们确定了三个潜在的 Hsc70 相互作用序列，并设计了包含这些序列的肽以测试在溶液中的结合。我们使用生化和生物物理技术的组合表征了 SNAP-25 衍生肽与 Hsc70 和 CSPα 的相互作用，包括天然 PAGE、荧光各向异性的结合亲和力、Hsc70 的 ATP 酶活性和 NMR。我们在 SNAP-25 中鉴定了一个 Hsc70 结合位点，它可能代表细胞中用于促进 SNARE 复合物形成的位点。