Bhasne Karishma, Bogoian-Mullen Antonia, Clerico Eugenia M, Gierasch Lila M
Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst Massachusetts, USA.
Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst Massachusetts, USA.
J Biol Chem. 2024 Dec;300(12):108001. doi: 10.1016/j.jbc.2024.108001. Epub 2024 Nov 16.
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 synaptosomal-associated 25 kDa protein (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 used 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.
神经递质释放过程中，突触小泡与质膜融合的复杂机制始于神经元突触前末端SNARE复合体的形成。SNARE复合体由三种蛋白质贡献的四条螺旋组成：一条来自Syntaxin（定位于质膜），一条来自突触小泡蛋白（定位于突触小泡），两条来自本质无序且易于聚集的突触体相关25 kDa蛋白（SNAP-25），该蛋白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控，需要组成型表达的Hsp70伴侣蛋白（Hsc70）及其J蛋白共伴侣蛋白CSPα。我们推测Hsc70和CSPα协同作用，对SNAP-25进行伴侣介导，抑制其聚集，并使其保持能够形成SNARE复合体的折叠状态。为了验证这一假设，我们采用自下而上的方法，在体外研究了Hsc70和CSPα与SNAP-25之间的相互作用。我们发现，在Hsc70和CSPα存在的情况下，SNAP-25的聚集被延迟。通过使用覆盖SNAP-25序列的肽阵列，我们鉴定出三个潜在的与Hsc70相互作用的序列，并设计了包含这些序列的肽来测试溶液中的结合情况。我们结合生化和生物物理技术，包括非变性聚丙烯酰胺凝胶电泳、荧光各向异性测定结合亲和力、Hsc70的ATP酶活性以及核磁共振，对源自SNAP-25的肽与Hsc70和CSPα的相互作用进行了表征。我们在SNAP-25内鉴定出一个Hsc70结合位点，该位点可能代表细胞中用于促进SNARE复合体形成的位点。
