Arc is a flexible modular protein capable of reversible self-oligomerization
Arc is a flexible modular protein capable of reversible self-oligomerization
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DOI：
10.1042/bj20141446
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发表时间：
2015-05-15
影响因子：
4.1
通讯作者：
Bramham, Clive R.
中科院分区：
文献类型：
--
作者：
Myrum, Craig;Baumann, Anne;Bramham, Clive R.
即时早期基因产物Arc（活性调节的细胞因子相关蛋白）被认为是长期突触可塑性和记忆的主要调节因子。然而，Arc的物理化学和结构特性尚未阐明。在本研究中，我们表达和纯化重组人Arc（hArc），并进行了第一个生化和生物物理分析的hArc的结构和稳定性。有限的蛋白水解测定和MS分析表明，hArc在中心更无序的接头区域的两侧具有两个主要结构域，与计算机结构预测一致。使用CD估计hArc的二级结构，并通过CD监测的热变性和差示扫描荧光法（DSF）分析稳定性。用动态光散射（DLS）和原子力显微镜（AFM）、电子显微镜（EM）研究了不同条件下的齐聚状态。生物物理分析表明，hArc是一个模块化的蛋白质，具有明确的二级结构和松散的三级结构。hArc作为单体看起来是U形的，并且能够可逆地自缔合，形成大的可溶性低聚物。hArc的N-末端结构域是高度碱性的，这可以促进与细胞骨架结构或其他聚阴离子表面的相互作用，而C-末端结构域是酸性的并且通过促进寡聚化的离子条件稳定。在结合早老素-1（PS1）肽后，hArc经历大的结构变化。hArc的非同义遗传变体（V231 G）显示出与野生型（WT）蛋白相似的性质。我们的结论是，hArc是一个灵活的多结构域蛋白，存在于单体和寡聚体的形式，兼容的多样性，枢纽样的可塑性相关的过程中的作用。
The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.