新型分子伴侣蛋白Spy内在无序区域的功能研究及机制探索

It has long been assumed that protein structure dictates function, however, this notion has been challenged by recent advance on the study of intrinsically disordered regions in proteins. Unstructured but functional, these regions can exist simultaneously in different, yet highly dynamic conformations and play important roles in key biological processes, such as regulation of transcription and translation, cellular signal transduction, and molecular recognition. Molecular chaperones are a large and diverse group of proteins that assist in the folding and assembly of macromolecules. It is becoming clear that structural disorder also exists in molecular chaperones, and its roles in the mechanism of chaperone's action have increasingly been explored in the last few years. The recently discovered chaperone Spy possesses long disordered regions that account for 30% of its residues. Our previous work on enhancing Spy's chaperone activity has showed that the activity-enhancing Spy variants were generally more unstable than is wild type Spy and had increased apparent flexibility, indicating that intrinsic disorder is important for determining Spy's activity. We decide to further investigate the functional contribution of Spy's intrinsically disordered regions, by focusing on their roles in substrate recognition, binding, and release using a combination of bacterial genetic, biochemical and biophysical techniques. We also aim to determine the mechanism of Spy by which it assists the folding/refolding of substrate proteins in an ATP-independent manner. The implementation of our proposed research will provide direct experimental evidence of the role of disorder in chaperone function and may also reveal novel chaperone mechanisms. Our result will also bring new insights in investigating more complex chaperone systems, such as the Hsp60 and Hsp70 chaperone systems, therefore advance our understanding of protein intrinsic disorder, the function of molecular chaperones and the mechanism of protein folding.

传统认为蛋白质的结构决定其功能，近年来对蛋白质中内在无序区域的研究挑战了这一观点。这些区域被认为是多种动态结构的集合，在转录与翻译调控、细胞信号转导、分子识别等关键过程中发挥重要作用。在分子伴侣蛋白这一大类帮助生物大分子折叠与组装的关键蛋白中也存在内在无序区域，探索其功能成为分子伴侣研究领域的一个热点问题。新型分子伴侣蛋白Spy约30%的残基位于无序区域，前期研究表明增加Spy内在无序性能够增加其分子伴侣活力，但作用机制还有待确定。本项目拟深入探讨Spy内在无序性区域在其识别、结合与释放底物中的作用，并提出Spy不依赖ATP和辅助因子促进底物蛋白折叠的分子机制。本项目将为分子伴侣内在无序区域的功能性研究提供直接的实验证据，揭示新型分子伴侣机制，为解析如Hsp60、Hsp70等复杂分子伴侣系统的机制提供新思路，从而显著提高对蛋白质内在无序、分子伴侣结构与功能的关系和蛋白质折叠机制的理解。

蛋白质中内在无序区域被认为是多种动态结构的集合，在转录与翻译调控、细胞信号转导、分子识别等关键过程中发挥重要作用。在分子伴侣蛋白这一大类帮助生物大分子折叠与组装的关键蛋白中也发现了内在无序区域，其存在被认为有助于增强分子伴侣结构灵活性，从而影响分子伴侣功能的发挥，但阐述其中具体机制的研究还屈指可数。本项目以新型分子伴侣蛋白Spy为研究对象，详细解析了其末端无序区域在结合与释放底物中的作用。我们发现Spy的C端对其功能的影响不大，但在去除Spy的N端26个残基之后，突变体的分子伴侣活力得到明显提升，释放底物的速率更慢，与底物的亲和力上升。进一步，我们发现Spy的无序N端能够采取与底物蛋白类似的方式，与Spy呈摇篮形结构的主体部分结合；针对第26位天冬氨酸的突变减弱了这种结合，揭示D26是介导Spy无序N端与自身结合的关键残基。在体外分子伴侣测活实验中，Spy N端肽段的添加减弱了Spy的分子伴侣活力，表明Spy N端肽段可直接与底物蛋白竞争Spy主体上的结合位点，我们推测这将促进折叠好的底物从Spy表面释放。将D26突变为A/K/R 之后，Spy N端肽段对Spy分子伴侣活力的抑制减弱，因此D26可能通过干扰底物与Spy之间的静电相互作用从而导致底物的释放。. 由此，我们提出了Spy利用自身无序片段竞争性释放底物的模型。这项研究揭示了一种全新的分子伴侣作用机制，为分子伴侣内在无序区域的功能性研究提供了直接的实验证据，也为解析其他复杂分子伴侣系统的机制提供了新思路。受该基金支助的SCI论文共计2篇（1篇为通讯作者），还有1篇文章在准备投稿中。