Structural and Functional Analysis of the Sigma-1 Receptor

描述(申请人提供)：Sigma受体是一个生物学上重要的整合膜受体家族，对学习、记忆、药物成瘾和老年性疾病(如阿尔茨海默病)至关重要。虽然这些受体的功能数据在过去的几十年里已经被报道，但对于完整的膜蛋白(IMP)的一个共同特征--结构上的理解是完全缺失的。对于这一提议最重要的是，sigma-1受体的初级序列不像任何其他哺乳动物蛋白质的序列，因此可能有一个新的IMP折叠。此外，Sigma-1被认为是GPCR介导的钙信号的配体调节的放大器，这是一种新的蛋白质功能。因此，为了进一步加深我们对sigma-1受体及其新活性和IMP折叠空间的了解，我们需要确定它的三维结构。这也将使我们能够实现我们的长期目标，使sigma受体家族成为明确和有效的药物靶点。在过去的几年里，核磁共振波谱已经成为IMPS结构研究的主要实验工具。我们将使用功能数据来指导我们的结构生物学努力，以使用核磁共振光谱来确定sigma-1受体的三维结构。我们将使用液态核磁共振波谱来表达、纯化、溶解和确定sigma-1受体的结构。为此，我们将仔细研究各种洗涤剂中重组表达和分解的sigma-1受体的活性。这将使我们能够找到用于活性和结构研究的最佳胶束。最后，我们将在确定的最佳条件下对sigma-1进行深入的药理学表征。然后将使用sigma-1的三维结构来进一步了解其关键的生物学功能。具体地说，我们将使用核磁共振光谱和其他生物物理技术来筛选结构和功能不同的sigma-1受体配体的小型文库，包括激动剂和拮抗剂，以确定负责结合的sigma-1残基和结构域。这将使我们能够对sigma-1受体上不同的激动剂和拮抗剂结合部位进行分类。总之，这项工作将为我们的目标奠定基础，使Sigma受体家族成为一个全面表征的药物靶标，对于寻找治疗药物成瘾和各种衰老疾病的方法至关重要。与公共健康相关的Sigma-1受体是细胞信号的有效增强剂，与药物成瘾、癌症和阿尔茨海默病等疾病直接相关。只有提供这种基于蛋白质结构的三维模型，药物设计才能成功应用，这项技术已经在艾滋病、流感、青光眼和关节炎方面取得了成功。因此，这项研究计划需要及时执行，以建立Sigma受体家族作为一个具有良好特征的药物靶点。

DESCRIPTION (provided by applicant): Sigma receptors are a biologically important family of integral membrane receptors that are critical for learning, memory, drug addiction and aging diseases such as Alzheimer's disease. While functional data for these receptors has been reported during the last decades, a structural understanding is completely missing, a common feature for integral membrane proteins (IMP). Most important for this proposal, the sigma-1 receptor primary sequence does not resemble that of any other mammalian protein and thus potentially have a novel IMP fold. Furthermore, sigma- 1 is known to act as a ligand-regulated amplifier of GPCR-mediated calcium signaling, a novel protein function. Therefore, to further enhance our understanding of the sigma-1 receptor, its novel activity and the IMP fold space in general, we need to determine its 3-dimensional structure. This will also allow us to achieve our long-term goal of making the sigma receptor family a well-defined and potent drug target. During the last few years, NMR spectroscopy has become a major experimental tool for structural investigations of IMPs. We will use functional data to guide our structural biology efforts to determine the 3-dimensional structure of the sigma-1 receptor using NMR spectroscopy. We will express, purify, solubilize and determine the structure of the sigma-1 receptor using liquid state NMR spectroscopy. To do this we will carefully characterize the activity of the recombinantly expressed and resolubilized sigma-1 receptor in a variety of detergents. This will enable us to find the optimal micelle for activity and structural studies. Lastly, we will pursue an in-depth pharmacological characterization of sigma- 1 in the identified optimal conditions. The sigma-1 3-dimensional structure will then be used to further the understanding of its critical biological functions. Specifically, we will use NMR spectroscopy and additional biophysical techniques to screen a small tailored library of structurally and functionally diverse sigma-1 receptor ligands, both agonists and antagonists, to identify sigma-1 residues and domains responsible for binding. This will enable us to categorize distinct agonist and antagonist binding sites on the sigma-1 receptor. Together, this work will form the foundation of our goal to make the sigma receptor family a comprehensively characterized drug target, critical for finding cures for drug addiction and a variety of aging diseases. PUBLIC HEALTH RELEVANCE Sigma-1 receptors are potent enhancers of cell signaling and directly linked with diseases such as drug addiction, cancer and Alzheimer's disease. Only by providing a 3-dimensional model of this protein structure based drug design can be successfully employed, a technique which has been a success story for AIDS, flu, glaucoma and arthritis. Therefore, this research plan needs to be executed in a timely manner to establish the sigma receptor family as a well-characterized drug target.