Structural and Functional Analysis of the Sigma-1 Receptor

描述（由申请人提供）：σ受体是生物学上重要的整合膜受体家族，其对于学习、记忆、药物成瘾和衰老疾病如阿尔茨海默病至关重要。虽然在过去的几十年中已经报道了这些受体的功能数据，但结构理解完全缺失，这是整合膜蛋白（IMP）的共同特征。对于该提议最重要的是，sigma-1受体一级序列不类似于任何其他哺乳动物蛋白，因此可能具有新的IMP折叠。此外，已知sigma- 1作为GPCR介导的钙信号传导的配体调节放大器，这是一种新的蛋白质功能。因此，为了进一步增强我们对sigma-1受体、其新活性和IMP折叠空间的理解，我们需要确定其三维结构。这也将使我们能够实现我们的长期目标，使sigma受体家族成为一个明确的和有效的药物靶点。在过去的几年中，核磁共振波谱已成为一个主要的实验工具的结构研究的IMP。我们将使用功能数据来指导我们的结构生物学工作，以确定使用NMR光谱的sigma-1受体的三维结构。我们将表达，纯化，溶解和确定的sigma-1受体的结构，使用液态核磁共振光谱。为此，我们将仔细表征重组表达和再溶解的σ-1受体在各种洗涤剂中的活性。这将使我们能够找到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。sigma-1的三维结构将用于进一步了解其关键的生物学功能。具体而言，我们将使用NMR光谱和其他生物物理技术来筛选一个小型定制的结构和功能多样的sigma-1受体配体库，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位点进行分类。总之，这项工作将构成我们目标的基础，使sigma受体家族成为一个全面表征的药物靶标，对于寻找药物成瘾和各种衰老疾病的治疗方法至关重要。Sigma-1受体是细胞信号传导的有效增强剂，与药物成瘾、癌症和阿尔茨海默病等疾病直接相关。只有通过提供这种蛋白质结构的三维模型，才能成功地采用基于药物设计的技术，这种技术已经成功地用于艾滋病、流感、青光眼和关节炎。因此，这项研究计划需要及时执行，以建立sigma受体家族作为一个充分表征的药物靶点。

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.