Structural Biology of Cancer Related Membrane Proteins Expressed in P. Pastoris

描述(由申请人提供)：细胞分化、黏附和运动、增殖和凋亡是对细胞外环境的反应。膜蛋白检测环境刺激，并引发决定细胞命运的分子和遗传反应。当这些膜蛋白协调的过程出现故障时，癌症就会发生。尽管人膜蛋白在癌症生物学中扮演着中心角色，但这些蛋白并不存在高分辨率结构。我们的目标是解决在了解癌症分子机制方面的这一差距。我们将启动癌症相关膜蛋白(CRIMP)结构生物学的全面研究。我们选择了参与信号通路、细胞间相互作用和转运的膜蛋白靶标，这些都是癌症生物学中不可或缺的部分。拟议研究的目标不是解决所有目标卷曲的结构。然而，我们提出的方法与庞大、多样化的目标集相结合，使得解决几种卷曲结构变得现实。任何靶点的结构都将对了解癌症生物学做出重大贡献。虽然很难预测膜蛋白结构在药物设计中的用途，但治疗潜力是我们选择靶点的一个重要因素，卷曲结构可能有助于治疗药物或方案的开发。我们还将产生关于人膜蛋白在巴斯德毕赤酵母中异源表达的有价值的数据。在拟议的研究过程中，我们将完成迄今为止对人膜蛋白在巴斯德毕赤酵母中表达的最全面的分析。这项研究的具体目的是：1.鉴定与癌症相关的、易结晶的人膜蛋白。我们将根据癌症的相关性和预测的紊乱程度，选择60种结构和功能不同的膜蛋白。选择具有有限无序的卷曲将提高成功结晶的几率。2.研究60种不同肿瘤相关膜蛋白在巴斯德毕赤酵母中的异源表达。我们将使用新的Gateway？1/2载体、10ml巴斯德毕赤酵母生长、斑点杂交和小规模洗涤剂增溶实验来快速评估目标CRIMPS的异源表达和洗涤剂溶解性。所有通过增溶的实验将在12个目标的批次上进行。3.解决与癌症相关的膜蛋白的过度表达和形成良好晶体的结构，而不需要进行显著的修饰。我们将使用一种新的膜蛋白溶解度筛选来确定结晶目标的最佳浓度。我们将使用结晶机器人来设置1024个条件，其中包含120微升蛋白质(每个实验~100 NL)。我们将使用实验室编写的软件和流体处理机器人来优化结晶。我们将使用自动平板成像仪来查看和记录结晶实验。我们将解决形成高质量晶体的卷曲结构。

DESCRIPTION (provided by applicant): Cell differentiation, adhesion and motility, proliferation and apoptosis occur in response to the extracellular environment. Membrane proteins detect environmental stimuli, and elicit molecular and genetic responses that determine cell fate. Cancer results when these membrane-protein-orchestrated processes malfunction. Despite the central roles of human membrane proteins in cancer biology, no high-resolution structures of these proteins exist. Our goal is to address this gap in the understanding of the molecular mechanisms of cancer. We will initiate a comprehensive study of the structural biology of cancer-related membrane proteins (CRIMPs). We selected membrane protein targets involved in signaling pathways, cell-cell interactions, and transport that are integral to cancer biology. The goal of the proposed research is not to solve the structures of all of the target CRIMPs. However, the combination of our proposed methods and the large, diverse target set makes solving several CRIMP structures realistic. Structures of any of the targets will contribute significantly to the understanding of cancer biology. While it is difficult to predict the utility of membrane protein structures in drug design, therapeutic potential was an important factor in our target selection, and structures of CRIMPs may facilitate therapeutic drug or protocol development. We will also generate valuable data on the heterologous expression of human membrane proteins in P. pastoris. During the course of the proposed research we will complete the most comprehensive analysis to date on the expression of human membrane proteins in P. pastoris. The specific aims of the proposed research are: 1. Identify cancer related human membrane proteins that are amenable to crystallization. We will select 60 structurally and functionally diverse membrane proteins based on cancer relevance and degree of predicted disorder. Selecting CRIMPs with limited disorder will enhance the probability of successful crystallization. 2. Evaluate the heterologous expression of sixty diverse cancer related membrane proteins in Pichia pastoris. We will use novel Gateway¿1/2 vectors, 10 ml P. pastoris growths, dot-blots, and small scale detergent solubilization experiments to rapidly evaluate the heterologous expression and detergent solubility of target CRIMPs. All experiments through solubilization will be performed on batches of twelve targets. 3. Solve the structures of cancer-related membrane proteins that overexpress and form good crystals without significant modification. We will use a novel membrane protein solubility screen to determine the optimal concentrations of targets for crystallization. We will use a crystallization robot to set up 1024 conditions with 120 microliters of protein (~100 nl per experiment). We will use lab-written software and a fluid-handling robot to optimize crystallization. We will use an automated plate imager to view and document crystallization experiments. We will solve the structures of CRIMPs that form high quality crystals.