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

毕赤酵母表达的癌症相关膜蛋白的结构生物学

• 批准号: 7313169
• 负责人: Michael Wiener
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313169
• 关键词: Address; Adhesions; Affinity Chromatography; Apoptosis; Biological; Biological Assay; Cancer Biology; Cell Communication; Cell Differentiation process; Cells; Computer Simulation; Computer software; Condition; Crystallization; Data; Databases; Detergents; Development; Disease; Dot Immunoblotting; Drug Design; Ensure; Environment; Gel Chromatography; Gene Targeting; Goals; Growth; Housing; Human; Image; In Vitro; Integral Membrane Protein; Laboratories; Liquid substance; Literature; Malignant Neoplasms; Medical; Membrane; Membrane Proteins; Metals; Methods; Modification; Molecular; Molecular Genetics; Pharmaceutical Preparations; Phase; Photons; Pichia; Polishes; Probability; Process; Protein Overexpression; Proteins; Protocols documentation; Publishing; Reagent; Recombinants; Research; Research Personnel; Resolution; Robot; Robotics; Role; Sampling; Screening procedure; Sequence Analysis; Signal Pathway; Solubility; Source; Stimulus; Structure; System; Therapeutic; Writing; base; beamline; cell motility; design; extracellular; glycosylation; in vivo; instrumentation; novel; polyhistidine; programs; protein structure; protocol development; research study; response; scale up; size; structural biology; tool; vector

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.

描述（由申请人提供）：细胞分化、粘附和运动、增殖和凋亡响应于细胞外环境而发生。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。当这些膜蛋白协调过程发生故障时，就会产生癌症。尽管人类膜蛋白在癌症生物学中发挥着重要作用，但这些蛋白质的高分辨率结构并不存在。我们的目标是解决癌症分子机制理解中的这一差距。我们将启动癌症相关膜蛋白（CRIMPs）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点，这些靶点是癌症生物学不可或缺的。所提出的研究的目标不是解决所有目标CRIMPs的结构。然而，我们提出的方法和大的，不同的目标集相结合，使解决几个CRIMP结构现实。任何靶点的结构都将对癌症生物学的理解做出重大贡献。虽然很难预测膜蛋白结构在药物设计中的效用，但治疗潜力是我们靶点选择的重要因素，CRIMPs的结构可能有助于治疗药物或方案的开发。我们还将获得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关于人膜蛋白在巴斯德毕赤酵母中表达的最全面的分析。本研究的具体目的是：1.鉴定与癌症相关的易于结晶的人类膜蛋白。我们将根据癌症相关性和预测的疾病程度选择60种结构和功能不同的膜蛋白。选择具有有限无序的CRIMP将提高成功结晶的概率。2.评估60种不同癌症相关膜蛋白在毕赤酵母中的异源表达。我们将使用新的网关？1/2载体、10 ml巴斯德毕赤酵母生长、斑点印迹和小规模去污剂溶解实验，以快速评估靶CRIMP的异源表达和去污剂溶解度。通过增溶的所有实验将对12个靶批次进行。3.解决癌症相关膜蛋白的结构，这些蛋白过度表达并形成良好的晶体，而没有显著的修饰。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设置120微升蛋白质的1024个条件（每个实验约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CRIMPs的结构，形成高质量的晶体。