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: 7683733
• 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/7683733
• 关键词: Address; Adhesions; Affinity Chromatography; Apoptosis; Biological; Biological Assay; Cancer Biology; Cell Communication; Cell Differentiation process; Cells; Computer Simulation; Computer software; 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; 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; overexpression; polyhistidine; programs; protein structure; protocol development; research study; response; scale up; structural biology; tool; vector

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¿ 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 tragets 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 imagerto view and document crystallization experiments. We will solve the structures of CRIMPs that form high quality crystals.

细胞分化、黏附和运动、增殖和凋亡都是对细胞外的反应。 环境。膜蛋白检测环境刺激，并引发分子和遗传反应 它们决定了细胞的命运。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起着中心作用，但没有高分辨率的结构 这些蛋白质是存在的。我们的目标是解决这一差距的分子机制的理解 癌症。我们将启动癌症相关膜蛋白结构生物学的全面研究 (卷边)。我们选择了与信号通路、细胞-细胞相互作用有关的膜蛋白靶标 运输对于癌症生物学来说是不可或缺的。拟议的研究的目标不是解决这些结构 所有的目标卷曲。然而，我们提出的方法和大的、多样化的目标相结合 SET使解算多个卷曲结构变得逼真。任何目标的结构都将有助于 对癌症生物学的理解具有重要意义。然而，膜的用途很难预测。 蛋白质结构在药物设计中，治疗潜力是我们选择靶点的一个重要因素，并且 卷曲的结构可能有助于治疗药物或方案的开发。我们还将生成 人膜蛋白在巴斯德毕赤酵母中异源表达的研究。在课程期间 在拟议的研究中，我们将完成迄今为止关于表达 巴斯德毕赤酵母中的人膜蛋白。拟议研究的具体目标是： 1.鉴定与癌症相关的、易结晶的人膜蛋白。我们将选出60个 基于癌症相关性和预测程度的结构和功能差异的膜蛋白 无序。选择具有有限无序的卷曲将提高成功结晶的几率。 2.鉴定60种不同肿瘤相关膜蛋白在毕赤酵母中的异源表达 牧羊人。我们将使用新型Gateway载体、10毫升巴斯德酵母生长、斑点杂交和小规模 洗涤剂增溶实验快速评价异源表达和洗涤剂溶解度 目标卷曲的数量。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过度表达并形成良好晶体的结构 重大修改。我们将使用一种新的膜蛋白溶解度筛选来确定最佳的 结晶的目标浓度我们将使用结晶机器人来设置1024个条件 120微升蛋白质(~100毫升/次实验)。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。