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; 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（由适用提供）：细胞分化，粘合剂和运动，增殖和凋亡是响应细胞外环境而发生的。膜蛋白检测到环境刺激，并引起确定细胞命运的分子和遗传反应。当这些膜 - 蛋白质原处理过程故障时，癌症会产生。尽管人类膜蛋白在癌症生物学中的主要作用，但这些蛋白质的高分辨率结构却没有。我们的目标是在理解癌症的分子机制方面解决这一差距。我们将对与癌症相关的膜蛋白（CIRMPS）的结构生物学进行全面研究。我们选择了涉及信号通路，细胞相互作用和运输的膜蛋白靶标，这些靶标与癌症生物学不可或缺。拟议的研究的目的不是解决所有目标压接的结构。但是，我们提出的方法和大的，不同的目标集的结合使得解决了几个压接结构现实。任何目标的结构都将有助于理解癌症生物学。尽管很难预测药物设计中膜蛋白结构的效用，但治疗潜力是我们目标选择的重要因素，压接结构可能促进治疗药物或方案的开发。我们还将生成有关P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. persology oferologiogy oferology oferologic ofer的有价值数据。在拟议的研究过程中，我们将完成有关P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p。拟议研究的具体目的是：1。鉴定与结晶相关的癌症相关的人膜蛋白。我们将根据癌症的相关性和预测性疾病程度来选择60个结构和功能多样的膜蛋白。选择有限障碍的压接会增强成功结晶的可能性。 2。评估Pichia Pastoris中六十种潜水癌症相关的膜蛋白的异源表达。我们将使用新颖的网关使用1/2个载体，10 ml P. P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p。p. p. p. p. p。p. p. p. p. p. p. p。p. p. p. p. p. p. p. p. p。p. p. p. p. p. p. p。blovents top-blots and Dot-blots和小规模确定溶液实验，以快速评估异源表达和确定目标压皮的溶液。通过解决方案的所有实验将在十二个目标的批次上进行。 3。解决与癌症相关的膜蛋白的结构，这些膜蛋白过表达并形成良好的晶体而没有显着修饰。我们将使用新型的膜蛋白溶液筛选来确定靶标的最佳浓度。我们将使用一个结晶机器人建立1024条蛋白质（每次实验约100 nL）的条件。我们将使用实验室写的软件和流体处理机器人来优化结晶。我们将使用自动板成像仪查看和记录结晶实验。我们将解决形成高质量晶体的压接的结构。