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

基本信息

批准号：
7489827
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
项目类别：
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/7489827
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

项目摘要

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，粘合剂和运动，增殖和凋亡是响应细胞外的环境。膜蛋白检测环境刺激，并引起分子和遗传反应这决定了细胞命运。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具有核心作用，但没有高分辨率结构这些蛋白质存在。我们的目标是在理解分子机制时解决这一差距癌症。我们将对与癌症相关的膜蛋白的结构生物学进行全面研究（压接）。我们选择了涉及信号通路，细胞 - 细胞相互作用和是癌症生物学不可或缺的运输。拟议研究的目的不是解决结构所有目标压接。但是，我们提出的方法和大型潜水员的目标的结合集合使解决几个压接结构现实。任何目标的结构都会贡献显着了解癌症生物学。虽然很难预测膜的效用药物设计中的蛋白质结构，治疗潜力是我们目标选择的重要因素，压接结构可能有助于治疗药物或方案开发。我们还将生成关于P. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p. p。在课程中在拟议的研究中，我们将完成有关表达的最全面的分析 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。我们将选择60 基于癌症相关性和预测程度的结构和功能上多样化的膜蛋白紊乱。选择有限障碍的压接会增强成功结晶的可能性。 2。评估pichia中六十个潜水癌膜蛋白的异源表达牧师。我们将使用新颖的网关媒介，10 ml P. P. P. P. P. 清洁剂溶解实验以快速评估异源表达并确定溶解度目标压接。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没有良好的晶体重大修改。我们将使用新型的膜蛋白溶解度筛选来确定最佳用于结晶的小ra虫的浓度我们将使用结晶机器人设置1024条条件蛋白质的120微晶（〜100 nl_per实验）。我们将使用实验室写的软件和流体处理机器人以优化结晶。我们将使用自动板图像视图和文档结晶实验。我们将解决形成高质量晶体的压接的结构。