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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：
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
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/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

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.

细胞的分化、粘附和运动、增殖和凋亡都与细胞外反应有关