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Regulation of eukaryotic membrane structure and function

真核细胞膜结构和功能的调节

基本信息

批准号：
8033388
负责人：
W Scott Moye-Rowley
金额：
$ 8.44万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-19 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8033388
关键词：
ATP-Binding Cassette Transporters Amino Acid Permease Anabolism Antifungal Agents Binding Candida albicans Carrier Proteins Cell Line Cell Membrane Permeability Cell membrane Cells Ceramides Data Defect Development Drug resistance Ensure Environment Enzymes Eukaryotic Cell Exhibits Funding Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription Goals Growth Human Integral Membrane Protein Interruption Lead Link Lipids Mammalian Cell Mammals Measures Mediating Membrane Membrane Lipids Membrane Microdomains Membrane Proteins Membrane Structure and Function Membrane Transport Proteins Modeling Molecular Multi-Drug Resistance Multidrug Resistance Gene Multidrug Resistance Induction Organism P-Glycoproteins Pathway interactions Pharmaceutical Preparations Phenotype Production Protein Kinase Proteins Regulation Reporting Research Personnel Resistance Saccharomyces cerevisiae Signal Pathway Signal Transduction Signal Transduction Pathway Site Sphingolipids Staging Sterols Toxic effect Work Yeasts Zinc Cluster base genetic analysis insight liquid chromatography mass spectrometry mutant overexpression phytosphingosine programs research study trafficking transcription factor tumor

项目摘要

DESCRIPTION (provided by applicant): Multidrug resistance often arises in human tumors due to overexpression of plasma membrane ATP-binding cassette (ABC) transporter proteins. Recent experiments have indicated that multidrug resistant cells frequently exhibit changes in the profile of lipid species known as sphingolipids along with increases in ABC transporter expression and activity. We have recently discovered that multidrug resistant Saccharomyces cerevisiae cells exhibit a similar molecular linkage between increases in ABC transporter expression and changes in sphingolipid biosynthesis. Work from our lab and others has shown that increases in the activity of the zinc cluster transcription factors Pdr1 p and Pdr3p lead to induction of multidrug resistant ABC transporters like Pdr5p and Yor1p but also the long chain base (LCB) transporter Rsb1p. LCBs are bioactive precursors in sphingolipid biosynthesis in eukaryotic cells. We hypothesize that the coregulation of the ABC transporters and LCB transporter Rsb1p is required for normal modulation and function of the plasma membrane. The Ypk protein kinases in S. cerevisiae are activated by LCBs and we will examine the ability of Ypk2p to control multidrug resistance and gene expression through regulation of Pdr3p. Loss of Pdr5p leads to induction of LCB resistance. Genetic analysis will be performed to identify and analyze components of this signaling pathway connecting loss of this ABC transporter with induction of LCB tolerance. Levels of LCBs will also be analyzed in cells lacking Pdr5p and Yor1p as well as in mutant strains with different levels of Pdr1p/Pdr3p transcriptional activity. This will allow us to determine both the identity of LCBs that are regulated by Pdr pathway function and to uncover sphingolipids that may serve to regulate activity of Pdr1p and/or Pdr3p. Finally, the effect of accumulation of sphingolipid precursors on ABC transporter function will be determined. Transporter activity and trafficking will be measured in strains exposed to high levels of LCBs or containing defects in sphingolipid synthesis. Our work indicates that drug resistant yeast cells co-regulate the biosynthesis of drug transporters with constituents of the membranes in which these transporters will function. This coordinate regulation is important to ensure normal membrane function and may allow new antifungal drugs to be targeted towards its interruption as later stages of this pathway are carried out by enzymes that are not found in mammals.

描述(申请人提供)：由于质膜三磷酸腺苷结合盒(ABC)转运蛋白的过度表达，人类肿瘤经常出现多药耐药。最近的实验表明，随着ABC转运蛋白表达和活性的增加，多药耐药细胞经常表现出被称为鞘脂的脂类的变化。我们最近发现，多药耐药的酿酒酵母细胞在ABC转运蛋白表达增加和鞘磷脂生物合成变化之间表现出类似的分子连锁。我们实验室和其他实验室的工作表明，锌簇转录因子Pdr1p和Pdr3p活性的增加会导致诱导多药耐药的ABC转运蛋白，如Pdr5p和York 1p，但也会诱导长链碱基(LCB)转运蛋白Rsb1p。LCB是真核细胞中鞘磷脂生物合成的活性前体。我们推测，ABC转运蛋白和LCB转运蛋白Rsb1p的共同调节是质膜正常调节和功能所必需的。酿酒酵母中的Ypk蛋白激酶被LCBs激活，我们将通过调节Pdr3p来检测Ypk2p控制多药耐药和基因表达的能力。Pdr5p的缺失导致LCB抗性的诱导。基因分析将被用来鉴定和分析这一信号通路的组成部分，该信号通路将ABC转运蛋白的缺失与LCB耐受性的诱导联系起来。还将分析缺乏Pdr5p和York 1p的细胞以及具有不同水平Pdr1p/Pdr3p转录活性的突变菌株中LCBs的水平。这将使我们能够确定受PDR途径功能调控的LCB的身份，并发现可能调节Pdr1p和/或Pdr3p活性的鞘磷脂。最后，神经鞘脂前体的积累对ABC转运蛋白功能的影响将被确定。将在暴露于高水平LCB或含有鞘脂合成缺陷的菌株中测量转运蛋白的活性和贩运。我们的工作表明，耐药酵母细胞与药物转运蛋白的膜成分共同调节药物转运蛋白的生物合成，这些转运蛋白将在其中发挥作用。这种协调调节对于确保正常的膜功能非常重要，并可能允许新的抗真菌药物靶向阻断它，因为这一途径的后期阶段是由哺乳动物中没有的酶来执行的。