MEMBRANE TRAFFIC AND PROTEIN TOXINS

膜运输和蛋白质毒素

• 批准号: 2684787
• 负责人: ROCKFORD Keith DRAPER
• 金额: $ 23.36万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684787
• 关键词: CHO cells; antisense nucleic acid; bacterial toxins; binding proteins; clathrin; endocytosis; endoplasmic reticulum; enzyme activity; eukaryote; exocytosis; exotoxins; gel filtration chromatography; gene complementation; intracellular transport; ion exchange chromatography; laboratory rabbit; membrane permeability; molecular cloning; nucleic acid chemical synthesis; nucleic acid sequence; protein transport; proteolysis; serine proteinases; temperature sensitive mutant; toxicant interaction

There are two long-term objectives in this proposal. One is to better comprehend how certain bacterial and plant protein toxins exploit pathways of membrane traffic to enter the cytosol of eukaryotic cells. Information about the entry process is important to understand how pathogenic toxins work and also may provide insight into basic cellular processes. The second objective is to capitalize on the properties of protein toxins to develop applications of therapeutic value. There are four specific aims: AIM 1, analysis of toxin entry into CHO cell mutants of the ldlF group. ldlF mutants express a temperature-sensitive defect in epsilon-COP, a subunit of non-clathrin coatomers. These cells serve as a model system to investigate whether toxin access to the cytosol requires epsilon-COP. AIM 2, the interaction of precleaved exotoxin A (ETA) with cells. ETA must be proteolytically cleaved to enter the cytosol. We are studying here the consequences of precleaving the toxin before adding it to cells. AIM 3, the effect of covalently attaching apyrase to ETA. The objective of this aim is to determine whether ETA enters the endoplasmic reticulum before entering the cytosol. If it does, then ETA should carry attached apyrase to the endoplasmic reticulum, which should have detectable consequences. AIM 4, studies of a variant of ETA that has a free sulfhydryl near the carboxyl terminus. ETA with a cysteine residue inserted near the carboxyl terminus has much-reduced cytotoxicity. The objective of this aim is to understand why.

该提案中有两个长期目标。 一个是更好 理解某些细菌和植物蛋白毒素如何利用途径 膜流量进入真核细胞的细胞质。 信息 关于进入过程对于了解致病性毒素很重要 工作，也可能提供对基本蜂窝过程的见解。 这 第二个目标是利用蛋白质毒素的特性 开发治疗价值的应用。 有四个具体目标： 目标1，分析毒素进入LDLF组的CHO细胞突变体。 LDLF突变体在Epsilon-cop中表达温度敏感的缺陷，A 非加拉蛋白外套的亚基。 这些单元充当模型系统 研究毒素是否可以进入细胞质需要Epsilon-cop。 AIM 2，脱离的外毒素A（ETA）与细胞的相互作用。 ETA 必须将蛋白水解切割才能进入细胞质。 我们正在学习 在此将毒素添加到细胞之前，将毒素排除的后果。 AIM 3，共价连接到ETA的效果。 目标 此目的是确定ETA是否进入内质网 进入细胞质之前。 如果是这样，那么ETA应该随身携带 内质网的apyrase，应检测到可检测到的 结果。 AIM 4，对ETA变体的研究 羧基末端。 ETA的半胱氨酸残留物在羧基附近插入 末端具有大量减少的细胞毒性。 这个目标的目的是 了解原因。