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.

这项建议有两个长期目标。 一个是为了更好 了解某些细菌和植物蛋白毒素如何利用 进入真核细胞的胞质溶胶。 信息 对于了解致病毒素如何 工作，也可以提供对基本细胞过程的洞察。 的 第二个目标是利用蛋白质毒素的特性， 开发具有治疗价值的应用。 有四个具体目标： 目的1，分析毒素进入LDL F组的CHO细胞突变体。 ldlF突变体在ε-COP中表达温度敏感性缺陷， 非网格蛋白被膜分子的亚基。 这些细胞作为模型系统， 研究毒素进入胞质溶胶是否需要ε-COP。 目的2.预脱膜外毒素A（ETA）与细胞的相互作用。 ETA 必须被蛋白水解裂解以进入胞质溶胶。 我们正在研究 这里是在将毒素加入细胞之前预先分离毒素的结果。 目的3，三磷酸腺苷双磷酸酶与ETA共价连接的效果。 客观 目的是确定ETA是否进入内质网， 在进入细胞质之前。 如果是这样，那么埃塔应该携带附加的 腺苷三磷酸双磷酸酶的内质网，这应该有可检测的 后果 目的4，研究ETA的一种变体，其在 羧基末端。 在羧基附近插入半胱氨酸残基的ETA 末端具有大大降低的细胞毒性。 这一目标的目的是 理解为什么。