Cellular Intoxication Pathway of Cytolethal Distending Toxin

细胞致死膨胀毒素的细胞中毒途径

• 批准号: 8607690
• 负责人: Kenneth Alan Bradley
• 金额: $ 2.9万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8607690
• 关键词: Address; Area; Bacterial Proteins; Bacterial Toxins; Binding; Biology; California; Carrier Proteins; Cell Cycle; Cell Cycle Arrest; Cell Line; Cell Nucleus; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Comparative Study; Complement; Complex; Cytokinesis; Cytosol; DNA Damage; DNA Repair; Data; Deoxyribonuclease I; Destinations; Endocytosis Pathway; Event; Family; Foundations; Future; Genes; Genetic; Genetic Screening; Gram-Negative Bacteria; Hemophilus ducreyi; Illinois; Immune Sera; Integration Host Factors; Intoxication; Intracellular Transport; Knowledge; Los Angeles; Mammalian Cell; Membrane Protein Traffic; Methodology; Modeling; Molecular; Mutagenesis; Nature; Operon; Organelles; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Process; Production; Property; Protein Subunits; Proteins; Reagent; Receptor Cell; Research; Research Personnel; Resolution; Role; Route; Signal Transduction; Structure; System; Time; Toxin; Translating; Universities; Vesicle; Vesicle Transport Pathway; Work; ataxia telangiectasia mutated protein; base; cytolethal distending toxin; design; extracellular; holotoxins; human H2AX protein; human disease; insight; member; molecular carrier; mutant; novel; novel therapeutics; pathogen; pathogenic bacteria; receptor; receptor binding; research study; tool; trafficking; uptake

DESCRIPTION (provided by applicant): The intracellular-acting bacterial protein toxins have evolved various means to interact with and enter mammalian cells, taking advantage of existing host cellular processes. Studies of the mechanisms by which bacterial toxins are internalized have provided fundamental advances in several areas of cell biology, including receptor biology, mechanisms and pathways of endocytosis, vesicle trafficking, and membrane translocation. Here, we propose studies focused on one member of an emerging family of toxins called the cytolethal distending toxins (CDTs), which are unusual in their intracellular localization, modulatory activities, and overall structures. CDTs are multi-subunit toxins that are generated by a diverse group of pathogenic Gram-negative bacteria and function by modulating the host cell cycle. Active CDT holotoxins are heterotrimeric complexes of three protein subunits, generally encoded by three contiguous genes (cdtA, cdtB, cdtC) in a single operon. To exert their cyclomodulatory effects on cells, CDTs must be taken up from the cell surface and transported intracellularly in a manner that ultimately results in localization of the enzymatic subunit CdtB to the nucleus. However, the molecular details and mechanism by which CDTs exploit existing uptake and transport pathways to gain access to the cytosol, and ultimately the nucleus, are poorly understood. This application addresses these current gaps in knowledge and represents a collaborative proposal between investigators at the University of California at Los Angeles (Dr. Bradley) and the University of Illinois (Dr. Blanke) to investigate the molecular basis of CDT cellular intoxication. Two Aims are proposed that address hypotheses regarding the cell surface binding (Aim 1) and intracellular transport (Aim 2) of CDTs required for cellular intoxication. Aim 1 investigates the molecular determinants of the initial CDT-cell binding contributed by both the host (Aim 1.1) and toxin (Aim 1.2). Aim 2 focuses squarely on the host cellular requirements for uptake and trafficking of CDT, utilizing hypothesis driven (Aim 2.1) and forward genetic (Aim 2.2) approaches. Importantly, the heterotrimeric nature of CDTs, as well as the localization of the catalytic CdtB subunit to the nucleus, are unique features of the CDTs. Therefore, identification of host cell determinants and trafficking pathways that are important for CDT intoxication is predicted to reveal novel host cell requirements and/or routes for transporting proteins from the cell surface to the nucleus and provide insight into toxin-host interactions.

描述（由申请人提供）：利用现有的宿主细胞过程，细胞内作用的细菌蛋白毒素已经进化了与哺乳动物细胞相互作用并进入哺乳动物细胞的各种方法。对细菌毒素被内在化的机制的研究在细胞生物学的几个领域，包括受体生物学，内吞作用的机制和途径，囊泡运输和膜易位提供了基本进步。在这里，我们提出的研究集中在一个新兴的毒素家族中，称为细胞降低的毒素（CDT），它们在其细胞内定位，调节活动和整体结构中是不寻常的。 CDT是由多元化的致病革兰氏阴性细菌产生的多生毒素，并通过调节宿主细胞周期来功能。活性CDT Holotoxin是三个蛋白亚基的异三聚体复合物，通常由单个操纵子中的三个连续基因（CDTA，CDTB，CDTC）编码。为了对细胞发挥其环形调节作用，必须从细胞表面取下CDT并以最终导致酶亚基CDTB定位的方式进行细胞内运输。然而，CDT利用现有摄取和传输途径以进入细胞质及其核的分子细节和机制知​​之甚少。该申请解决了这些当前知识的差距，并代表了加利福尼亚大学洛杉矶分校（布拉德利博士）和伊利诺伊大学（Blanke博士）的调查人员之间的合作提议，以研究CDT细胞中毒的分子基础。提出了两个目的，该目标是针对细胞表面结合（AIM 1）和细胞内转运（AIM 2）的假设。 AIM 1研究了由宿主（AIM 1.1）和毒素（AIM 1.2）贡献的初始CDT细胞结合的分子决定因素。 AIM 2直接集中在使用假设驱动（AIM 2.1）和正向遗传（AIM 2.2）方法的宿主细胞需求，以吸收和运输CDT。重要的是，CDTs的异三分体性质以及将催化CDTB亚基的定位是CDT的独特特征。因此，预计对CDT中毒很重要的宿主细胞决定因素和运输途径的鉴定可以揭示新型的宿主细胞需求和/或将蛋白质从细胞表面运输到细胞核的途径，并提供对毒素 - 宿主相互作用的见识。