Microbial Effects on Epithelial Ubiquitination Pathways

微生物对上皮泛素化途径的影响

• 批准号: 7077627
• 负责人: LAUREN S COLLIER-HYAMS
• 金额: $ 5.73万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7077627
• 关键词: HeLa cells; I kappa B beta; SDS polyacrylamide gel electrophoresis; Salmonella infections; Salmonella typhimurium; apoptosis; bacteria infection mechanism; bacterial proteins; biological signal transduction; gastrointestinal epithelium; gene induction /repression; mass spectrometry; nuclear factor kappa beta; protein protein interaction; protein transport; recombinant proteins; tissue /cell culture; transfection; ubiquitin

DESCRIPTION (provided by applicant): Salmonella typhimurium is a common cause of foodborne enterocolitis in the U.S. and developing countries. The clinical manifestations of S. typhimurium infection result from the presence of polymorphonuclear leukocytes (PMN) in the intestinal mucosa and lumen that are recruited along a gradient of chemotactic chemokines such as interleukin-8. These inflammatory mediators are activated at the transcriptional level by the action of DNA-binding transcription factors, of which NF-kappaB is the key member. We have described strains of human non-pathogenic Salmonellae that inhibit activation of inflammatory processes in vitro. Infected or colonized epithelial cells become refractory to inflammatory responses elicited by pathogens and cytokines because of inhibitory effects on the NF-kappaB signal transduction pathway. This inhibition appears to result from a block of IkappaBeta-a ubiquitination but not phosphorylation. We have identified a candidate bacterial protein, AvrA, which we hypothesize mediates this phenomenon. The goal of this proposal is to identify mechanisms by which bacteria inhibit ubiquitination to block NF-kappaB activation. The specific aims in this proposal are 1) to determine the mechanism of AvrA inhibition of NF-kappaB activation; 2) to define the biological role of AvrA in epithelia; and 3) to characterize other mechanisms by which Salmonella alter ubiquitination in epithelial signaling pathways. In these studies, we will examine 2 mechanisms that Salmonella or its effector protein AvrA may use to inhibit NF-kB activation: deubiquitination of IkappaBeta and interference of IkappaBeta ubiquitin ligase. We will use transfection and infection methods to introduce AvrA into epithelial cells to study the biological effects of AvrA on proinflammatory and apoptotic pathways. We will use novel biochemical reagents to determine if AvrA targets ubiquitin or ubiquitin-like modifications. Such studies will provide insights into the molecular basis of bacterial inhibition of ubiquitination in epithelial cell signaling pathways which may lead to therapeutic inventions in the treatment of acute inflammatory diseases of mucosal tissues.

描述（由申请人提供）： 鼠伤寒沙门氏菌是美国和发展中国家食源性小肠结肠炎的常见原因。鼠伤寒链球菌感染的临床表现是由于肠粘膜和腔内中存在多形核白细胞（PMN）而引起的，这些白细胞（PMN）沿着趋化性趋化因子（如白介素-8）的梯度募集。这些炎症介质通过DNA结合转录因子的作用在转录水平上激活，其中NF-kappab是关键成员。我们已经描述了人类非致病沙门氏菌的菌株，这些菌株在体外抑制炎症过程的激活。由于对NF-kappab信号转导途径的抑制作用，感染或定殖的上皮细胞对病原体和细胞因子引起的炎症反应难治性。这种抑制似乎是由一块iKappabeta-a泛素化而导致的，而不是磷酸化。我们已经确定了候选细菌蛋白AVRA，我们假设该蛋白介导了这种现象。该提案的目的是确定细菌抑制泛素化以阻断NF-kappab激活的机制。该提案中的具体目的是1）确定NF-kappab激活的AVRA抑制机制； 2）定义Avra在上皮中的生物学作用； 3）表征沙门氏菌在上皮信号通路中改变泛素化的其他机制。在这些研究中，我们将研究两种机制，即沙门氏菌或其效应蛋白AVRA可能用来抑制NF-KB激活：Ikappabeta的去泛素化和Ikappabeta ubiquitin rigase的干扰。我们将使用转染和感染方法将AVRA引入上皮细胞中，以研究AVRA对促炎和凋亡途径的生物学作用。我们将使用新型的生化试剂来确定AVRA是否靶向泛素或泛素样修饰。这些研究将提供有关上皮细胞信号通路中细菌抑制泛素化的分子基础的见解，这可能导致治疗粘膜组织急性炎性疾病的治疗性发明。