Epithelial specific ubiquitination events

描述(申请人提供)：肠道炎症反应在很大程度上是由分泌的细胞因子和其他炎症效应分子的合成上调所介导的。这些炎症介质在转录水平上被核因子-kB等DNA结合转录因子激活。核因子-kB本身是由一条快速的翻译后途径激活的，包括顺序的磷酸化、泛素化和抑制分子IKB的降解。肠道上皮在各种组织中是独一无二的，其功能与各种原核菌群密切接触。人们对这些细菌对宿主上皮细胞的有害和有益的影响越来越感兴趣。在这项建议中，我们描述了肠道共生细菌通过影响限速生化步骤泛素化来抑制关键的核因子-kB调节因子的激活。泛素化是一种可诱导的共价修饰，在蛋白质周转和降解中具有明确的作用，在信号转导、细胞内转运和其他细胞功能中也发挥着重要作用。细菌及其产物可能会影响激活负责IKB泛素化的酶所需的共价修饰，并可能影响参与上皮信号转导的其他蛋白质。在其他工作中，我们已经确定了肠道病原体中的候选效应分子，这些分子是NFkB的有效抑制剂，可能作为脱泛素酶发挥作用。我们推测，这些蛋白将对真核细胞的促炎途径和细胞凋亡的控制产生深远的影响。 我们的总体假设是，肠道细菌，既是肠道的共生居民，也是已知的病原体，已经进化出通过影响真核泛素系统来调节上皮信号通路的机制。根据特定的生物学背景，这些相互作用的后果可能是由传染性和特发性结肠炎引起的，和/或可能有助于肠道的生理和整体健康。这项建议描述了各种体外和体内实验方法，以研究细菌如何影响肠上皮细胞泛素化系统，特别是与核因子-kB的激活和凋亡途径有关。阐明这些宿主-微生物的相互作用将增加我们对人类小肠结肠炎，以及可能参与特发性炎症性肠病的上皮和细菌因素的了解。

DESCRIPTION (provided by applicant): The intestinal inflammatory response is mediated in large part by synthetic upregulation of secreted cytokines and other inflammatory effector molecules. These inflammatory mediators are activated at the transcriptional level by the action of DNA binding transcription factors such as NF-kB. NF-kB itself is activated by a rapid post translational pathway involving sequential phosphorylation, ubiquitination and degradation of the inhibitor molecule, IkB. Unique among tissues, the intestinal epithelia functions in close contact with a diverse prokaryotic flora. There is increasing interest in the effects, both deleterious and beneficial, of these bacteria on the host epithelia. In this proposal, we describe enteric commensal bacterial strains that repress activation of the key NF-kB regulator by influencing a rate-limiting biochemical step, ubiquitination. Ubiquitination is an inducible covalent modification with a well defined role in protein turnover and degradation, and emerging roles in signal transduction, endocytic trafficking and other cellular functions. Bacteria and their products may influence covalent modifications necessary for activation of the enzymes responsible for ubiquitination of IkB and potentially other proteins involved in epithelial signaling. In other work, we have identified candidate effector molecules in enteric pathogens that are potent inhibitors of NFkB and likely act as deubiquitinating enzymes. We hypothesize these proteins will have profound effects on eukaryotic proinflammatory pathways and control of cellular apoptosis. Our overall hypothesis is that enteric bacteria, both commensal inhabitants of the intestine and known pathogens, have evolved mechanisms to modulate epithelial signaling pathways by influencing the eukaryotic ubiquitin system. Depending the specific biological context, the consequences of these interactions may by causal of infectious and idiopathic colitis, and/or may contribute to the physiology and overall health of the intestine. This proposal describes a variety of in vitro and in vivo experimental methods to study how bacteria influence the ubiquitination system in the intestinal epithelia, especially as it relates to the activation of the NF-kB and apoptotic pathways. Elucidation of these host-microbe interactions will increase our understanding of the epithelial and bacterial factors involved in human enterocolitis, and potentially in idiopathic inflammatory intestinal disease.