HRF, an NFkB antagonist targeting multiple pathogenes

HRF，一种针对多种病原体的 NFkB 拮抗剂

• 批准号: 7162923
• 负责人: Malgorzata Simm
• 金额: $ 30.69万
• 依托单位: ST. LUKE'S-ROOSEVELT INST FOR HLTH SCIS
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7162923
• 关键词: Acute; Binding; Biological Assay; Campylobacter; Categories; Cell Line; Cell Nucleus; Cell physiology; Cells; Cloning; Coculture Techniques; Code; Complex; Consensus; DNA; Dengue; Development; Disease; Dose; Electrophoretic Mobility Shift Assay; Exposure to; Funding; Gene Expression; Genes; Genetic Transcription; Gram-Negative Bacteria; Human; Immunotherapeutic agent; Infection; Inflammatory; Inflammatory Response; Investigation; Length; Libraries; Lipopolysaccharides; Mediating; Molecular Cloning; Molecular Mechanisms of Action; Nuclear; Oligonucleotides; Pathogenesis; Physiological; Polymerase Chain Reaction; Production; Proteins; Recombinants; Salmonella; Shigella; Site-Directed Mutagenesis; Stimulus; System; T-Lymphocyte; TNFRSF5 gene; Testing; Transcript; Virus; base; expression vector; mutant; p65; pathogen; pathogenic bacteria; programs; protein aminoacid sequence; relisorm L; response

DESCRIPTION (provided by applicant): This application for funds is submitted in response to RFA AI-03-017 and proposes characterization and cloning of a human gene product as a broad spectrum immunotherapeutic for the treatment of infection with multiple Category A and B priority pathogens. Infection by or exposure to some viruses or Gram-negative bacteria induces inflammatory responses that contribute significantly to disease pathogenesis. Common to many such responses is the activation of nuclear factor kB (NFkB) - directed transcription. In our ongoing investigation of HRF, an anti-HIV-1 factor secreted by a transformed human T cell line, we found that it acts through interference with NFkB binding to kB on chromosomal DNA. In recent studies we confirmed that HRF blocks NFkB induction by lipopolysaccharides of several pathogenic bacteria and NFkB activation of inflammatory genes in human cells. Recent HRF peptide sequence findings enabled us to identify a homologous coding sequence, facilitating molecular cloning. This application is directed toward characterization of HRF as an antagonist of NFkB-directed inflammatory responses to several high priority pathogens and to completion of our ongoing program of cloning HRF. We propose to: 1) determine the activity of HRF in human cells in culture in NFkB-directed responses to Shigella, Salmonella, Campylobacter, and Dengue; 2) clone and express recombinant HRF and evaluate its activity in culture for treatment of common inflammatory response to different pathogens; 3) determine the molecular mechanism of action through which HRF antagonizes NFkB-directed transcription. These studies will employ several prokaryotic-eukaryotic coculture systems, molecular cloning, PCR site-directed mutagenesis, and electrophoretic mobility shift assays of protein-protein and protein-DNA complexes. This program will result in the development of a new broad spectrum immunotherapeutic based upon a human protein for the control of NFkB-directed acute inflammatory responses to multiple pathogens.

描述（由申请人提供）：该资金申请是针对RFA AI-03-017提交的，并提出了人类基因产物作为广泛的免疫治疗方法的表征和克隆，用于用多种类别A和B优先病原体治疗感染。通过某些病毒或革兰氏阴性细菌感染或暴露于某些病毒或革兰氏阴性细菌会引起炎症反应，从而对疾病发病机理产生重大贡献。许多这种反应共同的是核因子Kb（NFKB）的激活 - 定向转录。在我们对HRF的持续研究中，是由转化的人类T细胞系分泌的抗HIV-1因子，我们发现它通过干扰NFKB在染色体DNA上与Kb结合而起作用。在最近的研究中，我们证实HRF阻断了几种致病细菌的脂多糖和人类细胞中炎症基因的NFKB激活的NFKB诱导。最近的HRF肽序列发现使我们能够识别同源编码序列，从而促进分子克隆。该应用是针对HRF作为NFKB定向炎症反应的拮抗剂的表征，并完成了我们正在进行的克隆HRF计划。我们建议：1）确定NFKB对志贺氏菌，沙门氏菌，弯曲杆菌和登革热的NFKB定向反应中HRF在培养中的活性； 2）克隆并表达重组HRF，并评估其在治疗对不同病原体的常见炎症反应中培养的活性； 3）确定HRF通过拮抗NFKB定向转录的作用分子机制。这些研究将采用几种原核生物 - 核培养系统，分子克隆，PCR位置指导的诱变以及蛋白质蛋白质和蛋白质-DNA复合物的电泳迁移率转移测定法。该程序将基于人类蛋白质来控制新的广谱免疫治疗，以控制NFKB指导的急性炎症反应对多种病原体。