STUDIES OF ALPHA-DEFENSINS IN PRIMATE INNATE IMMUNITY

灵长类先天免疫中阿尔法防御素的研究

• 批准号: 7900547
• 负责人: Andre J. Ouellette
• 金额: $ 33.14万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900547
• 关键词: Accounting; Acids; Anabolism; Azurophilic Granule; Binding; Biological Assay; Defensins; Elements; Enteral; Enzymes; Event; Gene Expression; Genes; Genetic Transcription; Goals; Human; In Vitro; Individual; Intestines; Macaca; Macaca mulatta; Measures; Mediating; Membrane; Monkeys; Mucous Membrane; Mutagenesis; Mutation; Myelogenous; Myeloid Cells; Natural Immunity; Natural Killer Cells; Nucleic Acid Regulatory Sequences; Paneth Cells; Peptides; Phospholipids; Positioning Attribute; Primates; Probiotics; Process; Proteolysis; Research; Role; Secretory Vesicles; Site; Small Intestines; Small intestine mucous membrane; Solutions; Specificity; Structure; Testing; Tissues; Variant; alpha-Defensins; arginyl-glycyl-glutamic acid; bactericide; base; cis acting element; disulfide bond; in vivo; intestinal epithelium; microbicide; monocyte; mutant; neutrophil; peptide structure; promoter

DESCRIPTION (provided by applicant): The purpose of this research is to investigate the role of alpha-defensins in innate immunity of the rhesus macaque (Macaca mulatta). The goals of the proposed studies are to define the determinants of primate alpha-defensin bactericidal activity, their mechanisms of action, and to characterize mechanisms that regulate myeloid (RMADs) and Paneth cell (REDs) alpha-defensin biosynthesis at the transcriptional and posttranslational levels. We propose to quantitate the expression of RED and RMAD mRNAs and peptides in small bowel mucosa, neutrophils and NK cells, characterize and compare their mechanisms of action and the structural basis of that activity, investigate mechanisms of proRED and proRMAD activation in Paneth cells and myeloid cells, and identify cis-acting structural components of RMAD and RED genes that regulate the specificity of their transcription. In Aim #1, the microbicidal activities of REDs 1-6 will be tested against varied bacterial species, and membrane disruptive activities of natural and mutant REDs and RMADs will be characterized and quantitated. The hypothesis that mutagenesis alters peptide structure will be tested by determining NMR structures of native and mutant RED4 and RMAD4 peptides in solution and in the presence of rapidly-tumbling phospholipid bicelles to test the hypothesis that specific residue positions interact with phospholipid bilayers. In Aim #2, we will test the hypothesis that alpha-defensin precursor processing is mediated by lineage-specific proteolytic events by measuring the extent of RED and RMAD precursor processing in intact monkey small intestine and in rhesus neutrophils, NK cells, and monocytes. We will identify the respective processing enzymes, determine whether precursor proteolysis is coincident with activation of bactericidal activity, and determine the effect of mutations at conserved alpha-defensin positions on peptide folding, disulfide bond formation, activation, and membrane binding and disruption. Thus, in Aim #3, the hypotheses that primate alpha-defensin gene expression is regulated by lineage-specific, cis-acting elements will be tested by quantitating the expression and distribution of individual RED and RMAD mRNAs in enteric and myeloid tissues and by identifying determinants of lineage-specific alpha-defensin gene transcription. Potential lineage-specific gene regulatory regions will be assayed by functional analyses of RED and RMAD promoter constructs and chimeric minigenes in cultured monocytes.

描述（由申请人提供）：这项研究的目的是研究α-防御素在恒河猴先天免疫中的作用（Macaca Mulatta）。拟议的研究的目标是定义灵长类动物α-防御素的杀菌活性的决定因素，其作用机制，并表征调节髓样（RMADS）的机制和paneth细胞（REDS）α-德防御素的生物合成在转录和转换后水平。 We propose to quantitate the expression of RED and RMAD mRNAs and peptides in small bowel mucosa, neutrophils and NK cells, characterize and compare their mechanisms of action and the structural basis of that activity, investigate mechanisms of proRED and proRMAD activation in Paneth cells and myeloid cells, and identify cis-acting structural components of RMAD and RED genes that regulate the specificity of their transcription.在AIM＃1中，将针对各种细菌物种测试红色1-6的微生物活性，并且将对天然和突变的红色和RMADS的膜破坏活性进行表征和定量。通过确定溶液中天然和突变体RED4和RMAD4肽的NMR结构以及在存在迅速吸引的磷脂双皮细胞中测试特定残基与磷脂比脂脂相互作用的假说，可以通过确定溶液中天然和突变体Red4和RMAD4肽的NMR结构来改变肽结构的假说。在AIM＃2中，我们将检验以下假设：α-防御素前体的加工是通过谱系特异性蛋白水解事件介导的，通过测量完整的猴子小肠和恒河猴中性粒细胞，NK细胞和单一细胞中的红色和RMAD前体处理的程度。我们将确定各自的加工酶，确定前体蛋白水解是否与杀菌活性的激活相吻合，并确定在保守的α-防御素位置对肽折叠，二硫键键形成，激活，激活和膜结合和破坏的突变对突变的影响。因此，在AIM＃3中，灵长类动物α-防御素基因表达受到谱系特异性调节，将通过量化肠和髓样组织中单个红色和rmad mRNA的表达和分布来调节顺式作用元素，并通过识别谱系特异性α-脱甲素基因基因基因的确定性。潜在的谱系特异性基因调节区域将通过在培养的单核细胞中对红色和RMAD启动子构建体和嵌合微型烯的功能分析来测定。