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: 7640893
• 负责人: Andre J. Ouellette
• 金额: $ 33.03万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7640893
• 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）：本研究的目的是研究α-防御素在恒河猴（Macaca mulatta）先天免疫中的作用。拟议研究的目标是确定灵长类α-防御素杀菌活性的决定因素及其作用机制，并表征在转录和翻译后水平调节骨髓（RMAD）和潘氏细胞（RED）α-防御素生物合成的机制。我们建议定量小肠粘膜、中性粒细胞和 NK 细胞中 RED 和 RMAD mRNA 和肽的表达，表征和比较它们的作用机制和该活性的结构基础，研究潘氏细胞和骨髓细胞中 proRED 和 proRMAD 激活的机制，并鉴定调节其转录特异性的 RMAD 和 RED 基因的顺式作用结构成分。在目标 #1 中，将测试 RED 1-6 针对不同细菌种类的杀菌活性，并对天然和突变 RED 和 RMAD 的膜破坏活性进行表征和定量。通过测定溶液中天然和突变 RED4 和 RMAD4 肽的 NMR 结构以及在快速翻滚的磷脂双分子层存在下的情况，将测试诱变改变肽结构的假设，以测试特定残基位置与磷脂双层相互作用的假设。在目标 #2 中，我们将通过测量完整猴小肠以及恒河猴中性粒细胞、NK 细胞和单核细胞中 RED 和 RMAD 前体加工的程度来检验 α-防御素前体加工是由谱系特异性蛋白水解事件介导的假设。我们将鉴定各自的加工酶，确定前体蛋白水解是否与杀菌活性的激活同时发生，并确定保守的α-防御素位置的突变对肽折叠、二硫键形成、激活以及膜结合和破坏的影响。因此，在目标#3中，灵长类α-防御素基因表达受谱系特异性顺式作用元件调节的假设将通过定量肠和骨髓组织中各个RED和RMAD mRNA的表达和分布以及鉴定谱系特异性α-防御素基因转录的决定因素来测试。将通过对培养的单核细胞中的 RED 和 RMAD 启动子构建体和嵌合小基因进行功能分析来测定潜在的谱系特异性基因调控区。