STUDIES OF ALPHA-DEFENSINS IN PRIMATE INNATE IMMUNITY

描述(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。本研究的目的是确定灵长类动物α-防御素杀菌活性的决定因素及其作用机制，并在转录和翻译后水平上表征调节髓系细胞(RMADs)和潘氏细胞(Reds)α-防御素生物合成的机制。我们建议定量检测红色和RMAD的mRNAs和多肽在小肠粘膜、中性粒细胞和NK细胞中的表达，表征和比较它们的作用机制和活性的结构基础，研究前RED和RMAD在Paneth细胞和髓系细胞中的激活机制，并鉴定RMAD和RED基因的顺式作用结构成分，调节其转录的特异性。在目标1中，将测试Reds 1-6对各种细菌的杀菌活性，并对天然和突变的Reds和RMADs的膜破坏活性进行表征和定量。突变改变多肽结构的假设将通过测定天然和突变的RED4和RMAD4多肽在溶液中以及在快速翻滚的磷脂双链存在下的核磁共振结构来检验，以检验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的猴小肠以及恒河猴的中性粒细胞、NK细胞和单核细胞中红色和RMAD前体的加工程度，来验证阿尔法防御素前体加工是由谱系特异性的蛋白分解事件介导的假设。我们将确定各自的加工酶，确定前体蛋白降解是否与杀菌活性的激活一致，并确定α-防御素保守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控的假设将通过量化单个红色和RMAD mRNAs在肠道和髓系组织中的表达和分布以及通过识别谱系特异性α-防御素基因转录的决定因素来检验。潜在的谱系特异性基因调控区将通过对RED和RMAD启动子结构以及培养单核细胞中嵌合微基因的功能分析来分析。

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.