Physiologic Peptide Cyclization in Myeloid Cells

描述（由申请方提供）：防御素是三二硫键肽，参与针对潜在病原微生物的先天免疫。髓样防御素被包装在中性粒细胞和单核细胞的颗粒中，并且上皮防御素在多种粘膜组织中表达。最近发现的防御素，称为θ-防御素，是由三个平行的二硫键稳定的18个氨基酸的大环肽。 从恒河猴白细胞中分离出来的θ-防御素是一种非常有效的抗生素，可以杀死细菌和真菌，并抑制HIV-1。抗微生物活性通过主链环的打开而被废除。 动物中存在大环肽以前是未知的。此外，θ-防御素的生物合成是新颖的，因为环肽是由以头对尾构型拼接在一起的两个9-氨基酸区段合成的。虽然介导这种翻译后途径的细胞机制是未知的，我们假设在θ-防御素产生细胞中表达的酶负责成熟环状分子生物合成所必需的九肽切除和连接步骤。我们建议通过追求三个特定目标来表征θ-防御素加工途径的分子组分： 1.在具体目标1中，我们将分析在骨髓细胞中产生的前-θ-防御素中间体，并将确定所鉴定的分子中间体的亚细胞区室。 2.具体目的2是鉴定表达θ-防御素的细胞的提取物中的原θ-防御素转化活性。 对于这些研究，我们将使用合成和重组形式的假定底物参与切除/连接途径，并使用免疫沉淀，色谱，电泳和质谱方法检测和表征相关的酶活性。 3.具体目标3是表征与原θ-防御素和后续中间体相互作用的蛋白质，因为这些可能是θ-防御素生物合成中涉及的切除/连接步骤所必需的转化酶或分子伴侣。 从这些研究中获得的结果可能会揭示哺乳动物细胞中剪接和环化蛋白质的新机制。

DESCRIPTION (provided by applicant): Defensins are tridisulfide peptides implicated in innate immunity against potentially pathogenic microorganisms. Myeloid defensins are packaged in the granules of neutrophils and monocytes, and epithelial defensins are expressed in a wide variety of mucosal tissues. The most recently discovered defensins, termed theta-defensins, are 18-amino acid macrocyclic peptides that are stabilized by three parallel disulfide bonds. Isolated from rhesus monkey leukocytes, theta-defensins are remarkably potent antibiotics that kill bacteria and fungi, and they inactivate HIV-1. Antimicrobial activity is abrogated by opening of the backbone ring. The presence of macrocyclic peptides in animals was not previously known. Moreover, the biosynthesis of theta-defensins is novel, as the cyclic peptide is synthesized from two 9-amino acid segments that are spliced together in a head-to-tail configuration. While the cellular machinery that mediates this post-translational pathway is unknown, we hypothesize that enzymes expressed in theta-defensin-producing cells are responsible for the nonapeptide excision and ligation steps necessary for biosynthesis of the mature cyclic molecule. We propose to characterize the molecular components of the theta-defensin processing pathway by pursuing three specific Aims: 1. In Specific Aim 1, we will analyze the pro-theta-defensin intermediates produced in myeloid cells, and will determine the subcellular compartments of the molecular intermediates identified. 2. Specific Aim 2 is to identify pro-theta-defensin converting activities in extracts of theta-defensin-expressing cells. For these studies we will use synthetic and recombinant forms of putative substrates involved in the excision/ligation pathway, and use immunoprecipitation, and chromatographic, electrophoretic, and mass spectroscopic methods for detecting and characterizing the relevant enzymatic activities. 3. Specific Aim 3 is to characterize proteins that interact with pro-theta-defensins and subsequent intermediates, as these are likely to be convertases or chaperones necessary for the excision/ligation steps involved in theta-defensin biosynthesis. Results obtained from these studies are likely to disclose novel mechanisms that have evolved for splicing and cyclizing proteins in mammalian cells.