Physiologic Peptide Cyclization in Myeloid Cells

描述(申请人提供)：防御素是三硫基多肽，与对潜在致病微生物的先天免疫有关。髓系防御素包装在中性粒细胞和单核细胞的颗粒中，上皮防御素在多种粘膜组织中表达。最新发现的防御素，称为theta-防御素，是由18个氨基酸组成的大环肽，由三个平行的二硫键稳定。从恒河猴白细胞中分离出来的theta-Defensins是一种非常有效的抗生素，可以杀死细菌和真菌，并使HIV-1失活。主干环的打开使抗菌活性丧失。大环肽在动物体内的存在在以前是未知的。此外，theta-Defensins的生物合成是新颖的，因为环肽是由两个9-氨基酸片段以头尾相连的形式拼接而成的。虽然介导这一翻译后途径的细胞机制尚不清楚，但我们假设，在theta防御素产生细胞中表达的酶负责非肽的切除和连接步骤，这是成熟环状分子生物合成所必需的。我们建议通过追求三个特定的目标来表征theta-Defensin处理途径的分子组成： 1.在特定的目标1中，我们将分析髓系细胞产生的Theta-Defensin原中间体，并确定所鉴定的分子中间体的亚细胞室。 2.特定目的2鉴定表达theta防御素的细胞提取物中的theta防御素转化活性。在这些研究中，我们将使用参与切除/连接途径的合成和重组形式的假定底物，并使用免疫沉淀以及色谱、电泳和质谱学方法来检测和表征相关的酶活性。 3.具体目标3是确定与theta防御素原和随后的中间体相互作用的蛋白质的特征，因为这些可能是theta防御素生物合成所涉及的切除/连接步骤所必需的转换酶或伴侣。 这些研究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.