Physiologic Peptide Cyclization in Myeloid Cells

描述（由申请人提供）：防御素是一种与先天免疫有关的三二硫肽，涉及对潜在致病微生物的免疫。髓系防御素被包装在中性粒细胞和单核细胞的颗粒中，上皮防御素在各种粘膜组织中表达。最近发现的防御素，称为-防御素，是由三个平行二硫键稳定的18个氨基酸的大环肽。从恒河猴的白细胞中分离出来的-防御素是一种非常有效的抗生素，可以杀死细菌和真菌，并灭活HIV-1。主环的打开使抗菌活性丧失。大环肽在动物体内的存在以前并不为人所知。此外，防御素的生物合成是新颖的，因为环肽是由两个9个氨基酸片段以头对尾的形式拼接在一起合成的。虽然介导这一翻译后通路的细胞机制尚不清楚，但我们假设在产生β -防御素的细胞中表达的酶负责成熟环状分子生物合成所需的非肽切除和连接步骤。我们提出通过追求三个具体目标来表征-防御素加工途径的分子成分：

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.