Adenovirus Modulation of Lacrimal Gland Function

描述（由申请人提供）：泪液蛋白在对抗病毒和细菌感染以及支持角膜和结膜方面发挥关键作用。泪腺腺泡细胞的主要功能是产生多种蛋白质并将其释放到泪液中，所述蛋白质包括激素、乳铁蛋白和溶酶体水解酶。这些蛋白质中的许多储存在大的亚顶端分泌囊泡中，一旦适当的细胞内信号通路被激活，这些囊泡就在顶端膜处释放其内容物。 我们已经发现，原代培养的兔泪腺腺泡与腺病毒血清型5（Ad 5）载体的转导减少了促分泌素刺激的泪液蛋白的释放，与消耗的rab 3D-丰富的成熟分泌囊泡在次顶端的细胞质平行。Ad 5衣壳蛋白，五邻体和纤维，分别通过与整合素和柯萨奇病毒和腺病毒受体的相互作用介导病毒结合和内吞作用。整合素连接与细胞内信号传导途径的主要变化相关，包括那些可能调节生物合成/分泌膜运输的信号传导途径。 五邻体蛋白也可能通过募集宿主膜运输蛋白参与内化病毒的细胞内运输，从而减少这些蛋白对其他基本膜运输功能的可用性。利用原代培养的兔泪腺泡作为我们的实验系统，提出了两个具体的目标：1）是腺病毒5介导的损害的泪腺泡分泌引起的衣壳蛋白和2）是腺病毒5介导的损害的泪腺泡分泌由于螯合网格蛋白和衔接蛋白的五邻体二亮氨酸基序？ 这些独特的假设将进行测试，使用重组组装能力野生型和突变体Ad 5 penton和纤维蛋白，并将利用各种技术，包括共聚焦荧光和电子显微镜分析分泌囊泡的内容，亚细胞膜分离，免疫沉淀和测量刺激的蛋白质分泌。最近相当大的兴趣集中在使用Ad 5或Ad 5衍生材料的眼部基因治疗上。然而，暴露的泪腺广告衍生材料可能会严重损害其能力，以保持足够的蛋白质分泌能力。这里提出的研究将是必不可少的，在评估使用广告衍生材料的眼部基因治疗的可行性，并在提高这种输送系统的安全性。

DESCRIPTION (provided by applicant): Tear proteins play key roles in combating viral and bacterial infections and in supporting cornea and conjunctiva. A primary function of the acinar cells of the lacrimal gland is the production and release of a variety of proteins including hormones, lactoferrin and lysosomal hydrolases into tear fluid. Many of these proteins are stored in large sub-apical secretory vesicles which release their contents at the apical membrane once appropriate intracellular signaling pathways have been activated. We have found that transduction of primary cultured rabbit lacrimal acini with adenovirus serotype 5 (Ad5) vector decreases the secretagogue-stimulated release of tear proteins in parallel with depletion of rab3D-enriched mature secretory vesicles in the sub-apical cytoplasm. The Ad5 capsid proteins, penton and fiber, mediate virus binding and endocytosis through interactions with `v integrins and coxsackievirus and adenovirus receptor, respectively. Integrin ligation is associated with major changes in intracellular signaling pathways including those which may regulate biosynthetic/secretory membrane traffic. Penton proteins may also participate in the intracellular trafficking of internalized virus by recruitment of host membrane trafficking proteins, diminishing the availability of these proteins for other essential membrane trafficking functions. Two specific aims are proposed utilizing primary cultured rabbit lacrimal acini as our experimental system: 1) Is Ad5-mediated impairment of lacrimal acinar secretion caused by capsid proteins and 2) Is Ad5-mediated impairment of lacrimal acinar secretion due to sequestration of clathrin and adaptor proteins by the penton dileucine motif? These unique hypotheses will be tested using recombinant assembly-competent wild type and mutant Ad5penton and fiber proteins, and will utilize a variety of techniques including confocal fluorescence and electron microscopy analysis of secretory vesicle content, subcellular membrane fractionation, immunoprecipitation and measurements of stimulated protein secretion. Considerable recent interest has focused on ocular gene therapy using Ad5 or Ad5-derived materials. However, exposure of the lacrimal gland to Ad-derived materials may severely compromise its ability to maintain adequate protein secretory capacity. The studies proposed here will be essential in evaluating the feasibility of ocular gene therapy using Ad-derived materials and in improving the safety of such delivery systems.