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。泪腺腺泡细胞的一个主要功能是产生和释放多种蛋白质，包括激素、乳铁蛋白和溶酶体水解酶到泪液中。许多这些蛋白质储存在大的亚根尖分泌囊泡中，一旦适当的细胞内信号通路被激活，它们的内容物就会在根尖膜上释放出来。我们发现用腺病毒血清型5 （Ad5）载体转导原代培养的兔泪腺，可减少分泌刺激的泪液蛋白释放，同时减少亚顶细胞质中富含rab3d的成熟分泌囊泡的消耗。Ad5衣壳蛋白penton和fiber分别通过与' v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通路的主要变化有关，包括那些可能调节生物合成/分泌膜交通的信号通路。Penton蛋白也可能通过招募宿主膜转运蛋白参与内化病毒的细胞内转运，从而降低了这些蛋白在其他基本膜转运功能中的可用性。利用原代培养的兔泪腺作为实验系统，我们提出了两个具体的目的：1)ad5介导的泪腺分泌损伤是由衣壳蛋白引起的；2)ad5介导的泪腺分泌损伤是由于戊二苯胺基序对泪蛋白和接头蛋白的隔离引起的？这些独特的假设将使用重组组装能力野生型和突变型Ad5penton和纤维蛋白进行测试，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含量，亚细胞膜分离，免疫沉淀和刺激蛋白分泌的测量。近年来，人们对使用Ad5或Ad5衍生材料进行眼部基因治疗非常感兴趣。然而，泪腺暴露于ad源性物质可能严重损害其维持足够蛋白质分泌能力的能力。本文提出的研究对于评估使用ad衍生材料进行眼部基因治疗的可行性以及提高此类传递系统的安全性至关重要。

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.