Ad5 Fiber Entry and Trafficking in Lacrimal Acini

Ad5 纤维在泪腺泡中的进入和运输

• 批准号: 7075776
• 负责人: Sarah F Hamm-Alvarez
• 金额: $ 39.14万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-03 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7075776
• 关键词: Adenoviridae; acinar cell; proteins; receptor

DESCRIPTION: A major contribution to ocular morbidity is lacrimal dysfunction, affecting over 10 million Americans. The principal cell of the lacrimal gland and primary contributor of proteins into ocular surface fluid is the lacrimal acinar cell, which is the target of much of the ocular research into the etiology of dry eye diseases including the severe autoimmune disease, Sjogren's syndrome. Ongoing studies are now shedding insights into the precise mechanisms involved in initiation, development and progression of disease, suggesting that identification of prospective therapeutic targets is likely in the not so distant future. However, we are still very limited in our ability to specifically target the next generation of macromolecular drugs, particularly DNA-, protein- or peptide based drugs, to the lacrimal gland, raising the possibility that we may soon identify advanced therapies for treatment of severe dry eye diseases but will be unable to deliver these drugs to the target site. Our focus here is to explore the unusual and possibly unique uptake mechanism utilized for lacrimal acinar internalization of adenovirus serotype 5 (Ad5). We have demonstrated in lacrimal acinar cells that Ad5 utilizes a unique fiber-dependent internalization pathway, in contrast to the penton-dependent internalization described in other systems. We hypothesize that Ad5 may use multiple fiber receptors for binding and entry in lacrimal acini, and further that one or more of these entry pathways is either unusually robust or unique to lacrimal acini, explaining the unusual fiber-dependence of entry in tandem with the high efficiency of viral transduction in lacrimal acini. We propose to characterize the participants in this novel fiber-dependent internalization pathway at the molecular level, with a particular focus on coxsackievirus adenovirus receptor, major histocompatibility complex class 1 and heparin sulfate-glycosaminoglycan receptors, and their different modes of endocytosis. The aims are as follows: Aim #1. Does coxsackievirus adenovirus receptor mediate fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? Aim #2. What other receptors participate in fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? Aim #3. What intracellular trafficking pathways mediate fiber-dependent Ad5 internalization or free fiber internalization in lacrimal acini? At the conclusion of this work, we will have elucidated the contributions of specific receptors and endocytic internalization pathways responsible for fiber-dependent internalization of Ad5. We will have determined to what extent the recombinant fiber or knob, the terminal region of the fiber protein, can recapitulate these pathways. Finally, we will have tested proof-of-principle experiments to determine whether fiber or knob can facilitate entry of antisense oligonucleotides and proteins into lacrimal acinar cytosol.

描述：眼部疾病的一个主要原因是泪液功能障碍，影响了1000多万美国人。泪腺的主要细胞和蛋白质进入眼表液体的主要贡献者是泪腺泡细胞，它是包括严重的自身免疫性疾病干燥综合征在内的许多干眼疾病的眼部研究的目标。目前正在进行的研究正在深入了解疾病的发生、发展和发展所涉及的确切机制，这表明在不远的将来有可能确定预期的治疗靶点。然而，我们在针对泪腺的下一代大分子药物，特别是DNA、蛋白质或多肽药物方面的具体能力仍然非常有限，这增加了我们可能很快就会找到治疗严重干眼病的先进疗法，但无法将这些药物输送到目标部位的可能性。我们在这里的重点是探索不寻常的，可能是独特的摄取机制，用于泪腺病毒5型(Ad5)的腺泡内化。我们已经在泪腺泡细胞中证明，Ad5利用独特的纤维依赖的内化途径，而不是其他系统中描述的五酮依赖的内化。我们假设Ad5可能使用多个纤维受体与泪腺泡结合和进入，而且这些进入途径中的一个或多个要么异常强健，要么是泪腺泡所特有的，这解释了泪腺泡中异常的纤维依赖进入和高效的病毒转导。我们建议在分子水平上描述这种新的纤维依赖内化途径的参与者，特别是柯萨奇病毒腺病毒受体、主要组织相容性复合体1类和硫酸肝素-糖胺聚糖受体，以及它们不同的内吞模式。目的：1.柯萨奇病毒腺病毒受体在泪腺泡中是否介导纤维依赖性Ad5内化或游离纤维内化？目的#2.泪腺泡中还有哪些受体参与纤维依赖的Ad5内化或游离纤维内化？目的#3.泪腺泡细胞内转运途径是介导纤维依赖的Ad5内化还是游离纤维内化？在这项工作的结论，我们将阐明特定的受体和内胞内化途径负责纤维依赖的内化Ad5的作用。我们将确定重组纤维或结节，即纤维蛋白的末端区域，能在多大程度上概括这些途径。最后，我们将测试原理验证实验，以确定纤维或结节是否可以促进反义寡核苷酸和蛋白质进入泪腺泡胞浆。