INTRACELLULAR TRAFFICKING OF TROPOELASTIN

弹性纤维组装是一个复杂的过程，涉及到 原弹性蛋白单体在微纤维支架上的组织和 随后将这些单体交联成不溶的弹性蛋白 矩阵。在管壁中，弹性纤维形成同心片层。 对结构完整性和功能至关重要的 船只。越来越多的证据表明，在几乎所有的血管 疾病，弹性纤维组件或结构的某些方面是 受影响。然而，我们研究这些异常变化的能力是 目前受限于我们对弹性纤维如何 通常在发育中的组织中组装。尽管取得了重大进展 已经在表征弹性纤维成分方面取得了进展，我们 对其所涉及的细胞内事件知之甚少 靶向并最终从细胞分泌。最近，我们展示了 在分泌途径中，原弹性蛋白是65-kD的配体 FK506结合蛋白(FKBP65)。由于对FKBP65知之甚少，我们的 第一个特定的目标是通过确定 它的时间和组织特异性分布，建立了它的 蜂窝内的位置和地形，并识别其他 蛋白质的配体。由于FKBP家族的成员都是多肽- 脯氨酰顺式-反式异构酶参与折叠和 人口贩运事件，原弹性蛋白与FKBP65的关联提高 耐人寻味的假设是FKBP65在折叠和/或 原弹性蛋白向膜组装部位的运输。我们的第二个 因此，特定的目标是通过研究 原弹性蛋白与FKBP65在体外实验中的直接结合 如果FKBP65在原弹性蛋白特异性多肽上具有异构化活性， 并研究FKBP65在原弹性蛋白表达中的作用， 用免疫抑制剂处理细胞的分泌和组装 药物FK506，以破坏FKBP65-原弹性蛋白的相互作用。在我们的 第三个具体目标，我们将扩大我们目前对 原弹性蛋白在细胞内的运输 与原弹性蛋白相关的蛋白质和伴侣蛋白 分泌途径和通过表征高尔基体后转运 基于我们初步数据的蛋白质支持酸性物质的作用 原弹性蛋白运输到细胞表面的隔室。 这些研究的结果不仅将提供基本的信息 关于正常弹性纤维所需的细胞内事件 大会，但最终将为未来奠定重要基础 弹性纤维在许多血管疾病中的研究，如 主动脉瓣上狭窄、高血压和主动脉瘤。

Elastic fiber assembly is a complicated process that involves the organization of tropoelastin monomers on a microfibril scaffold and the subsequent crosslinking of these monomers into an insoluble elastin matrix. In the vessel wall, elastic fibers form concentric lamellae that are critical for the structural integrity and function of the vessel. Increasing evidence suggests that in virtually all vascular diseases, some aspect of elastic fiber assembly or structure is affected. Our ability to study these aberrant changes, however, is presently restricted by our limited knowledge of how elastic fibers normally assemble in developing tissues. Although significant advances have been made in the characterization of elastic fiber components, we know remarkably little about the intracellular events involved in their targeting and eventual secretion from the cell. Recently, we have shown that within the secretory pathway, tropoelastin is a ligand for a 65-kD FK506 binding protein (FKBP65). Since little is known about FKBP65, our first specific aim is to fully characterize the protein by determining its temporal and tissue specific distribution, establishing its intracellular location and topography, and identifying additional ligands for the protein. Since members of the FKBP family are peptidyl- prolyl cis-trans isomerases that have been implicated in folding and trafficking events, the association of tropoelastin with FKBP65 raises the intriguing hypothesis that FKBP65 plays a role in the folding and/or transport of tropoelastin to membrane assembly sites. Our second specific aim, therefore, is to test this hypothesis by studying the direct binding of tropoelastin to FKBP65 in in vitro assays, determining if FKBP65 has isomerization activity on tropoelastin-specific peptides, and investigating the role of FKBP65 in tropoelastin expression, secretion and assembly by treating cells with the immunosuppressant drug, FK506, to disrupt the FKBP65-tropoelastin interaction. In our third specific aim, we will extend our present studies on the intracellular trafficking of tropoelastin by identifying additional proteins and chaperones that associate with tropoelastin in the secretory pathway and by characterizing the post-Golgi transport of the protein based on our preliminary data that supports a role for an acidic compartment in the transport of tropoelastin to the cell surface. Results from these studies will not only provide fundamental information concerning the intracellular events required for normal elastic fiber assembly, but will ultimately establish an important basis for future studies on elastic fibers in a number of vascular diseases, such as supravalvular aortic stenosis, hypertension and aortic aneurysms.