INTRACELLULAR TRAFFICKING OF TROPOELASTIN

弹性纤维组装是一个复杂的过程， 在微纤维支架上的原弹性蛋白单体的组织和 这些单体随后交联成不溶性弹性蛋白 矩阵 在血管壁中，弹性纤维形成同心圆状的层 这对结构的完整性和功能至关重要， 容器。 越来越多的证据表明，在几乎所有的血管 疾病时，弹性纤维组件或结构的某些方面被 影响。 然而，我们研究这些异常变化的能力， 目前受限于我们对弹性纤维 通常在发育中的组织中组装。 虽然取得了重大进展， 已经在弹性纤维成分的表征，我们 对细胞内的事件知之甚少， 靶向并最终从细胞分泌。 最近，我们发现 在分泌途径中，弹性蛋白原是65 kD的配体， FK 506结合蛋白（FKBP 65）。 由于对FKBP 65知之甚少，我们的 第一个具体目标是通过测定蛋白质的分子量来充分表征蛋白质， 其时间和组织特异性分布，建立其 细胞内的位置和地形，并确定额外的 蛋白质的配体。 由于FKBP家族的成员是肽基- 脯氨酰顺-反异构酶已经涉及折叠， 在贩运事件中，弹性蛋白原与FKBP 65的关联提高了 有趣的假设是，FKBP 65在折叠和/或 将原弹性蛋白运输至膜组装位点。 我们的第二 因此，具体的目的是通过研究 体外测定中原弹性蛋白与FKBP 65的直接结合，测定 如果FKBP 65对原弹性蛋白特异性肽具有异构化活性， 并研究FKBP 65在原弹性蛋白表达中的作用， 通过用免疫抑制剂处理细胞的分泌和组装 药物FK 506破坏FKBP 65-原弹性蛋白相互作用。 在我们 第三个具体目标，我们将扩大我们目前的研究， 原弹性蛋白的细胞内运输 蛋白质和分子伴侣与原弹性蛋白在 分泌途径，并通过表征后高尔基体运输的 蛋白质基于我们的初步数据，支持酸性蛋白质的作用， 在将弹性蛋白原运输到细胞表面的过程中，细胞膜是细胞膜的一部分。 这些研究的结果不仅将提供基本信息， 关于正常弹性纤维所需的细胞内事件 大会，但最终将建立一个重要的基础， 研究弹性纤维在一些血管疾病，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.