Corneal-Epithelial Nuclear Ferritin and U.V. Protection

描述（由申请人提供）：数据表明，鸟类角膜上皮（CE）细胞已经进化出一种新的机制，用于预防紫外线诱导的氧化损伤。这涉及使铁螯合分子铁蛋白位于细胞核中，而不是在其他细胞类型中的细胞质中。这种铁蛋白的核定位涉及称为类铁蛋白的组织特异性核转运分子。最近已经表明，类铁蛋白不仅作为铁蛋白的核转运蛋白，而且一旦在细胞核内，类铁蛋白保留其与铁蛋白的结合，在那里它们一起形成稳定的铁蛋白-类铁蛋白复合物。该复合物具有使其在真核生物铁蛋白中独特的结构和功能特性，包括：（1）其大小-大约是“典型的”细胞质铁蛋白的一半，（2）其固有的低铁含量-这可能使其在螯合铁方面特别有效，从而防止铁介导的氧化损伤，和（3）其与DNA结合的能力--在这种情况下，其通过螯合DNA相关的铁和通过与DNA的物理结合来最有效地防止损伤。提出的研究的一个目的是进一步检查CE细胞的核铁蛋白-类铁蛋白复合物的结构和功能特性。这些分析将涉及确定复合物是否是单一分子类型或是否存在多种类型的复合物。这些分析将利用柱色谱法、分析性超离心和电子显微镜。此外，由于复合物提供的一种拟议保护机制涉及消除游离铁的有害影响，因此还将对其铁吸收进行分析。研究的另一个目的是检查铁蛋白-类铁蛋白复合物的紫外线防护。这些研究中的某些将采用整个角膜器官培养系统，其中CE细胞保持其正常的分层排列，并重现正常发育期间观察到的铁蛋白和类铁蛋白产生的事件。该器官培养系统还允许通过使用铁螯合剂去铁胺来操纵CE细胞中铁蛋白和类铁蛋白的合成-去铁胺可逆地阻断两种组分的合成。其他研究将使用人CE细胞进行。这些研究将评估内源性杂聚核铁蛋白-类铁蛋白复合物和铁蛋白和类铁蛋白的均聚复合物的紫外线防护作用。最后，将检查核铁蛋白在保护细胞免受紫外线损伤和死亡方面可能具有额外功能的可能性-这涉及影响细胞信号传导。拟议的实验包括分析信号活性的发育变化（之前和之后的发展收购核铁蛋白）和增益和功能丧失的实验。这些操作将涉及UV-B照射，然后评估细胞损伤和死亡。 公共卫生相关性：紫外线（UV）光构成了对身体所有暴露组织的主要环境损害-因为这种辐射源可以破坏各种各样的大分子，包括DNA。然而，角膜上皮细胞似乎对这种损伤不敏感，这表明这些细胞已经进化出了防御机制，可以防止这种DNA损伤。通过确定如何提供这种保护，可以将其传达给其他细胞类型。

DESCRIPTION (provided by applicant): Data suggests that avian corneal epithelial (CE) cells have evolved a novel mechanism for preventing UV-induced oxidative damage. This involves having the iron-sequestering molecule ferritin in a nuclear localization rather than the cytoplasmic location it has in other cell types. This nuclear localization of ferritin involves a tissue-specific nuclear transport molecule termed ferritoid. Recently it has been shown that ferritoid not only serves as the nuclear transporter for ferritin, but once within the nucleus, ferritoid retains its association with ferritin, where together they form a stable ferritin-ferritoid complex(es). This complex has structural and functional properties that make it unique among eukaryotic ferritins, including: (1) its size - which is approximately half that of a "typical", cytoplasmic ferritin, (2) its intrinsic low content of iron - which may make it exceptionally effective in sequestering iron and thus preventing iron-mediated oxidative damage, and (3) its ability to bind to DNA - where it could be most effective in preventing damage by sequestering DNA-associated iron and through physical association with the DNA. One aim of the proposed studies is to examine further the structural and functional properties of the nuclear ferritin-ferritoid complex(es) of CE cells. These analyses will involve determining whether the complex(es) are a singular molecular type or whether there are multiple types of complexes. These analyses will utilize column chromatography, analytical ultracentrifugation, and electron microscopy. Also, as one proposed mechanism of protection afforded by the complex(es) involves abrogating the deleterious effects of free iron, analyses will also be performed on their uptake of iron. Another aim of the studies will be to examine UV protection by ferritin-ferritoid complexes. Certain of these studies will employ a whole corneal organ culture system in which the CE cells maintain their normal, stratified arrangement and recapitulate the events observed for ferritin and ferritoid production during normal development. This organ culture system also allows the manipulation of synthesis of ferritin and ferritoid in CE cells by using the iron chelator deferoxamine - which reversibly blocks the synthesis of both components. Other studies will be performed using human CE cells. The studies will evaluate UV protection by the endogenous heteropolymeric nuclear ferritin-ferritoid complexes and by homopolymeric complexes of ferritin and ferritoid. Lastly, the possibility will be examined that nuclear ferritin may have an additional function(s) in protecting cells from UV damage and death - which involves affecting cell signaling. The proposed experiments include analysis of developmental changes in signaling activity (before and after the developmental acquisition of nuclear ferritin) and gain- and loss-of-function experiments. These manipulations will involve UV-B irradiation followed by evaluation of cell damage and death. PUBLIC HEALTH RELEVANCE: Ultraviolet (UV) light constitutes a major environmental insult to all exposed tissues of the body - as this source of radiation can damage a wide variety of macromolecules, including DNA. However, corneal epithelial cells seem to be refractory to such damage, suggesting that these cells have evolved defense mechanisms that prevent such damage to their DNA. By determining how this protection is afforded, it may be possible to convey this to other cell types.