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来防止紫外线诱导的氧化损伤。这涉及到将铁隔离分子铁蛋白定位在核中，而不是它在其他细胞类型中的细胞质位置。铁蛋白的这种核定位涉及一种被称为铁蛋白的组织特异性核运输分子。最近的研究表明，类铁蛋白不仅作为铁蛋白的核转运体，而且一旦进入细胞核，就会与铁蛋白保持联系，在那里它们共同形成稳定的铁蛋白-铁蛋白复合体(ES)。这种复合体的结构和功能特性使其在真核细胞铁蛋白中独一无二，包括：(1)它的大小-大约是“典型”细胞质铁蛋白的一半，(2)其固有的低铁含量-这可能使其在隔离铁从而防止铁介导的氧化损伤方面非常有效，以及(3)它与DNA结合的能力-通过隔离与DNA相关的铁以及通过与DNA的物理结合来最有效地防止损伤。拟议研究的目的之一是进一步研究CE细胞的铁蛋白-铁蛋白复合体(ES)的结构和功能特性。这些分析将涉及确定络合物是否为单一分子类型或是否存在多种类型的络合物。这些分析将利用柱层析、分析超速离心法和电子显微镜。此外，由于一种被提议的保护机制涉及消除游离铁的有害影响，因此还将对它们对铁的吸收进行分析。这项研究的另一个目的是检查铁蛋白-铁酸盐复合体的紫外线防护作用。其中一些研究将采用完整的角膜器官培养系统，在该系统中，CE细胞保持其正常的分层排列，并总结在正常发育过程中观察到的铁蛋白和铁类物质产生的事件。这个器官培养系统还允许通过使用铁络合剂去铁胺在CE细胞中操纵铁蛋白和铁类化合物的合成--它可逆地阻止这两种成分的合成。其他研究将使用人类CE细胞进行。这项研究将评估内源杂多核铁蛋白-铁类化合物以及铁蛋白和铁类化合物的均聚体的紫外线防护作用。最后，将研究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(S)，这涉及到影响细胞信号传递。拟议的实验包括分析信号活性的发育变化(在发育获得核铁蛋白之前和之后)以及获得和丧失功能的实验。这些操作将包括UV-B辐射，然后评估细胞损伤和死亡。 与公共健康相关：紫外线(UV)对身体所有暴露的组织构成了一种主要的环境侮辱--因为这种辐射源可以破坏包括DNA在内的各种大分子。然而，角膜上皮细胞似乎对这种损伤难以抵抗，这表明这些细胞已经进化出防御机制，可以防止对其DNA的这种损伤。通过确定这种保护是如何提供的，就有可能将这一点传达给其他类型的细胞。

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.