Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Studies

眼晶状体中的胆固醇晶域功能：EPR 自旋标记研究

• 批准号: 7994801
• 负责人: WITOLD K SUBCZYNSKI
• 金额: $ 32.83万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-02 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994801
• 关键词: Affect; Aging; Animals; Appearance; Attention; Biological; Blindness; Buffers; Cataract; Cell Fraction; Cell Nucleus; Cell membrane; Cholesterol; Crystalline Lens; Developing Countries; Discrimination; Electron Spin Resonance Spectroscopy; Goals; Human; Hydrophobicity; Lateral; Lecithin; Lens Fiber; Lipid Bilayers; Lipids; Measurement; Membrane; Methodology; Methods; Modeling; Mole the mammal; Nuclear; Oxygen; Pathology; Phospholipids; Physiological; Play; Prevention; Process; Property; Research; Role; Sampling; Solubility; Sphingomyelins; Spin Labels; Structure; Surface; System; Techniques; Testing; Wisconsin; age related; aged; base; design; fiber cell; fluidity; insight; lens; lens transparency; medical schools; membrane model; molecular dynamics; oxygen transport; physical property; prevent; public health relevance; research study

DESCRIPTION (provided by applicant): Fiber-cell membranes of the human eye lens are saturated with cholesterol (showing cholesterol-to- phospholipid mole ratios from 1 in the cortex to as high as 4 in the nucleus), which leads to the formation of immiscible cholesterol crystalline domains (CCDs) within these membranes. At the highest cholesterol content, up to 50% of the cell-membrane surface can be occupied by CCDs. These conditions exist in the lens nucleus, where the cholesterol content can significantly exceed the solubility threshold of the membrane. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens can CCDs play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. The long-term objective of this proposal is to achieve a greater understanding of cholesterol's function in fiber-cell membranes. In the short-term, we will (i) examine how high cholesterol content affects the lateral organization of phospholipid lens membranes, with special attention paid to the formation of CCDs, and (ii) test the hypothesis that the presence of CCDs determines the properties of the surrounding bulk phospholipid- cholesterol membranes. (iii) Additionally, experiments will reveal if the phospholipid composition of the lens membrane affects the formation, size, and stability of CCDs. (iv) Finally, the methodology developed and tested in model-membrane systems will be applied to detect and characterize coexisting cholesterol crystalline and bulk phospholipid-cholesterol domains in lens fiber-cell membranes during maturation, aging, and cataract formation. For studies of coexisting domains in membranes isolated from the eye lens, as well as in models of fiber-cell membranes, the discrimination by oxygen transport (DOT) method will be used. The DOT method, which is based on electron paramagnetic resonance (EPR) spin-labeling techniques, permits discrimination of different membrane domains and gives information about structure and molecular dynamics as a function of the membrane depth in coexisting domains without the need for their separation. It also allows information about oxygen transport within and across membrane domains to be obtained. PUBLIC HEALTH RELEVANCE: Age-related cataracts are a major cause of blindness in developing countries. The reason for the onset of cataracts is unknown, but a great deal of evidence suggests that the presence of the cholesterol crystalline domain in the eye lens helps to maintain transparency and prevent cataract formation. The proposed studies will generate important fundamental information about topographical and age-related differences in cholesterol- membrane interactions in the eye lens that will increase our understanding of the role cholesterol plays and, in turn, help contribute to the prevention of age-related nuclear cataracts.

描述（由申请人提供）：人眼晶状体的纤维细胞膜被胆固醇饱和（显示胆固醇与磷脂的摩尔比从皮质中的 1 到细胞核中高达 4），这导致形成这些膜内有不混溶的胆固醇结晶结构域 (CCD)。当胆固醇含量最高时，CCD 可以占据高达 50% 的细胞膜表面。这些情况存在于晶状体核中，其中胆固醇含量可显着超过膜的溶解度阈值。 CCD的出现通常是病理的征兆；然而，只有在眼睛的晶状体中，CCD 才能发挥积极的生理功能，保持晶状体的透明度，并可能防止白内障的形成。 该提案的长期目标是更好地了解胆固醇在纤维细胞膜中的功能。短期内，我们将 (i) 研究高胆固醇含量如何影响磷脂晶状体膜的横向组织，特别关注 CCD 的形成，以及 (ii) 检验 CCD 的存在决定特性的假设周围的大量磷脂-胆固醇膜。 (iii) 此外，实验将揭示晶状体膜的磷脂成分是否影响 CCD 的形成、尺寸和稳定性。 (iv)最后，在模型膜系统中开发和测试的方法将用于检测和表征在成熟、老化和白内障形成过程中晶状体纤维细胞膜中共存的胆固醇结晶和大量磷脂-胆固醇结构域。为了研究与眼晶状体分离的膜中的共存域以及纤维细胞膜模型中的共存域，将使用氧传输（DOT）方法进行区分。 DOT 方法基于电子顺磁共振 (EPR) 自旋标记技术，可以区分不同的膜域，并提供有关结构和分子动力学的信息，作为共存域中膜深度的函数，而无需将它们分离。它还允许获得有关膜域内和跨膜域的氧运输的信息。 公共卫生相关性：与年龄相关的白内障是发展中国家失明的主要原因。白内障发病的原因尚不清楚，但大量证据表明，眼睛晶状体中胆固醇结晶结构域的存在有助于保持透明度并防止白内障形成。拟议的研究将产生有关眼晶状体中胆固醇膜相互作用的地形和年龄相关差异的重要基本信息，这将增加我们对胆固醇作用的理解，进而有助于预防与年龄相关的核性白内障。