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

描述(申请人提供)：人眼晶状体的纤维细胞膜被胆固醇饱和(显示出皮质中胆固醇与磷脂的摩尔比从1到细胞核中高达4的比率)，这导致在这些膜中形成不相容的胆固醇结晶域(CCDs)。在胆固醇含量最高时，高达50%的细胞膜表面可以被CCDs占据。这些情况存在于晶状体核中，那里的胆固醇含量可能显著超过膜的溶解阈值。CCDs的出现通常是一种病理征象；然而，只有在眼睛的晶状体中，CCDs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。这项提议的长期目标是更好地理解胆固醇在纤维细胞膜中的作用。在短期内，我们将(I)研究高胆固醇含量如何影响磷脂晶状体膜的侧向组织，特别关注CCDs的形成，以及(Ii)检验CCDs的存在决定周围大量磷脂-胆固醇膜性质的假设。(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、大小和稳定性。(Iv)最后，在模型膜系统中开发和测试的方法将被用于检测和表征成熟、老化和白内障形成过程中晶状胆固醇和大量磷脂-胆固醇结构域共存的晶状体纤维细胞膜。为了研究从眼晶状体分离的膜中的共存区域，以及在纤维细胞膜的模型中，将使用氧传输区分(DOT)方法。基于电子顺磁共振(EPR)自旋标记技术的DOT方法可以区分不同的膜结构域，并提供关于共存结构域中膜深度的结构和分子动力学信息，而不需要分离它们。它还允许获得关于膜结构域内和跨膜结构域的氧运输的信息。 公共卫生相关性：老年性白内障是发展中国家失明的主要原因。白内障的发病原因尚不清楚，但大量证据表明，眼球晶状体中胆固醇结晶域的存在有助于保持透明度，防止白内障形成。拟议的研究将产生关于眼晶状体中胆固醇-膜相互作用的地形和年龄相关差异的重要基本信息，这些信息将增加我们对胆固醇所起作用的理解，并反过来有助于预防与年龄相关的核性白内障。

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.