Quantitation of protein turnover in the human adult lens using the 14C bomb-pulse

描述（由申请人提供）：在健康人体中研究大分子的合成和修复是困难的。1955年至1963年的地面核武器试验产生了一个巨大的14 CO2爆炸脉冲，它迅速分布在地球仪周围，并内在地标记了生物圈中所有可交换的碳。自1963年《禁核试条约》以来，大气中的14 C炸弹脉冲呈指数级下降，平均寿命约为16年，这不是由于放射性衰变，而是由于扩散和与海洋和生物圈的平衡。所有活着的人都被标记了，所有有机大分子中的14 C浓度都可以灵敏地识别它们是何时合成的。事实上，我们都是长期准稳定同位素示踪研究的对象，在这项研究中，可以通过使用加速器质谱（AMS）来计算亚毫克样品中的单个14 C原子来确定分子合成的日期。 我们试图利用这种有效的分子计时器来建立，量化，并确定在健康的成年人的眼睛晶状体蛋白质营业额的具体性质。来自60岁以上晶状体的初步数据表明，老化核纤维细胞的晶状体蛋白质含有比细胞本身明显年轻的碳。这作为人核纤维细胞中体内蛋白质周转的直接证据，并验证了有争议的假设，即人眼透镜通过从头蛋白质产生在其核心（至少部分）保持稳态。我们将测量在爆炸脉冲峰值后形成的晶状体核芯中纯化蛋白质的碳周转率，再加上一些老化的对照。与从老年供体收集的数据相比，年轻晶状体中的蛋白质掺入将提供直接证据，证明当我们达到中年时，该速率是否会减弱。更好地了解透镜的维护可以提供一个有用的新工具，以了解和最终预防最常见的透镜病理形式，年龄相关性核性（ARN）白内障。 白内障手术的费用和需求已经使这种疾病成为我们陷入困境的医疗保健系统的最大负担之一。虽然手术干预是恢复视力的一种非常有效的手段，但如果我们能将白内障的发病时间推迟十年，那么对手术的需求将减少一半。我们将建立健康成人透镜中特定蛋白质周转的基线，并将周转率与年龄联系起来。白内障可被归类为与年龄相关的蛋白质折叠疾病，需要长期（~50年）稳定同位素示踪评估以揭示健康透镜用于维持其极长寿蛋白质的活力的基本机制。AMS使我们能够使用大气核试验产生的14 C的升高的组织负荷作为评估健康透镜组织中蛋白质的长期动态的手段。我们的初步数据表明，透镜核心的可溶性晶体蛋白质中蛋白质的周转率约为每年1%，表明透镜在以前被认为是衰老的组织区域中具有蛋白质修复机制。本研究的目的是牢固地建立、量化和鉴定健康晶状体中这种蛋白质交换的特定靶点，并引起全世界白内障研究人员对这种透镜生理学未知但似乎关键特征的关注。

DESCRIPTION (provided by applicant): Studying synthesis and repair of macromolecules in healthy humans is difficult. Above ground testing of nuclear weapons from 1955-1963 produced a large bomb-pulse of 14CO2, which was quickly distributed around the globe and intrinsically labeled all exchangeable carbon in the biosphere. Since the Test Ban Treaty in 1963, the atmospheric 14C bomb-pulse has been decreasing exponentially with a mean life of ~ 16 years, not due to radioactive decay, but due to diffusion and equilibration with the oceans and biosphere. All living humans have been labeled, and the 14C concentration in all organic macromolecules can sensitively identify when they were synthesized. In reality, we are all subjects in a long-term quasi-stable isotope tracer study in which molecular synthesis can be dated through the use of accelerator mass spectrometry (AMS) to count individual 14C atoms in sub-milligram samples. We seek to utilize this effective molecular chronometer to establish, quantify, and identify the specific nature of protein turnover in healthy adult human eye lenses. Preliminary data from lenses age 60 and greater suggests that the crystallin proteins of aged nuclear fiber cells contain carbon significantly younger than the cells themselves. This serves as direct evidence of in vivo protein turnover in human nuclear fiber cells and verifies the controversial hypothesis that the human eye lens maintains homeostasis at its core (at least in part) by de novo protein production. We will measure carbon turnover in purified proteins from the nuclear core of lenses formed after the peak of the bomb-pulse plus some aged controls. Compared with the data gathered from older donors, dating protein incorporation in younger lenses will provide direct evidence if this rate is attenuated when we reach middle age. A better understanding of lens maintenance could provide a useful new tool to understand and ultimately prevent the most common form of lens pathology, age-related nuclear (ARN) cataract. The expense and the demand for cataract surgery have catapulted this disease to become among the single largest burdens to our beleaguered healthcare system. Although surgical intervention is a highly effective means of restoring vision, if we could delay the onset of cataract by a decade the need for the procedure would drop by half. We will establish the baseline for specific protein turnover in the healthy adult human lens and relate the turnover rate to age. Cataracts, which can be classified as an age-related protein folding disease, require a long-term (~50 year) stable isotope tracer assessment to reveal the fundamental mechanisms a healthy lens employs to maintain the viability of its exceedingly long-lived proteins. AMS has enabled us to use elevated tissue loads of 14C produced by atmospheric nuclear testing as a means to evaluate the long term dynamics of protein in healthy lens tissue. Our preliminary data has demonstrated protein turnover at approximately 1% per year by mass in the soluble crystalline proteins of the lens core, indicating that the lens possesses a mechanism for protein repair in a region of tissue previously believed to be senescent. The aim of this study is to firmly establish, quantify, and identify the specific targets of this protein exchange in healthy lenses and bring the existence of this unknown but seemingly key feature of lens physiology to the attention of cataract researchers worldwide.