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

描述(申请人提供)：研究健康人体内大分子的合成和修复是困难的。1955-1963年的地面核武器试验产生了14CO2的大爆炸脉冲，迅速分布在全球各地，并在本质上标记了生物圈中所有可交换的碳。自1963年《禁止核试验条约》以来，大气14C炸弹脉冲一直呈指数级下降，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。所有活着的人类都被标记了，所有有机大分子中的14C浓度可以灵敏地识别它们是在什么时候合成的。事实上，我们都是长期准稳定同位素示踪剂研究的对象，在该研究中，分子合成可以通过使用加速器质谱仪(AMS)来计算亚毫克样品中的单个14C原子来确定。 我们试图利用这种有效的分子计时器来建立、量化和鉴定健康成人眼晶状体中蛋白质周转的特定性质。来自60岁及以上的晶状体的初步数据表明，老化的核纤维细胞的晶体蛋白含有比细胞本身年轻得多的碳。这是人类核纤维细胞体内蛋白质周转的直接证据，并验证了有争议的假说，即人眼晶状体通过从头产生蛋白质来维持其核心的动态平衡(至少部分)。我们将测量原子弹脉冲峰值后形成的晶状体核心的纯化蛋白质中的碳周转率，以及一些老化的对照组。与从老年捐献者那里收集的数据相比，年轻晶状体中的测年蛋白掺入将提供直接证据，如果我们到了中年，这一比率会减弱。更好地了解晶状体的维护可以提供一个有用的新工具来了解并最终预防最常见的晶状体病理形式-年龄相关核性白内障。白内障手术的费用和需求使这种疾病成为我们陷入困境的医疗体系的最大负担之一。虽然手术干预是恢复视力的一种非常有效的方法，但如果我们可以将白内障的发病推迟十年，那么对手术的需求将减少一半。我们将建立健康成人晶状体中特定蛋白质周转率的基线，并将周转率与年龄联系起来。AMS使我们能够利用大气核试验产生的高14C组织负荷作为评估健康晶状体组织中蛋白质长期动态的一种手段。我们的初步数据表明，晶状体核心可溶结晶蛋白中的蛋白质周转率每年约为1%，这表明晶状体在先前被认为是衰老的组织区域具有蛋白质修复机制。这项研究的目的是确定、量化和确定健康晶状体中这种蛋白质交换的特定靶点，并使这一未知但似乎是关键的晶状体生理学特征的存在引起世界各地的白内障研究人员的注意。

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.