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

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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.