The human lens acts as a UV filter and to focus light onto the retina. Transparency and high refractive index are key biophysical properties that allow the lens to function properly. In order to maintain transparency, fiber cells must maintain close packing and protein solubility. Given the lack of blood supply to the lens, transport of water, ions, and neutral solutes are critical processes for maintenance of lens homeostasis. Moreover, since there is little turnover or repair of lens proteins, changes in protein structure, including transport proteins, accumulate with age. Our hypothesis is that posttranslational modifications of the lens water channel, MIP, increase with age resulting in impairment of protein function and cataract. The long-term objective of this work is to understand the etiology of senile nuclear cataracts on the molecular level so that prophylactic therapies can be developed. The design of methods for preventing these changes can only begin after i) understanding the changes in protein structure during normal fiber cell maturation, ii) identifying the modifications to lens proteins that are unique to diseased tissue, and iii) determining the structures responsible for impaired function. We propose to identify and quantify age-specific and cataract-specific lens protein modifications and to determine whether those molecular changes cause functional alterations. A multidisciplinary approach, including clinicians and basic scientists, is advanced in this proposal in which state-of-the-art mass spectrometric methods will be employed to characterize structural modifications of lens MIP isolated from aged and cataractous lenses. In addition, molecular biology approaches are presented to test for functional importance of modified residues.
Specific aims i nclude: 1) to identify posttranslational modifications in lens MIP isolated from dissected sections from normal lenses, 2) to identify MIP modifications in membranes isolated from clear and opaque tissue from single diseased lenses, and 3) to determine the role of specific modifications in impairment of water permeability. The results of this work are expected to provide new detailed information on the structure and function of the most abundant integral membrane protein in the lens, a critical need in the fundamental understanding of normal lens maturation and senile cataractogenesis.
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