The specific aims of this proposal are to determine the biochemical and biophysical properties of the eye lens crystallins and their involvement in the processes of cataractogenesis. Our main effort will focus on the effect of high hydrostatic pressure on the function, structure, and interactions of alpha crystallins. The alpha crystallins are key members of the small heat-shock protein family that in addition to their critical function in the lens, are also involved in many neurodegenerative diseases and cancers. High hydrostatic pressure is currently being used as a valuable tool for studying protein aggregation and amyloidosis. A variety of spectroscopical techniques are used at high hydrostatic pressure. However, circular dichroism (CD), a most useful, powerful and readily available technique could not be used at high pressure until we were successful in building a system by which we can perform CD measurements up to pressure of 2,500 bars. Our approach will allow us to obtain thermodynamic data from which we can get information about the state of cavities in alpha crystallin. Our hypothesis is that cavities and voids within the structure of alpha crystallin are essential to its stability and function.
The specific aims are: (1-i). Elucidate the secondary, tertiary, and quaternary structure of native alpha crystallins using high pressure CD spectroscopy. (1-ii). Determine under high hydrostatic pressures the conformational transitions that will occur in the alpha crystallins with the mutations that cause cataract, and obtain thermodynamic data for calculating changes in molar volume that reflect the state of the cavities within the protein. (2-i). Determine the chaperone-like properties of the native alpha crystallin, WT alpha A and B and phosphorylated mimics of alpha A and B using insulin and alcohol dehydrogenase as target proteins. (2-ii). Determine using high hydrostatic pressure, the interactions of native and WT alpha crystallins with mutant gamma crystallins that are known to cause cataract. Health relatedness of the project: The involvement of alpha crystallin in cataractogenesis and other diseases, and especially its chaperone properties for controlling aggregation make it a prime candidate to be used as a therapeutic agent. This project will add critical information for understanding its complex and dynamic structure and it's inherit capabilit to control amyloidosis as well as the potential of its cavities to deliver therapeutic molecules.
The goal of this project is to get structural information about a class of proteins (alpha crystallin) that are intimately involved in cataractogenesis and many other neurodegenerative diseases. The project utilizes a newly developed system that provides structural information that will help explain the properties of these proteins. The alpha crystallis have a potential of being used as therapeutic agents for controlling protein aggregation that take place in cataractogenesis and other diseases.
|Lerch, Michael T; LÃ³pez, Carlos J; Yang, Zhongyu et al. (2015) Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure. Proc Natl Acad Sci U S A 112:E2437-46|
|Lerch, Michael T; Horwitz, Joseph; McCoy, John et al. (2013) Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin. Proc Natl Acad Sci U S A 110:E4714-22|