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E. Posokhova, H. Song, M. Belcastro, L. R. Bigley, K. A. Martemyanov, M. Sokolov; Disruption of the Chaperonin CCT Function in Rods Causes Retinal Degeneration by Affecting Multiple Protein Networks. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1111.
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It was demonstrated in yeast- and cell culture-based assays that about 7% of newly translated cytosolic proteins interact with CCT (Chaperonin-Containing TCP-1) for proper folding and posttranslational stabilization. To address CCT function in specialized neurons, such as retinal photoreceptors, we created a CCT-deficiency in mouse rods, and identified the CCT-dependent pathways using quantitative proteomics.
The CCT-deficient mouse rods were created by the transgenic expression of N-terminally-truncated phosducin-like protein (PhLP1), an established dominant-negative CCT inhibitor, under the control of a rhodopsin promoter. The morphology of the CCT-deficient photoreceptors was monitored at different stages of postnatal development, using light- and electron microscopy. Photoreceptor responses to light stimulation were assessed by ERG. Proteins affected by transgenic manipulation were identified by comparative proteomics using both 2D-DIGE and iTRAQ labeling approaches followed by MALDI-TOF/TOF mass-spectrometric analysis. Differentially expressed proteins were validated by Western blotting.
We found that CCT-deficient photoreceptors were incapable of maintaining the outer segment compartments, which apparently was the cause of their rapid death. Investigation of the underlying causes by comparative proteomic analyses revealed that, prior to massive photoreceptor death, the CCT inhibition affected expression levels of ~ 200 proteins. We will describe groups of proteins encompassing distinct functional networks exhibiting significant up- or down-regulation in response to the CCT inhibition. Among those were selected phototransduction proteins, including rhodopsin, transducin and phosducin, and the outer segment structural proteins, peripherin and ROM, indicating that CCT is essential for outer segment maintenance and suggesting a potential mechanism of the photoreceptor degeneration.
Our studies reveal photoreceptor proteins, which depend on the chaperonin CCT for posttranslational stabilization, and that are essential for the formation of the rod outer segment and phototransduction. In addition, we present the first proteomic analysis of the changes in protein expression upon retina degeneration. These data provide important insights into the function of CCT in neurons and the mechanisms of retinal degeneration.
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